U.S. patent application number 11/751261 was filed with the patent office on 2008-12-18 for meth1 and meth2 polynucleotides and polypeptides.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to James A. Fornwald, Gregg A. Hastings, Luisa Iruela-Arispe, Zdenka L. Jonak, Steven M. Ruben, Jonathan A. Terrett, Stephen H. Trulli.
Application Number | 20080312146 11/751261 |
Document ID | / |
Family ID | 46279490 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080312146 |
Kind Code |
A1 |
Iruela-Arispe; Luisa ; et
al. |
December 18, 2008 |
METH1 and METH2 Polynucleotides and Polypeptides
Abstract
The present invention relates to novel anti-angiogenic proteins,
related to thrombospondin. More specifically, isolated nucleic acid
molecules are provided encoding human METH1 and METH2. METH1 and
METH2 polypeptides are also provided, as are vectors, host cells
and recombinant methods for producing the same. Also provided are
diagnostic methods for the prognosis of cancer and therapeutic
methods for treating individuals in need of an increased amount of
METH1 or METH2. Also provided are methods for inhibiting
angiogenesis using METH1 or METH2.
Inventors: |
Iruela-Arispe; Luisa; (Los
Angeles, CA) ; Hastings; Gregg A.; (Westlake Village,
CA) ; Ruben; Steven M.; (Brookeville, MD) ;
Jonak; Zdenka L.; (Devon, PA) ; Trulli; Stephen
H.; (Havertown, PA) ; Fornwald; James A.;
(Norristown, PA) ; Terrett; Jonathan A.;
(Abingdon, GB) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
Beth Israel Deaconess Medical Center
Boston
MA
SmithKline Beecham Corp.
Philadelphia
PA
|
Family ID: |
46279490 |
Appl. No.: |
11/751261 |
Filed: |
May 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09373658 |
Aug 13, 1999 |
7220557 |
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11751261 |
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09318208 |
May 25, 1999 |
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09373658 |
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09235810 |
Jan 22, 1999 |
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09318208 |
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08845496 |
Apr 24, 1997 |
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09373658 |
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60098539 |
Aug 28, 1998 |
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60072298 |
Jan 23, 1998 |
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60147823 |
Aug 10, 1999 |
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60144882 |
Jul 20, 1999 |
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Current U.S.
Class: |
514/1.1 ;
435/320.1; 435/325; 435/455; 435/69.1; 514/13.3; 530/350;
536/23.1 |
Current CPC
Class: |
C07K 14/4702 20130101;
A61P 9/00 20180101; C07K 2319/00 20130101; C12N 9/6489 20130101;
A61K 48/00 20130101; C07K 14/78 20130101; C12N 2799/026 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
536/23.1; 435/320.1; 435/325; 435/455; 435/69.1; 530/350 |
International
Class: |
A61K 38/00 20060101
A61K038/00; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 5/06 20060101 C12N005/06; A61P 9/00 20060101
A61P009/00; C12N 15/87 20060101 C12N015/87; C12P 21/04 20060101
C12P021/04; C07K 14/00 20060101 C07K014/00 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
selected from the group consisting of: (a) a polynucleotide
encoding amino acids 1 to 950 in SEQ ID NO:2; (b) a polynucleotide
encoding amino acids 2 to 950 in SEQ ID NO:2; (c) a polynucleotide
encoding amino acids 29 to 950 in SEQ ID NO:2; (d) a polynucleotide
encoding amino acids 30 to 950 in SEQ ID NO:2; (e) a polynucleotide
encoding the complete amino acid sequence encoded by the cDNA clone
contained in ATCC.TM. Deposit No. 209581; (f) a polynucleotide
encoding the mature amino acid sequence encoded by the cDNA clone
contained in ATCC.TM. Deposit No. 209581; (g) a polynucleotide
encoding amino acids 1 to 968 of SEQ ID NO:125; (h) a
polynucleotide encoding amino acids 235 to 459 in SEQ ID NO:2; (i)
a polynucleotide encoding amino acids 460 to 544 in SEQ ID NO:2;
(j) a polynucleotide encoding amino acids 545 to 598 in SEQ ID
NO:2; (k) a polynucleotide encoding amino acids 841 to 894 in SEQ
ID NO:2; (l) a polynucleotide encoding amino acids 895 to 934 in
SEQ ID NO:2; (m) a polynucleotide encoding amino acids 536 to 613
in SEQ ID NO:2; and (n) a polynucleotide encoding amino acids 549
to 563 in SEQ ID NO:2.
2. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector in operable
linkage to a promoter.
3. A recombinant vector produced by the method of claim 2.
4. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 3 into a host cell.
5. A recombinant host cell produced by the method of claim 4.
6. A recombinant method for producing a polypeptide, comprising
culturing the recombinant host cell of claim 5 under conditions
such that said polypeptide is expressed and recovering said
polypeptide.
7. (canceled)
8. An isolated nucleic acid molecule comprising a polynucleotide
selected from the group consisting of: (a) a polynucleotide
encoding amino acids 1 to 890 in SEQ ID NO:4; (b) a polynucleotide
encoding amino acids 2 to 890 in SEQ ID NO:4; (c) a polynucleotide
encoding amino acids 24 to 890 in SEQ ID NO:4; (d) a polynucleotide
encoding amino acids 112 to 890 in SEQ ID NO:4; (e) a
polynucleotide encoding the complete amino acid sequence encoded by
the cDNA clone contained in ATCC.TM. Deposit No. 209582; (f) a
polynucleotide encoding the mature amino acid sequence encoded by
the cDNA clone contained in ATCC.TM. Deposit No. 209582; (g) a
polynucleotide encoding amino acids 214 to 439 in SEQ ID NO:4; (h)
a polynucleotide encoding amino acids 440 to 529 in SEQ ID NO:4;
(i) a polynucleotide encoding amino acids 530 to 583 in SEQ ID
NO:4; (j) a polynucleotide encoding amino acids 837 to 890 in SEQ
ID NO:4; (k) a polynucleotide encoding amino acids 280 to 606 in
SEQ ID NO:4; and (l) a polynucleotide encoding amino acids 529 to
548 in SEQ ID NO:4.
9. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 8 into a vector in operable
linkage to a promoter.
10. A recombinant vector produced by the method of claim 9.
11. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 10 into a host
cell.
12. A recombinant host cell produced by the method of claim 11.
13. A recombinant method for producing a polypeptide, comprising
culturing the recombinant host cell of claim 12 under conditions
such that said polypeptide is expressed and recovering said
polypeptide.
14. (canceled)
15. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) amino acids 1 to 950 in
SEQ ID NO:2; (b) amino acids 2 to 950 in SEQ ID NO:2; (c) amino
acids 29 to 950 in SEQ ID NO:2; (d) amino acids 30 to 950 in SEQ ID
NO:2; (e) the complete amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 209581; (f) the a mature
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209581; (g) amino acids 1 to 968 of SEQ ID NO:125; (h)
amino acids 235 to 459 in SEQ ID NO:2; (i) amino acids 460 to 544
in SEQ ID NO:2; (j) amino acids 545 to 598 in SEQ ID NO:2; (k)
amino acids 841 to 894 in SEQ ID NO:2; (l) amino acids 895 to 934
in SEQ ID NO:2; (m) amino acids 536 to 613 in SEQ ID NO:2; and (n)
amino acids 549 to 563 in SEQ ID NO:2.
16. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: (a) amino acids 1 to 890 in
SEQ ID NO:4; (b) amino acids 2 to 890 in SEQ ID NO:4; (c) amino
acids 24 to 890 in SEQ ID NO:4; (d) amino acids 112 to 890 in SEQ
ID NO:4; (e) the complete amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 209582; (f) the mature
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209582; (g) amino acids 214 to 439 in SEQ ID NO:4; (h)
amino acids 440 to 529 in SEQ ID NO:4; (i) amino acids 530 to 583
in SEQ ID NO:4; (j) amino acids 837 to 890 in SEQ ID NO:4; (k)
amino acids 280 to 606 in SEQ ID NO:4; and (l) amino acids 529 to
548 in SEQ ID NO:4.
17-18. (canceled)
19. A method for inhibiting angiogenesis in an individual,
comprising administering an effective amount of METH1 or METH2.
20. The method of claim 19, wherein said method is used to treat
cancer, benign tumors, an ocular angiogenic disease, rheumatoid
arthritis, psoriasis, delayed wound healing, endometriosis,
vasculogenesis, granulations, hypertrophic scars, nonunion
fractures, scleroderma, trachoma, vascular adhesions, myocardial
angiogenesis, coronary collaterals, cerebral collaterals,
arteriovenous malformations, ischemic limb angiogenesis,
Osler-Webber Syndrome, plaque neovascularization, telangiectasia,
hemophiliac joints, angiofibroma, fibromuscular dysplasia, wound
granulation, Crohn's disease or atherosclerosis.
21. The method of claim 19, wherein said method is used in birth
control.
22. The method of claim 19, further comprising administering
another angiogenic compound.
23. The method of claim 19, wherein said METH1 or METH2 is
administered by cell or gene therapy means wherein cells have been
modified to produce and secrete METH1 or METH2.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/373,658, filed Aug. 13, 1999, which is a
continuation-in-part of U.S. application Ser. No. 09/318,208, filed
May 25, 1999 (now abandoned), which is a continuation-in-part of
U.S. application Ser. No. 09/235,810, filed Jan. 22, 1999 (now
abandoned), which claims benefit of U.S. Provisional Application
No. 60/098,539, filed Aug. 28, 1998, and U.S. Provisional
Application No. 60/072,298, filed Jan. 23, 1998. Said application
Ser. No. 09/373,658 is also a continuation-in-part of U.S.
application Ser. No. 08/845,496, filed Apr. 24, 1997 (now
abandoned). Said application Ser. No. 09/373,658 also claims
benefit of U.S. Provisional Application No. 60/147,823, filed Aug.
10, 1999 and U.S. Provisional Application No. 60/144,882, filed
Jul. 20, 1999. Each of the above-identified applications is
incorporated herein by reference in its entirety.
STATEMENT UNDER 37 C.F.R. .sctn. 1.77(b)(5)
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text document. The document is
entitled "PF453P2D1_SeqList.txt" (483,402 bytes, created May 18,
2007), and is hereby incorporated by reference herein in its
entirety.
FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
[0003] Part of the work performed during development of this
invention utilized U.S. Government funds. The U.S. Government has
certain rights in this invention.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention relates to novel anti-angiogenic
proteins, related to thrombospondin. More specifically, isolated
nucleic acid molecules are provided encoding human METH1 and METH2
(ME, for metalloprotease, and TH, for thrombospondin). METH1 and
METH2 polypeptides are also provided, as are vectors, host cells
and recombinant methods for producing the same. Also provided are
diagnostic methods for the prognosis of cancer and therapeutic
methods for treating individuals in need of an increased amount of
METH1 or METH2. Also provided are methods for inhibiting
angiogenesis using METH1 or METH2.
[0006] 2. Related Art
[0007] Angiogenesis, the formation of new blood vessels from
pre-existing vasculature, is a tightly regulated process in normal
adults. Under physiological circumstances, growth of new
capillaries is tightly controlled by an interplay of growth
regulatory proteins which act either to stimulate or to inhibit
blood vessel growth. Normally, the balance between these forces is
tipped in favor of inhibition and consequently blood vessel growth
is restrained. Under certain pathological circumstances, however,
local inhibitory controls are unable to restrain the increased
activity of angiogenic inducers. Angiogenesis is a key step in the
metastasis of cancer (Folkman, Nature Med. 1:27-31 (1995)) and in
abnormal wound healing, inflammation, rheumatoid arthritis,
psoriasis, and diabetic retinopathy, it is integral to the
pathology (Folkman et al., Science 235:442-447 (1987)), engendering
the hope that these pathological entities could be regulated by
pharmacological and/or genetic suppression of blood vessel growth
(Iruela-Arispe et al., Thromb. Haem. 78:672-677 1997)).
[0008] Thrombospondin-1 (TSP-1) is a 450 kDa, anti-angiogenic
adhesive glycoprotein released from activated platelets and
secreted by growing cells (reviewed in Adams, Int. J. Biochem.
Cell. Biol. 29:861-865 (1997)). TSP-1 is a homotrimer, with each
subunit comprised of a 1152 amino acid residue polypeptide,
post-translationally modified by N-linked glycosylation and
beta-hydroxylation of asparagine residues.
[0009] TSP-1 protein and mRNA levels are regulated by a variety of
factors. TSP-1 protein levels are downregulated by IL-1 alpha and
TNF alpha. TSP-1 mRNA and protein levels are upregulated by
polypeptide growth factors including PDGF, TGF-beta, and bFGF
(Bornstein, Faseb J. 6: 3290-3299 (1992)) and are also regulated by
the level of expression of the p53 tumor suppressor gene product
(Dameron et al., Science 265:1582-1584 (1994)). At least four other
members of the thrombospondin family have been identified: TSP-2,
TSP-3, TSP-4, and TSP-5 (also called COMP). There is a need in the
art to identify other molecules involved in the regulation of
angiogenesis.
SUMMARY OF THE INVENTION
[0010] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding the METH1
polypeptide having the amino acid sequence shown in SEQ ID NO:2 or
the amino acid sequence encoded by the cDNA clone deposited in a
bacterial host as ATCC.TM. Deposit Number 209581 on Jan. 15,
1998.
[0011] The present invention also provides isolated nucleic acid
molecules comprising a polynucleotide encoding the METH2
polypeptide having the amino acid sequence shown in SEQ ID NO:4 or
the amino acid sequence encoded by the cDNA clone deposited in a
bacterial host as ATCC.TM. Deposit Number 209582 on Jan. 15,
1998.
[0012] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of METH1 or METH2 polypeptides or
peptides by recombinant techniques.
[0013] The invention further provides an isolated METH1 or METH2
polypeptide having an amino acid sequence encoded by a
polynucleotide described herein.
[0014] The invention further provides a diagnostic method useful
during diagnosis or prognosis of cancer.
[0015] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of METH1
or METH2 activity in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an isolated METH1 or METH2 polypeptide of the invention
or an agonist thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino
acid (SEQ ID NO:2) sequences of METH1. The protein has a predicted
leader sequence of about 28 amino acid residues (underlined).
[0017] FIG. 2 shows the nucleotide (SEQ ID NO:3) and deduced amino
acid (SEQ ID NO:4) sequences of METH2. The protein has a predicted
leader sequence of about 23 amino acid residues (underlined).
[0018] FIG. 3 shows a comparison of the amino acid sequence of
METH1 (SEQ ID NO:2) and METH2 (SEQ ID NO:4) with that of their
closest homologue, a bovine metalloprotease (pNPI) (SEQ ID NO:5).
Identical amino acids are boxed. Functional domains predicted by
sequence and structural homology are labeled, including the signal
peptide (single line), the potential cleavage site for mammalian
subtilisin (double underlined), the zinc-binding-site (dotted line;
amino acids 383-395 in METH1 and 363-375 in METH2) in the
metalloprotease domain, and the putative disintegrin loops
(arrows).
[0019] FIG. 4 shows the primary structure of METH1, METH2 and pNPI
which includes a prodomain, a catalytic metalloprotease domain, a
cysteine rich disintegrin domain, a TSP-like domain, a spacer
region and a different number of TSP-like domains, three for METH1,
two for METH2, and four for pNPI.
[0020] FIG. 5 shows a comparison of the TSP-like domain of METH1
(SEQ ID NO:2) and METH2 (SEQ ID NO:4) with those of TSP1 (SEQ ID
NOs:6, 7, and 8) and TSP2 (SEQ ID NOs:9, 10, and 11), cysteines are
numbered 1 to 6, tryptophans are marked by asterisks.
[0021] FIG. 6 shows that peptides and recombinant protein derived
from the TSP-like domain of METH1 and METH2 block VEGF-induced
angiogenesis. Angiogenesis was induced on CAMs from 12-14-day-old
embryos using a nylon mesh containing VEGF casted on matrigel and
in the presence or absence of the peptides or recombinant protein.
Capillary density was evaluated as described in Example 4. Positive
and negative control included VEGF alone and vehicle alone,
respectively. (A) Quantification of the angiogenic response induced
by VEGF in the presence of recombinant proteins. TSP1, purified
platelet TSP1, GST, purified GST, GST-TSP1, GST-METH1, and
GST-METH2 are described in Example 4. (B) Quantification of the
angiogenic response induced by VEGF in the presence or absence of
the peptides; P-TSP1, P-METH1, and P-METH2 (peptide derived from
the Type I repeats of TSP, METH1 and METH2, respectively); SC1 and
SC2 are scramble peptides used as controls. (C) Dose-response of
the VEGF-induced angiogenesis in the presence of GST-METH1. (D)
Dose-response of the VEGF-induced angiogenesis in the presence of
GST-METH2. The angiogenic index was expressed considering the
vascular response from the VEGF-matrigel as 100% and subtracting
the background levels (matrigel alone). Assays were repeated, at
least, twice. Each treatment was done in triplicate. Values
represent the mean, bars indicate standard deviations.
*p<0.001.
[0022] FIG. 7 shows the effect of METH1 and METH2 recombinant
proteins on bFGF-stimulated cell proliferation. Cells were cultured
on 24-well plates in media containing bFGF and the recombinant
protein to be tested (3 .mu.g/ml, unless indicated in the graph).
Controls included vehicle or GST recombinant protein alone. (A),
HDEC, human dermal endothelial cells; (B), HMEC, human mammary
epithelial cells; (C), HDF, human dermal fibroblasts; (D), SMC,
smooth muscle cells; (E) Dose-response of GST-METH1 and GST-METH2
on HDEC proliferation. Experiments were repeated, at least, twice.
Each treatment was done in triplicate. Values represent the mean,
bars indicate standard deviations. *p<0.01.
[0023] FIG. 8 shows a schematic representation of the pHE4-5
expression vector (SEQ ID NO:12) and the subcloned METH1 or METH2
cDNA coding sequence. The locations of the kanamycin resistance
marker gene, the METH1 or METH2 coding sequence, the oriC sequence,
and the lacIq coding sequence are indicated.
[0024] FIG. 9 shows the nucleotide sequence of the regulatory
elements of the pHE promoter (SEQ ID NO:13). The two lac operator
sequences, the Shine-Delgarno sequence (S/D), and the terminal
HindIII and NdeI restriction sites (italicized) are indicated.
[0025] FIG. 10 shows an analysis of the METH1 amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown, and all were generated
using the default settings. In the "Antigenic Index or
Jameson-Wolf" graph, the positive peaks indicate locations of the
highly antigenic regions of the METH1 or METH2 protein, i.e.,
regions from which epitope-bearing peptides of the invention can be
obtained. The domains defined by these graphs are contemplated by
the present invention. Tabular representation of the data
summarized graphically in FIG. 10 can be found in Table 1.
[0026] FIG. 11 shows an analysis of the METH2 amino acid sequence.
Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown, and all were generated
using the default settings. In the "Antigenic Index or
Jameson-Wolf" graph, the positive peaks indicate locations of the
highly antigenic regions of the METH1 or METH2 protein, i.e.,
regions from which epitope-bearing peptides of the invention can be
obtained. The domains defined by these graphs are contemplated by
the present invention. Tabular representation of the data
summarized graphically in FIG. 11 can be found in Table 2.
TABLE-US-00001 TABLE 1 Garni . . . Garni . . . Garni . . . Garni .
. . Eisen . . . Karpl . . . Emini Res Pos. Alpha Chou- . . . Alpha
Beta Chou- . . . Beta Turn Chou- . . . Turn Coil Kyte- . . . Hydro
. . . Alpha Eisen . . . Beta Flexi . . . James . . . Antig . . .
Surfa . . . Met 1 A A . . . . . 0.41 * . . -0.30 0.60 Gly 2 . A . .
. . C 0.91 * . . 0.50 0.81 Asn 3 A A . . . . . 0.71 * . . 0.75 1.24
Ala 4 A A . . . . . 0.89 * . . 1.09 1.26 Glu 5 A A . . . . . 0.93 *
. F 1.58 1.97 Arg 6 . A B . . . . 1.23 . . F 1.92 1.21 Ala 7 . . B
. . T . 1.69 . . F 2.66 1.61 Pro 8 . . . . T T . 1.39 . . F 3.40
1.82 Gly 9 . . . . T T . 1.28 . . F 3.06 1.25 Ser 10 . . . . T T .
0.93 . . F 2.42 1.07 Arg 11 . . . . T T . 0.61 . * F 1.93 0.68 Ser
12 . . . . T T . 0.34 * . F 1.74 1.07 Phe 13 . . B . . T . 0.34 * .
F 0.25 0.59 Gly 14 . . B . . T . 0.38 * . F 0.25 0.47 Pro 15 . . B
B . . . -0.13 * . F -0.45 0.50 Val 16 . . B B . . . -1.06 * . F
-0.45 0.48 Pro 17 . . B B . . . -1.57 . . F -0.45 0.40 Thr 18 . A B
. . . . -1.68 . . F -0.45 0.21 Leu 19 . A B . . . . -1.92 . . .
-0.60 0.24 Leu 20 A A . . . . . -2.30 . . . -0.60 0.15 Leu 21 A A .
. . . . -2.03 . . . -0.60 0.11 Leu 22 A A . . . . . -2.63 . . .
-0.60 0.13 Ala 23 A A . . . . . -3.13 . . . -0.60 0.13 Ala 24 A A .
. . . . -2.91 . . . -0.60 0.13 Ala 25 A A . . . . . -2.96 . . .
-0.60 0.16 Leu 26 A A . B . . . -2.44 . . . -0.60 0.12 Leu 27 A A .
B . . . -1.63 . . . -0.60 0.16 Ala 28 A A . B . . . -1.63 . . .
-0.30 0.26 Val 29 A A . B . . . -1.86 . . . -0.30 0.32 Ser 30 A A .
. . . . -1.61 * * . -0.30 0.32 Asp 31 A A . . . . . -0.69 * * F
-0.15 0.31 Ala 32 A A . . . . . -0.09 * F 0.75 0.83 Leu 33 . A . .
. . C 0.20 * . F 1.55 0.96 Gly 34 . A . . . . C 1.06 * * F 1.85
0.77 Arg 35 . . . . . T C 1.36 * * F 2.70 1.32 Pro 36 . . . . . T C
1.36 * * F 3.00 2.76 Ser 37 . . . . . T C 1.94 * . F 2.70 4.66 Glu
38 A . . . . T . 2.76 * . F 2.20 4.12 Glu 39 A A . . . . . 2.29 * *
F 1.50 4.61 Asp 40 A A . . . . . 1.32 * * F 1.20 2.84 Glu 41 A A .
. . . . 0.68 . . F 0.90 1.22 Glu 42 A A . . . . . 0.77 . . F 0.75
0.52 Leu 43 A A . . . . . 0.77 . . . 0.60 0.48 Val 44 A A . . . . .
-0.04 . . . 0.60 0.48 Val 45 A A . . . . . -0.04 * . . -0.30 0.23
Pro 46 A A . . . . . 0.07 * . . -0.30 0.48 Glu 47 A . . . . . .
-0.52 * . F 1.10 1.27 Leu 48 A . . . . . . 0.08 * . F 1.41 1.73 Glu
49 A . . . . . . 0.59 * . F 1.72 1.73 Arg 50 A . . . . . . 1.41 * .
F 1.88 0.99 Ala 51 A . . . . T . 1.28 * . F 2.24 1.64 Pro 52 . . .
. T T . 0.97 * . F 3.10 0.93 Gly 53 . . . . T T . 1.47 * * F 2.49
0.69 His 54 . . . . . T C 1.58 * * F 1.38 0.98 Gly 55 . . . . . . C
0.66 * * F 1.62 1.25 Thr 56 . . . . . . C 1.36 . * F 0.71 1.04 Thr
57 . A B . . . . 0.76 . * F 0.60 1.49 Arg 58 . A B . . . . 1.07 . *
F 0.60 1.25 Leu 59 . A B . . . . 0.51 . * . 0.45 1.17 Arg 60 . A B
. . . . 0.16 . * . 0.30 0.82 Leu 61 . A B . . . . 0.47 . * . -0.30
0.36 His 62 . A B . . . . 0.78 . * . -0.30 0.74 Ala 63 A A . . . .
. 0.67 . * . 0.30 0.65 Phe 64 A A . . . . . 0.67 . * . -0.15 1.37
Asp 65 A A . . . . . 0.56 . * F -0.15 0.83 Gln 66 A A . . . . .
0.56 . * F 0.60 1.37 Gln 67 A A . . . . . 0.59 . * F 0.60 1.30 Leu
68 A A . . . . . 0.37 * * F 0.90 1.35 Asp 69 A A . . . . . 1.18 * *
. 0.30 0.64 Leu 70 . A B . . . . 0.97 . * . 0.94 0.73 Glu 71 . A B
. . . . 0.97 . * . 1.43 1.37 Leu 72 . A B . . . . 0.67 . * . 1.77
1.37 Arg 73 . . . . . T C 1.18 * * F 2.86 2.22 Pro 74 . . . . T T .
0.48 * * F 3.40 1.72 Asp 75 . . . . T T . 0.48 . * F 2.76 1.80 Ser
76 . . . . . T C -0.11 . * F 2.07 0.76 Ser 77 . . B . . . . 0.49 *
* F 0.73 0.50 Phe 78 . B . . . . 0.03 * * . 0.24 0.46 Leu 79 . . B
. . . . -0.46 . . . -0.40 0.34 Ala 80 . . B . . T . -0.77 . . .
-0.20 0.22 Pro 81 . . B . . T . -1.28 . . . -0.20 0.37 Gly 82 . . .
. T T . -0.98 . . . 0.20 0.37 Phe 83 . . B . . T . -0.28 . . .
-0.20 0.63 Thr 84 . . B B . . . -0.32 . . . -0.60 0.65 Leu 85 . . B
B . . . -0.08 * * . -0.60 0.49 Gln 86 . . B B . . . 0.24 * . .
-0.29 0.56 Asn 87 . . B . . T . 0.63 * . F 0.87 0.76 Val 88 . . B .
. T . 1.03 * * F 1.93 1.84 Gly 89 . . . . . T C 1.00 * . F 2.74
1.42 Arg 90 . . . . T T 1.51 * . F 3.10 0.87 Lys 91 . . . . . T C
1.51 * . F 2.74 1.58 Ser 92 . . . . . T C 1.20 * . F 2.43 2.76 Gly
93 . . . . . T C 1.84 . . F 2.38 2.04 Ser 94 . . . . . T C 1.38 . .
F 2.33 1.57 Glu 95 . . . . . . C 1.06 . . F 1.63 0.97 Thr 96 . . .
. . . C 1.01 . . F 2.04 1.51 Pro 97 . . . . . . C 1.00 . . F 2.60
1.96 Leu 98 . . . . . . C 1.34 . . F 2.04 1.63 Pro 99 A . . . . . .
0.83 . . F 1.58 1.89 Glu 100 A A . . . . . 0.24 . . F 1.12 1.01 Thr
101 A A . . . . . 0.52 . . F 0.86 1.23 Asp 102 A A . . . . . 0.07 .
. F 0.60 1.08 Leu 103 A A . . . . . 0.18 . . . 0.30 0.34 Ala 104 A
A . . . . . 0.14 . . . -0.60 0.20 His 105 . A B . . . . -0.16 * . .
-0.60 0.19 Cys 106 . A B . . . . -0.19 * . . -0.60 0.31 Phe 107 . A
B . . . . -0.50 * . . -0.60 0.30 Tyr 108 . . B . . T . -0.54 . . .
-0.20 0.32 Ser 109 . . . . T T . 0.04 . * F 0.35 0.44 Gly 110 . . .
. T T . -0.27 . * F 0.35 0.82 Thr 111 . . . . T T . 0.40 . * F 0.59
0.52 Val 112 . . B B . . . 0.89 . * F 0.93 0.65 Asn 113 . . . B T .
. 0.83 . * F 1.72 1.01 Gly 114 . . . B . . C 0.83 . * F 1.61 0.94
Asp 115 . . . . . T C 0.59 . * F 2.40 1.69 Pro 116 . . . . . T C
0.31 . * F 2.16 1.06 Ser 117 . . . . . T C 0.58 . * F 1.92 1.08 Ser
118 A . . . . T . -0.23 . . F 1.33 0.66 Ala 119 A A . . . . . -0.19
. . . -0.06 0.35 Ala 120 A A . . . . . -1.00 . . . -0.30 0.35 Ala
121 A A . . . . . -1.46 . . . -0.60 0.22 Leu 122 A A . . . . .
-1.16 . . . -0.60 0.11 Ser 123 A A . . . . . -1.20 . . . -0.30 0.20
Leu 124 A A . . . . . -1.47 * * . -0.30 0.19 Cys 125 . A B . . . .
-0.77 * * . -0.30 0.17 Glu 126 . A B . . . . -0.52 * * . 0.30 0.25
Gly 127 A . . . . . . -0.30 * * F 0.65 0.30 Val 128 A . . . . .
-0.70 * * F 0.65 0.57 Arg 129 . . B . . . . -0.13 * * F 0.65 0.29
Gly 130 . . B B . . . -0.28 * * . -0.60 0.45 Ala 131 . . B B . . .
-1.09 * * . -0.60 0.50 Phe 132 . . B B . . . -1.09 * * . -0.60 0.21
Tyr 133 . . B B . . . -0.23 * * . -0.60 0.21 Leu 134 . A B B . . .
-0.93 * * . -0.60 0.36 Leu 135 . A B B . . . -0.83 . * . -0.60 0.42
Gly 136 A A . B . . . -0.94 . . . -0.60 0.42 Glu 137 A A . . . . .
-1.13 . . . -0.60 0.44 Ala 138 A A . B . . . -0.89 . . . -0.60 0.38
Tyr 139 . . B B . . . -0.29 . . . -0.60 0.66 Phe 140 . . B B . . .
-0.29 . . . -0.60 0.59 Ile 141 . . B B . . . -0.16 . . . -0.60 0.48
Gln 142 . . B B . . . -0.74 . . . -0.60 0.48 Pro 143 . . B B . . .
-0.74 . . . -0.60 0.55 Leu 144 . A . . . . C -0.80 * . . -0.40 0.80
Pro 145 . A . . . . C -0.10 * . . -0.10 0.62 Ala 146 A A . . . . .
0.90 * * . 0.30 0.69 Ala 147 A A . . . . . 0.09 * . . 0.75 1.64 Ser
148 A A . . . . . -0.29 * . F 0.75 0.88 Glu 149 A A . . . . . 0.21
* . F 0.45 0.88 Arg 150 A A . . . . . -0.17 * . F 0.60 1.25 Leu 151
A A . . . . . -0.17 * . . 0.30 0.94 Ala 152 A A . . . . . 0.21 * *
. 0.30 0.55 Thr 153 A A . . . . . 0.17 * * . 0.04 0.43 Ala 154 A A
. . . . . 0.17 . . . 0.08 0.52 Ala 155 . . . . . T C 0.10 . * F
2.07 0.89 Pro 156 . . . . . T C 0.70 . . F 2.86 1.24 Gly 157 . . .
. T T . 1.08 . . F 3.40 1.90 Glu 158 . . . . . T C 0.80 . . F 2.86
2.90 Lys 159 . . . . . . C 1.18 . . F 2.32 1.90 Pro 160 . . . . . .
C 0.96 . * F 1.98 2.97 Pro 161 . . . . . . C 1.17 . * F 1.64 1.41
Ala 162 A A . . . . . 0.81 . * F 0.60 1.22 Pro 163 A A . . . . .
0.78 . * . -0.60 0.68 Leu 164 A A . . . . . -0.08 . * . -0.60 0.60
Gln 165 A A . . . . . -0.68 * * . -0.60 0.49 Phe 166 . A B . . . .
-0.36 * * . -0.60 0.26 His 167 . A B . . . . 0.34 * * . -0.26 0.62
Leu 168 . A B . . . . 0.56 * * . 0.38 0.70 Leu 169 . A B . . . .
1.48 * * . 0.87 1.31 Arg 170 . . . . T T . 1.48 * . F 3.06 1.88 Arg
171 . . . . T T . 1.83 * . F 3.40 3.96 Asn 172 . . . . T T . 1.87 *
. F 3.06 4.75 Arg 173 . . . T T . 1.82 * . F 2.72 4.05 Gln 174 . .
. . T . . 2.29 . . F 2.43 1.53 Gly 175 . . . . T . . 1.83 . . F
2.19 0.94 Asp 176 . . . . T T . 1.41 . * F 2.30 0.48 Val 177 . . B
. . T 0.74 * . F 1.85 0.40 Gly 178 . . . . T T . 0.29 * . F 2.50
0.22 Gly 179 . . B . . T . -0.57 . * F 1.85 0.13 Thr 180 . . B B .
. . -1.08 . * F 0.30 0.13 Cys 181 . . B B . . . -1.08 . . . -0.10
0.10 Gly 182 . . B B . . . -0.22 . . . -0.05 0.16 Val 183 . . B B .
. . 0.12 . . . 0.30 0.19 Val 184 . . B B . . . 0.26 * * . 0.90 0.60
Asp 185 . . B . . T . 0.68 * * F 1.75 0.94 Asp 186 . . B . . T .
1.13 * * F 2.20 2.49 Glu 187 . . B . . T . 1.17 * * F 2.50 5.18 Pro
188 . . . . . T C 1.68 * * F 3.00 4.48 Arg 189 . . . . . T C 2.58 *
* F 2.70 2.66 Pro 190 . . . . . T C 1.99 * * F 2.40 3.07 Thr 191 .
. . . . T C 1.99 * * F 2.10 2.00 Gly 192 . . . . . T C 1.68 * * F
1.80 1.77 Lys 193 A A . . . . . 1.89 * * F 0.90 1.65 Ala 194 A A .
. . . . 1.78 * * F 0.90 1.98 Glu 195 A A . . . . . 1.99 . * F 0.90
3.35 Thr 196 A A . . . . . 2.30 . * F 0.90 2.90 Glu 197 A A . . . .
. 2.64 . * F 0.90 4.79 Asp 198 A A . . . . . 2.26 . * F 0.90 4.79
Glu 199 A A . . . . . 2.53 . . F 0.90 3.29 Asp 200 A . . . . T .
2.53 . . F 1.30 2.74 Glu 201 A . . . . T . 2.50 . . F 1.30 2.84 Gly
202 A . . . . T . 2.50 . . F 1.30 1.62 Thr 203 A . . . . T . 2.50 .
. F 1.30 1.68 Glu 204 A A . . . . . 2.50 * . F 0.90 1.62 Gly 205 A
A . . . . . 2.16 * . F 1.20 2.84 Glu 206 A A . . . . . 1.94 * . F
1.50 1.95 Asp 207 . A . . T . . 2.29 * . F 2.20 1.74 Glu 208 . A .
. . . C 2.31 * . F 2.30 3.04 Gly 209 . . . . . T C 2.01 * . F 3.00
1.85 Pro 210 . . . . T T . 2.14 . . F 2.60 1.48 Gln 211 . . . . T T
. 2.14 . . F 2.30 1.32 Trp 212 . . . . . T C 2.14 . . F 1.44 2.32
Ser 213 . . . . . . C 1.93 . . F 1.78 2.50 Pro 214 . . . . T T .
1.69 . . F 2.12 2.23 Gln 215 . . . . . T C 1.09 . . F 1.56 2.15 Asp
216 . . . . . T C 1.09 . * F 2.40 1.32 Pro 217 . . . . . T C 1.03 .
. F 2.16 1.48 Ala 218 . . . . T . . 0.48 . . F 1.77 0.85 Leu 219 .
. B . . . . 0.34 * . F 0.53 0.38 Gln 220 . . B . . . . 0.34 * . F
-0.01 0.24 Gly 221 . . B . . T . 0.13 * * F -0.05 0.41 Val 222 . .
B . . T . 0.03 * . F -0.05 0.77 Gly 223 . . B . . T . 0.28 * . F
0.25 0.64 Gln 224 . . B . . T . 0.78 * * F 0.25 0.64 Pro 225 . . B
. . . . 0.43 . . F 0.20 1.25 Thr 226 . . . . T . . 0.48 . * F 0.60
1.25 Gly 227 . . . . . T C 0.44 * * F 0.45 0.97 Thr 228 . . B . . T
. 0.90 * * F 0.25 0.44 Gly 229 . . B . . T . 0.94 . * F 0.85 0.60
Ser 230 . . B . . T . 1.20 . * F 1.30 1.20 Ile 231 . A B . . . .
1.62 . * F 0.90 1.67 Arg 232 . A B . . . . 1.27 . * F 0.90 3.30 Lys
233 . A B . . . . 0.72 . . F 0.90 2.13 Lys 234 . A B B . . . 0.77 .
. F 0.90 2.26 Arg 235 . A B B . . . 0.77 . . F 0.90 1.55 Phe 236 .
. B B . . . 1.62 . * . 0.75 1.04 Val 237 . . B B . . . 1.62 . * .
0.30 0.71 Ser 238 . . B . . T . 1.33 * * . 0.70 0.71 Ser 239 . . .
. . T C 0.43 * . . 0.15 1.28 His 240 . . . . . T C 0.32 * * . 0.45
1.28 Arg 241 . . . . . T C 0.71 * . . 1.05 1.65
Tyr 242 A . . B . . . 0.97 * . . 0.45 1.78 Val 243 A . . B . . .
0.46 * . . 0.45 1.29 Glu 244 . . B B . . . -0.10 * . . -0.30 0.54
Thr 245 . . B B . . . -0.66 * . . -0.60 0.26 Met 246 A . B B . . .
-0.77 * . . -0.60 0.35 Leu 247 A . . B . . . -0.52 . . . 0.30 0.34
Val 248 A . . B . . . 0.03 . . . -0.30 0.41 Ala 249 A . . B . . .
-0.57 . . . -0.30 0.55 Asp 250 A . . . . T . -0.84 . . F 0.25 0.66
Gln 251 A . . . . T . -0.24 . . F 0.25 0.90 Ser 252 A . . . . T .
-0.13 . . F 1.30 1.54 Met 253 A . . . . T . 0.69 . * . 0.70 0.80
Ala 254 A . . . . . . 0.93 . * . -0.10 0.63 Glu 255 A . . . . . .
0.63 . * . -0.10 0.46 Phe 256 A . . . . . . 0.29 . * . -0.10 0.63
His 257 A . . . . T . -0.22 * . . 0.10 0.61 Gly 258 A . . . . T .
0.42 * . F 0.25 0.29 Ser 259 A . . . . T . 0.98 * * F 0.25 0.68 Gly
260 A . . . . T . 0.73 * * F 0.85 0.68 Leu 261 A A . . . . . 0.62 .
. F 0.00 1.07 Lys 262 A A . . . . . -0.16 . . . -0.60 0.66 His 263
. A B . . . . -0.12 * . . -0.60 0.55 Tyr 264 . A B . . . . -0.63 *
. . -0.60 0.96 Leu 265 . A B . . . . -0.99 * . . -0.60 0.40 Leu 266
. A B . . . . -0.48 * . . -0.60 0.25 Thr 267 . A B . . . . -1.38 *
. . -0.60 0.22 Leu 268 . A B . . . . -1.93 * . . -0.60 0.19 Phe 269
A A . . . . . -2.28 * * . -0.60 0.24 Ser 270 A A . . . . . -1.36 *
* . -0.60 0.17 Val 271 A A . . . . . -1.36 * * . -0.60 0.39 Ala 272
A A . . . . . -1.29 * * . -0.60 0.38 Ala 273 A A . . . . . -0.43 *
* . -0.60 0.44 Arg 274 A A . . . . . 0.23 * * . -0.15 1.18 Leu 275
A A . . . . . 0.32 * * . 0.45 1.59 Tyr 276 . . . . T . . 0.88 * * .
1.39 2.44 Lys 277 . . B . . . . 0.58 * * F 1.48 1.67 His 278 . . B
. . T . 1.28 . * F 1.12 1.42 Pro 279 . . B . . T . 1.17 . * F 2.36
1.77 Ser 280 . . . . T T . 1.68 . * F 3.40 1.43 Ile 281 . . B . . T
. 1.07 . * F 2.36 1.41 Arg 282 . . B B . . . 0.72 . * F 1.47 0.67
Asn 283 . . B B . . . -0.06 * * F 1.13 0.67 Ser 284 . . B B . . .
-0.70 * * F 0.19 0.79 Val 285 . . B B . . . -1.26 * * . -0.30 0.30
Ser 286 . . B B . . . -1.22 . * . -0.60 0.14 Leu 287 . . B B . . .
-1.29 . * . -0.60 0.08 Val 288 . . B B . . . -2.18 * . . -0.60 0.21
Val 289 . . B B . . . -2.69 . * . -0.60 0.11 Val 290 . . B B . . .
-2.69 . . . -0.60 0.11 Lys 291 . . B B . . . -3.28 . . . -0.60 0.11
Ile 292 . . B B . . . -2.50 . . . -0.60 0.10 Leu 293 . . B B . . .
-1.64 . * . -0.60 0.19 Val 294 . . B B . . . -0.79 . . . -0.30 0.16
Ile 295 . . B B . . . 0.07 . * . 0.00 0.39 His 296 A . . B . . .
0.07 . * . 0.90 0.81 Asp 297 A . . B . . . 0.61 . . F 1.80 2.19 Glu
298 A . . . . . . 1.21 * . F 2.30 3.09 Gln 299 . . . . T . . 2.07 *
. F 3.00 3.51 Lys 300 . . . . . . C 2.10 . . F 2.50 3.64 Gly 301 .
. . . . T C 1.82 . . F 2.40 1.56 Pro 302 . . . . . T C 1.52 . . F
2.10 1.30 Glu 303 . . B . . T . 1.52 * . F 1.45 0.87 Val 304 A . .
. . T . 0.93 * . F 1.00 1.42 Thr 305 A . . . . T . 0.30 . * F 0.85
0.93 Ser 306 A . . . . T . -0.17 . * F 0.85 0.54 Asn 307 A . . . .
T . -0.27 . * F -0.05 0.60 Ala 308 A . . . . T . -1.08 * * . -0.20
0.60 Ala 309 A . . . . . . -0.11 * * . -0.40 0.37 Leu 310 A . . . .
. . 0.20 * * . -0.10 0.45 Thr 311 . . B . . . . -0.20 * * . -0.10
0.72 Leu 312 . . B . . . . -0.87 * * . -0.40 0.61 Arg 313 . . B . .
. . -0.28 * * . -0.40 0.40 Asn 314 . . . . T . . 0.02 * * . 0.30
0.44 Phe 315 . . . . T T . 0.83 * * . 0.20 0.57 Cys 316 . . . . T T
. 1.19 * * . 0.20 0.50 Asn 317 . . . . T T . 2.00 * * . 0.20 0.62
Trp 318 . . . . T T . 1.86 * . . 0.35 1.25 Gln 319 . . . . T . .
1.86 . . . 0.45 3.16 Lys 320 . . . . T . . 2.34 * . F 0.60 3.16 Gln
321 . . . . T . . 2.80 . . F 0.94 4.65 His 322 . . . . . . C 2.50 *
. F 1.68 4.15 Asn 323 . . . . . . C 2.79 * . F 2.02 2.78 Pro 324 .
. . . . T C 2.90 . . F 2.56 2.68 Pro 325 . . . . T T . 2.86 * . F
3.40 3.86 Ser 326 . . . . . T C 2.27 . . F 2.86 4.01 Asp 327 . . .
. . T C 2.30 . . F 2.52 2.62 Arg 328 A A . . . . . 2.27 . . F 1.58
2.94 Asp 329 A A . . . . . 2.23 * . F 1.24 2.98 Ala 330 A A . . . .
. 2.44 * . . 0.90 2.80 Glu 331 A A . . . . . 2.43 * . . 0.75 2.38
His 332 A . . . . T . 1.84 * . . 1.15 2.06 Tyr 333 A . . . . T .
0.84 * . . 0.85 2.06 Asp 334 A . . . . T . 0.03 . . . 0.70 0.83 Thr
335 A . . . . T . -0.08 . . . -0.20 0.51 Ala 336 A A . . . . .
-0.39 * . . -0.60 0.28 Ile 337 A A . . . . . -0.24 * . . -0.60 0.24
Leu 338 . A B . . . . 0.00 . . . -0.60 0.33 Phe 339 . A B . . . .
0.00 . * . -0.60 0.56 Thr 340 . A B . . . . -0.50 . . F 0.00 1.34
Arg 341 . A B . . . . -0.58 . * F 0.25 1.34 Gln 342 . A . . T . .
-0.03. . * F 1.35 0.83 Asp 343 . A . . T . . 0.48 . * F 1.60 0.57
Leu 344 . A . . T . . 1.18 * . F 2.15 0.39 Cys 345 . . . . T T .
1.18 . * F 2.50 0.39 Gly 346 . . . . T T . 0.40 . * F 2.25 0.34 Ser
347 . . . . T T . 0.40 . . F 1.10 0.22 Gln 348 . . B . . T . 0.09 .
. F 1.35 0.68 Thr 349 . . B . . . . 0.09 . . F 0.90 0.99 Cys 350 .
. B . . . . 0.41 . . F 0.05 0.61 Asp 351 . . B . . T . 0.16 * . F
0.25 0.35 Thr 352 . . B . . T . -0.13 . . F 0.25 0.24 Leu 353 . . B
. . T . -0.13 . . . 0.10 0.45 Gly 354 . . B . . T . -0.68 . . .
0.70 0.45 Met 355 . . B . . . . -0.36 . . . -0.10 0.23 Ala 356 . .
B . . . . -0.67 . . . -0.10 0.28 Asp 357 . . B . . T . -1.21 . . .
0.10 0.41 Val 358 . . B . . T . -1.07 . . . 0.10 0.30 Gly 359 . . B
. . T . -0.72 . . . 0.10 0.16 Thr 360 . . B . . T -0.33 . . . 0.70
0.16 Val 361 . . B . . . . -0.04 . * . 0.24 0.34 Cys 362 . . B . .
. . 0.07 * . . 1.18 0.46 Asp 363 . . B . . T . 0.62 * . F 1.87 0.62
Pro 364 . . . . T T . 0.30 * . F 3.06 1.12 Ser 365 . . . . T T .
0.31 * . F 3.40 1.12 Arg 366 . . . . T T . 0.31 * . F 2.91 0.90 Ser
367 . . . B T . . 0.09 * . F 1.87 0.43 Cys 368 . . B B . . . 0.09 *
. . 0.38 0.22 Ser 369 . . B B . . . 0.30 * . . 0.64 0.20 Val 370 .
. B B . . . 0.60 * . . 0.30 0.25 Ile 371 . . B B . . . 0.14 * . .
0.60 0.77 Glu 372 . . B B . . . -0.37 . . . 0.60 0.57 Asp 373 A . .
. . T . 0.30 . . F 1.15 0.63 Asp 374 A . . . . T . 0.01 * . . 1.30
1.56 Gly 375 A . . . . T . 0.28 . . . 1.00 0.91 Leu 376 A . . . . T
. 0.47 * . . 0.70 0.55 Gln 377 A A . . . . . 0.16 . . . -0.30 0.29
Ala 378 A A . . . . . -0.16 * . . -0.60 0.42 Ala 379 A A . . . . .
-0.74 * . . -0.60 0.73 Phe 380 A A . . . . . -0.43 * . . -0.60 0.43
Thr 381 A A . . . . . 0.38 * * . -0.60 0.57 Thr 382 A A . . . . .
-0.43 * . . -0.30 0.98 Ala 383 A A . . . . . -0.19 * . . -0.60 0.94
His 384 A A . . . . . 0.37 * . . -0.30 0.64 Glu 385 A A . . . . .
0.21 * . . -0.30 0.61 Leu 386 A A . . . . . -0.18 * . . -0.30 0.45
Gly 387 A . . B . . . 0.13 * . . -0.60 0.28 His 388 A . . B . . .
0.12 * . . -0.60 0.26 Val 389 A . . B . . . -0.06 * . . -0.60 0.32
Phe 390 A . . B . . . -0.09 * . . -0.60 0.49 Asn 391 . . B B . . .
0.72 * . . -0.60 0.49 Met 392 . . B . . T . 1.07 * . . 0.25 1.11
Pro 393 A . . . . T . 0.51 * . . 0.85 2.14 His 394 . . . . T T .
1.41 * . F 1.70 1.34 Asp 395 A . . . . T . 2.11 * . F 1.30 2.72 Asp
396 A A . . . . . 1.44 * . F 0.90 3.04 Ala 397 A A . . . . . 1.46 *
. F 0.90 1.20 Lys 398 A A . . . . . 1.37 * * F 0.75 0.73 Gln 399 A
A . . . . . 0.59 . * . 0.60 0.58 Cys 400 . A B . . . . 0.59 . * .
-0.30 0.48 Ala 401 . A B . . . . 0.24 . * . 0.30 0.38 Ser 402 . . B
. . T . -0.02 . * . 0.10 0.22 Leu 403 . . B . . T . -0.07 . . .
0.04 0.30 Asn 404 . . . . T T . -0.07 . . . 0.68 0.48 Gly 405 . . .
. T T . 0.60 . . F 1.37 0.62 Val 406 . . . . . . C 0.89 . . F 1.96
1.26 Asn 407 . . . . . T C 1.16 . . F 2.40 1.05 Gln 408 A . . . . T
. 1.37 * . F 1.96 1.44 Asp 409 A . . . . T . 0.77 * . F 1.72 1.92
Ser 410 A . . . . T . 0.52 . . . 1.33 1.18 His 411 A A . . . . .
1.08 . * . -0.06 0.69 Met 412 A A . . . . . 0.48 . . . 0.30 0.55
Met 413 A A . . . . . -0.33 . . . -0.60 0.41 Ala 414 A A . . . . .
-0.63 . . . -0.60 0.25 Ser 415 A A . . . . . -0.33 * . . -0.60 0.34
Met 416 A A . . . . . -1.11 * * . -0.60 0.55 Leu 417 A . . . . T .
-0.51 * . . -0.20 0.45 Ser 418 A . . . . T . 0.06 * . . 0.38 0.56
Asn 419 A . . . . T . 0.34 . . . 0.66 0.76 Leu 420 . . . . . T C
0.64 . . . 1.29 1.24 Asp 421 . . . . T T . 1.03 . . . 2.37 1.60 His
422 . . . . T T . 1.56 . . F 2.80 1.54 Ser 423 . . . . . T C 1.56 .
. F 1.72 1.97 Gln 424 . . . . . T C 1.34 . . F 1.44 1.58 Pro 425 .
. . . T . . 1.49 . . F 0.86 1.79 Trp 426 . . . . T . . 1.19 . . F
0.43 0.72 Ser 427 . . . . . T C 0.63 . . F 0.15 0.55 Pro 428 . . .
. T T . 0.69 . . F 0.35 0.36 Cys 429 . . . . T T . 0.09 . . . 0.20
0.54 Ser 430 . . B . . T . -0.59 . . . -0.20 0.40 Ala 431 . . B B .
. . -0.61 . . . -0.60 0.18 Tyr 432 . . B B . . . -0.61 . . . -0.60
0.49 Met 433 . . B B . . . -1.10 . . . -0.60 0.49 lle 434 . . B B .
. . -1.24 * . . -0.60 0.42 Thr 435 . . B B . . . -0.94 * . . -0.60
0.22 Ser 436 . . B B . . . -0.36 * . . -0.60 0.37 Phe 437 . . B B .
. . -0.46 * . . -0.60 0.85 Leu 438 . . B . . T . 0.11 * . F 0.56
0.58 Asp 439 . . . . T T . 0.66 * . F 1.27 0.59 Asn 440 . . . . . T
C 0.97 . . F 1.38 0.68 Gly 441 . . . . T T . 0.60 . . F 2.94 1.42
His 442 . . . . T T . 0.49 . . F 3.10 0.46 Gly 443 A . . . . T .
0.70 . . F 1.49 0.23 Glu 444 A . . . . T . 0.70 . . . 1.03 0.23 Cys
445 . . B . . T . 0.74 . * . 1.32 0.29 Leu 446 . A B . . . . 0.88 .
. . 1.25 0.58 Met 447 . A B . . . . 0.91 * . . 1.28 0.52 Asp 448 .
A . . T . . 1.26 * . F 2.02 1.67 Lys 449 . A . . . . C 1.04 * . F
2.16 3.26 Pro 450 . . . . T T . 0.82 * * F 3.40 5.10 Gln 451 . . .
. T T . 1.63 * * F 3.06 2.14 Asn 452 . . B . . T . 1.42 * * F 2.02
1.85 Pro 453 . . B . . T . 1.21 * * F 0.63 0.99 lle 454 . . B . . .
. 0.82 * * F 0.09 0.88 Gln 455 . . B . . . . 1.03 * * F -0.25 0.54
Leu 456 . . B . . T . 0.22 * * F 0.25 0.59 Pro 457 . . B . . T .
0.01 * * F 0.25 0.69 Gly 458 . . B . . T . -0.12 . * F 0.25 0.62
Asp 459 . . B . . T . 0.46 . * F 0.25 0.74 Leu 460 . . . . . T C
0.16 . * F 1.05 0.69 Pro 461 . . B . . T . 0.72 . * F 0.85 0.93 Gly
462 . . B . . T . 0.93 . . F 0.25 0.88 Thr 463 . . B . . T . 0.69 .
* F 0.74 1.78 Ser 464 . . B . . . . 0.69 * . F 1.48 1.16 Tyr 465 .
. . . T . . 1.61 * . F 2.22 1.88 Asp 466 . . . . T T . 1.82 . . .
2.61 2.56 Ala 467 . . . . T T . 1.50 * . F 3.40 3.31 Asn 468 . . .
. T T . 1.81 . * F 2.76 1.13 Arg 469 . . B . . T . 1.41 . * F 2.32
1.17 Gln 470 . . B B . . . 1.34 * * . 0.53 1.01 Cys 471 . . B B . .
. 0.64 * * . 0.04 0.90 Gln 472 . . B B . . . 0.89 . . . -0.60 0.40
Phe 473 . . B B . . . 0.89 . . . -0.26 0.23 Thr 474 . . B B . . .
0.78 . . . 0.08 0.74 Phe 475 . . . B T . . 0.48 . * . 1.72 0.71 Gly
476 . . . . T T . 1.19 . * F 2.76 1.10 Glu 477 . . . . T T . 1.16 .
* F 3.40 1.52 Asp 478 . . . . T T . 1.19 * . F 3.06 2.39 Ser 479 .
. . . T T . 1.29 * . F 2.72 1.30 Lys 480 . . . . T . . 1.99 * . F
2.43 1.16 His 481 . . . . T . . 1.74 * . F 2.34 1.16 Cys 482 . . .
. . T C 1.16 * . F 2.10 0.87 Pro 483 A . . . . T . 0.86 . . F 2.15
0.44 Asp 484 . . . . T T . 0.84 * . F 2.50 0.43 Ala 485 A . . . . T
. 0.13 * . F 2.00 1.17 Ala 486 A . . . . . . -0.13 . . F 1.40 0.41
Ser 487 . . B . T T . 0.22 . . F 1.75 0.33 Thr 488 . . B . . T .
-0.38 * . F 0.50 0.46 Cys 489 . . B . . T . -0.67 * . F -0.05 0.38
Ser 490 . . B . . T . -0.74 . . F -0.05 0.30 Thr 491 . . B B . . .
-0.47 . . . -0.60 0.11 Leu 492 . . B B . . . -0.51 . . . -0.60
0.30
Trp 493 . . B B . . . -0.51 . . . -0.60 0.22 Cys 494 . . B B . . .
-0.14 . . . -0.60 0.22 Thr 495 . . B B T . . -0.19 . . F -0.05 0.36
Gly 496 . . . B T . . -0.22 . . F -0.05 0.34 Thr 497 . . . . T T .
-0.27 . . F 0.65 0.62 Ser 498 . . . . T T . -0.79 . . F 0.65 0.32
Gly 499 . . . . T T . -0.98 . . F 0.35 0.27 Gly 500 . . . . T T .
-1.33 . . F 0.35 0.14 Val 501 . . B B . . . -0.99 . . . -0.60 0.05
Leu 502 . . B B . . . -0.99 . . . -0.60 0.10 Val 503 . . B B . . .
-0.64 . . . -0.60 0.14 Cys 504 . . B . . T . -0.33 . . . -0.20 0.38
Gln 505 . . B . . T . -0.69 . . . 0.10 0.62 Thr 506 . . B . . T .
-0.04 . . F 0.25 0.72 Lys 507 . . B . . T . 0.48 . . F 0.40 2.09
His 508 . . . . . . C 0.74 . . . -0.05 1.27 Phe 509 . . B . . . .
1.41 . . . -0.40 0.89 Pro 510 . . . . T . . 1.07 . . . 0.30 0.74
Trp 511 . . . . T T . 1.07 . . . 0.20 0.54 Ala 512 . . . . T T .
0.72 . . . 0.51 0.90 Asp 513 . . . . T T . 0.09 . . F 1.27 0.78 Gly
514 . . . . T T . 0.44 . . F 1.58 0.40 Thr 515 . . . . T T . 0.66 .
. F 2.49 0.39 Ser 516 . . . . T T . 0.60 . * F 3.10 0.40 Cys 517 .
. . . T T . 1.23 . .* F 2.49 0.40 Gly 518 . . . . T T . 0.94 . * F
2.48 0.56 Glu 519 . . . . T . . 0.62 . * F 1.67 0.44 Gly 520 . . .
. T . . 0.04 . * F 1.36 0.44 Lys 521 . . . . T . . 0.34 . * F 0.45
0.31 Trp 522 . . . . T . . 0.67 . * . 0.90 0.29 Cys 523 . . B . . T
. 1.06 . * . -0.20 0.29 lle 524 . . B . . T . 0.39 . * . 0.70 0.29
Asn 525 . . . . T T . -0.12 . * . 0.20 0.15 Gly 526 . . . . T T .
-0.17 * * F 0.65 0.20 Lys 527 . . . . T . . 0.17 * * F 0.45 0.47
Cys 528 . . . . T T . 0.52 . * . 1.40 0.58 Val 529 . . B . . T .
1.41 * * . 1.04 0.85 Asn 530 . . B . . T . 1.52 * . F 1.83 0.71 Lys
531 . . B . . T . 1.91 * . F 2.32 2.58 Thr 532 . . B . . T . 1.83 *
. F 2.66 6.96 Asp 533 . . . . T T . 1.80 * . F 3.40 5.89 Arg 534 .
. . . T T . 2.66 * . F 3.06 2.55 Lys 535 . . B . . T . 2.34 * . F
2.32 2.95 His 536 . . B . . . . 2.09 * . F 1.78 2.55 Phe 537 . . B
. . . . 1.70 * . F 1.44 2.01 Asp 538 . . B . . . . 1.67 * . F 0.65
0.87 Thr 539 . . B . . . . 1.21 * . F -0.25 0.87 Pro 540 . . . . .
. C 0.87 * * F -0.05 1.00 Phe 541 . . . . T . . 0.61 . * F 0.45
0.80 His 542 . . . . T T . 0.97 . * . 0.20 0.58 Gly 543 . . . . T T
. 0.37 . * . 0.20 0.37 Ser 544 . . . . T T . 0.39 . * . 0.20 0.43
Trp 545 . . . . T T . 0.26 . * . 0.20 0.33 Gly 546 . . . . . . C
0.74 . * . -0.20 0.33 Met 547 . . . . T . . 0.49 . . . 0.00 0.38
Trp 548 . . . . T . . 0.49 . . . 0.00 0.38 Gly 549 . . . . . T C
0.79 . . . 0.00 0.38 Pro 550 . . . . T T . 0.41 . . F 0.35 0.64 Trp
551 . . . . T T . 0.46 * . F 0.66 0.33 Gly 552 . . . . T T . 1.17 *
. F 1.27 0.44 Asp 553 . . . . T . . 1.14 * . F 1.98 0.56 Cys 554 .
. . . T T . 0.82 * . F 2.49 0.77 Ser 555 . . . . T T . 0.69 * . F
3.10 0.42 Arg 556 . . . . T T . 0.63 * . F 2.79 0.25 Thr 557 . . .
. T T . 0.63 * . F 2.18 0.46 Cys 558 . . . . T T . -0.22 * . F 1.87
0.34 Gly 559 . . . . T T . 0.44 * . F 1.56 0.13 Gly 560 . . . . T T
. 0.50 * . F 0.65 0.15 Gly 561 . . . . T T . 0.08 * * F 0.35 0.45
Val 562 . . B B . . . -0.21 * * . -0.60 0.65 Gln 563 . . B B . . .
0.57 * * . -0.60 0.65 Tyr 564 . . B B . . . 0.91 * * . -0.15 1.29
Thr 565 . . B B . . . 0.59 * * . 0.79 3.01 Met 566 . . B B . . .
0.93 * * . 0.98 0.93 Arg 567 . . B B . . . 1.79 * * . 1.62 0.99 Glu
568 . . . . T . . 1.58 * * F 2.86 1.11 Cys 569 . . . . T T . 0.97 *
. F 3.40 1.73 Asp 570 . . . . T T . 1.07 * .. F 2.91 0.66 Asn 571 .
. . . . T C 1.71 * . F 2.37 0.59 Pro 572 . . . . . T C 1.60 * . F
2.52 2.18 Val 573 . . . . . . C 1.26 * . F 2.32 2.10 Pro 574 . . .
. T T . 1.58 * . F 2.42 1.29 Lys 575 . . . . T T . 1.62 * . F 2.61
0.83 Asn 576 . . . . T T . 1.38 * . F 3.40 2.23 Gly 577 . . . . T T
. 0.92 * . F 3.06 2.26 Gly 578 . . . . T T . 1.78 * . F 2.27 0.61
Lys 579 . . B . . T . 1.64 . . F 1.53 0.65 Tyr 580 . . B . . T .
1.64 . . F 1.19 0.65 Cys 581 . . B . . T . 1.76 . . F 1.30 1.32 Glu
582 . . B . . . . 1.24 . * F 1.10 1.29 Gly 583 . . B B . . . 1.70 .
* F 0.75 0.61 Lys 584 . . B B . . . 1.41 . * F 0.90 2.24 Arg 585 .
. B B . . . 1.77 . * F 1.15 2.02 Tyr 586 . . B B . . . 2.13 . * .
1.25 4.01 Arg 587 . . B B . . . 1.47 * * . 1.50 2.68 Tyr 588 . . B
. . T . 1.81 * * . 2.00 0.73 Arg 589 . . . . T T . 0.96 * * . 2.50
1.59 Ser 590 . . . . T T . 0.84 * * . 2.10 0.67 Cys 591 . . . . T T
. 1.70 . * . 1.85 0.74 Asn 592 . A . . T . . 0.92 . * . 1.50 0.63
Leu 593 . A B . . . . 0.96 . . . 0.89 0.25 Glu 594 . A B . . . .
0.84 . . F 1.13 0.73 Asp 595 . A . . T . . 1.17 . . F 2.17 0.76 Cys
596 . . B . . T . 1.81 . . F 2.66 1.48 Pro 597 . . . . T T . 1.47 *
* F 3.40 1.37 Asp 598 . . . . T T . 2.32 * * F 2.91 0.81 Asn 599 .
. . . T T . 2.01 * . F 3.02 3.03 Asn 600 . . . . T T . 1.31 * . F
2.98 2.83 Gly 601 . . . . T T . 2.09 * . F 2.94 1.47 Lys 602 . . .
. . T C 2.30 * * F 2.70 1.79 Thr 603 . . . . . T C 2.30 * . F 3.00
1.92 Phe 604 A A . . . . . 2.30 * . F 2.10 3.37 Arg 605 A A . . . .
. 1.63 . . F 1.80 2.91 Glu 606 A A . . . . . 1.98 * . F 1.50 1.08
Glu 607 A A . . . . . 1.34 * . F 1.20 2.17 Gln 608 A A . . . . .
1.62 * . F 0.90 1.12 Cys 609 A A . . . . . 2.32 * * . 0.60 0.88 Glu
610 A A . . . . . 2.21 . * . 0.60 0.81 Ala 611 A A . . . . . 1.51 .
* . 0.60 0.81 His 612 A A . . . . . 1.21 * . . 0.45 1.32 Asn 613 A
A . . . . . 1.26 * * . 0.45 1.02 Glu 614 A A . . . . . 1.33 * . .
0.45 2.02 Phe 615 A A . . . . . 1.03 * * F 0.60 1.50 Ser 616 A A .
. . . . 0.92 . . F 0.90 1.25 Lys 617 A A . . . . . 0.61 . . F 0.45
0.62 Ala 618 . A . . T . . 0.31 . . F 0.25 0.71 Ser 619 . A . . T .
. -0.03 . . F 0.85 0.71 Phe 620 . . . . T . . 0.46 . . F 1.26 0.35
Gly 621 . . . . T T . 0.17 . . F 1.07 0.54 Ser 622 . . . . . T C
-0.73 . * F 1.08 0.41 Gly 623 . . . . . T C -0.14 . . F 0.99 0.35
Pro 624 . . . . . T C -0.13 . . F 2.10 0.61 Ala 625 . A . . . . C
-0.32 . . F 0.89 0.48 Val 626 . A B . . . . -0.19 * . . 0.03 0.34
Glu 627 . A B . . . . 0.16 * . . -0.18 0.34 Trp 628 . A B . . . .
0.26 * . . -0.09 0.67 Ile 629 . . B . . . . -0.12 * . . -0.25 1.42
Pro 630 . . B . . T . 0.12 * . . 0.10 0.83 Lys 631 . . . . T T .
0.12 * . . 0.20 0.78 Tyr 632 . . . . T T . -0.18 * . . 0.20 0.82
Ala 633 . . . . T T . -0.10 * . . 0.84 0.71 Gly 634 . . . . T . .
0.83 * . . 0.98 0.55 Val 635 . . B . . . . 1.04 . * . 0.92 0.70 Ser
636 . . B . . T . 1.11 . * F 2.66 1.17 Pro 637 . . . . T T . 0.69 .
* F 3.40 2.31 Lys 638 . . . . T T . 1.32 . * F 3.06 1.67 Asp 639 .
. . . T T . 0.86 . * F 2.72 2.49 Arg 640 A A . . . . . 0.82 . * F
1.58 1.33 Cys 641 A A . . . . . 0.46 * * F 1.09 0.46 Lys 642 . A B
. . . . 0.67 * * . 0.30 0.15 Leu 643 . A B . . . . 0.03 . * . 0.30
0.13 lle 644 . A B . . . . 0.08 . * . -0.60 0.25 Cys 645 . A B . .
. . -0.38 . * . 0.30 0.25 Gln 646 . A B . . . . -0.60 * * . -0.30
0.30 Ala 647 . A B . . . . -0.99 * * . -0.30 0.30 Lys 648 . A B . .
. . -0.42 * * F -0.15 0.55 Gly 649 . . . . T T . -0.23 * . F 0.65
0.50 lle 650 . . . . T T . -0.27 . * . 0.20 0.43 Gly 651 . . B . .
T . -1.12 . * . -0.20 0.18 Tyr 652 . . B . . T . -1.34 . . . -0.20
0.14 Phe 653 . . B B . . . -1.39 . . . -0.60 0.16 Phe 654 . . B B .
. . -1.26 . * . -0.60 0.29 Val 655 . . B B . . . -0.32 . * . -0.60
0.28 Leu 656 . . B B . . . -0.83 . * . -0.60 0.65 Gln 657 . . B . .
T . -1.44 . . . -0.20 0.56 Pro 658 . . B . . T . -0.74 * . F -0.05
0.56 Lys 659 . . . . T T . -0.39 . * F 1.40 1.13 Val 660 . . B . .
T . 0.16 . . F 0.85 0.65 Val 661 . . B . . T . 0.76 . * F 0.85 0.60
Asp 662 . . B . . T . 0.09 . . F 1.06 0.47 Gly 663 . . B . . T .
0.00 * . F 0.67 0.34 Thr 664 . . B . . T . -0.26 * . F 1.48 0.61
Pro 665 . . B . . . . 0.60 . . F 1.49 0.56 Cys 666 . . . . T . .
1.16 . . F 2.10 0.95 Ser 667 . . . . . T C 0.84 . . F 1.89 0.88 Pro
668 . . . . T T . 0.89 . . F 1.88 0.82 Asp 669 . . . . T T . 0.34 .
. F 1.82 2.06 Ser 670 . . . . T T . -0.11 . . F 1.61 1.14 Thr 671 .
. . B T . . -0.30 . * F 0.85 0.39 Ser 672 . . B B . . . 0.00 . * F
-0.15 0.18 Val 673 . . B B . . . -0.13 . * . -0.60 0.23 Cys 674 . .
B B . . . -0.13 . * . -0.60 0.16 Val 675 . . B B . . . -0.50 . * .
-0.60 0.20 Gln 676 . . B B . . . -1.04 . * F -0.45 0.15 Gly 677 . .
B B . . . -0.70 . * F -0.45 0.20 Gln 678 . . B B . . . -0.43 . * F
-0.15 0.54 Cys 679 . . B B . . . -0.11 . . . 0.30 0.32 Val 680 . .
B B . . . 0.08 * * . 0.30 0.32 Lys 681 . . B . . T . 0.08 * . .
0.10 0.10 Ala 682 . . B . . T . 0.53 * . . 0.70 0.30 Gly 683 . . B
. . T . -0.36 * . . 1.00 0.80 Cys 684 . . B . . T . -0.58 * . .
1.00 0.28 Asp 685 A . . B . . . 0.28 * . . 0.30 0.20 Arg 686 A . .
B . . . -0.07 * . . 0.60 0.33 lle 687 A . . B . . . 0.57 * . . 0.60
0.82 lle 688 A . . B . . . 0.96 * . F 0.75 0.99 Asp 689 A . . . . T
. 1.67 * * F 1.30 1.01 Ser 690 A . . . . T . 0.97 * * F 1.30 2.88
Lys 691 A . . . . T . 0.86 * . F 1.61 3.55 Lys 692 . . . . T T .
1.79 * * F 2.32 3.55 Lys 693 . . . . T . . 2.01 * * F 2.43 5.30 Phe
694 . . . . T . . 1.67 * * F 2.74 1.42 Asp 695 . . . . T T . 1.11 *
. F 3.10 0.70 Lys 696 . . B . . T . 0.40 * . F 2.39 0.26 Cys 697 .
. B . . T . 0.01 * . . 1.63 0.16 Gly 698 . . B . . T . -0.38 * . .
1.32 0.10 Val 699 . . B . . . . 0.32 * . . 0.21 0.05 Cys 700 . . .
. T . . -0.02 . . . 0.00 0.14 Gly 701 . . . . T T . -0.37 . . F
0.65 0.14 Gly 702 . . . . T T . -0.01 . . F 0.65 0.26 Asn 703 . . .
. T T . -0.33 . . F 0.65 0.69 Gly 704 . . . . T T . 0.57 . . F 0.65
0.37 Ser 705 . . . . T T . 1.28 . . F 1.25 0.76 Thr 706 . . B . . T
. 0.73 . . F 1.41 0.94 Cys 707 . . B . . T . 0.78 . * F 1.37 0.67
Lys 708 . . B . . T . 0.43 . * F 1.63 0.67 Lys 709 . . B . . . .
0.48 * * F 1.69 0.46 Ile 710 . . B . . T . -0.08 * * F 2.60 1.14
Ser 711 . . B . . T . -0.08 * * F 1.89 0.42 Gly 712 . . B . . T .
0.29 * * F 1.03 0.31 Ser 713 . . B . . T . -0.34 * * F 0.77 0.58
Val 714 . . B B . . . -0.34 . * F 0.11 0.44 Thr 715 . . B B . . .
0.33 . . F 0.73 0.89 Ser 716 . . B B . . . 0.29 . . F 1.16 1.03 Ala
717 . . B . . . . 0.39 . . F 1.64 1.37 Lys 718 . . . . . T C 0.66 .
. F 2.32 1.49 Pro 719 . . . . T T . 1.51 * . F 2.80 1.51 Gly 720 .
. . . T T . 0.93 * . F 2.52 2.50 Tyr 721 . . B . . T . 0.34 * . .
1.54 0.88 His 722 . . B B . . . 0.62 * . . -0.04 0.40 Asp 723 . . B
B . . . -0.31 * . . -0.32 0.58 Ile 724 . . B B . . . -0.31 * . .
-0.60 0.26 Ile 725 . . B B . . . -0.28 * . . -0.60 0.29 Thr 726 . .
B B . . . -0.38 * . . -0.60 0.25 Ile 727 . . B . . T . -0.93 * . .
-0.20 0.36 Pro 728 . . B . . T . -1.24 * . F -0.05 0.52 Thr 729 . .
. . . T C -0.36 * . F 0.15 0.52 Gly 730 . . . . . T C -0.36 . * F
0.30 1.19 Ala 731 . . . B . . C -0.04 . * F -0.25 0.54 Thr 732 . .
. B . . C -0.01 . * F 0.65 0.65 Asn 733 . . B B . . . 0.24 . * F
-0.15 0.48 Ile 734 . . B B . . . 0.56 . * F 0.45 0.96 Glu 735 . . B
B . . . 1.01 . * F 0.60 1.15 Val 736 . . B B . . . 1.60 . * F 0.90
1.40 Lys 737 . . B B . . . 1.91 . * F 1.24 3.21 Gln 738 . . B . . .
. 2.02 . * F 1.78 3.21 Arg 739 . . B . . . . 2.57 * * F 2.12 8.48
Asn 740 . . B . . T . 2.27 * * F 2.66 4.20 Gln 741 . . . . T T .
3.23 * * F 3.40 3.25 Arg 742 . . . . T T . 3.19 * . F 3.06 3.25 Gly
743 . . . . T T . 3.19 * . F 3.00 3.25
Ser 744 . . . . T . . 2.73 * . F 2.74 3.02 Arg 745 . . . . . . C
2.43 * * F 2.48 1.52 Asn 746 . . . . T T . 1.73 * . F 2.82 2.06 Asn
747 . . . . T T . 0.81 * . F 2.80 1.33 Gly 748 . . . . . T C 0.57 .
* F 1.57 0.56 Ser 749 . . B . . T . -0.02 . * F 0.79 0.35 Phe 750 .
A B . . . . -0.09 . * . -0.04 0.15 Leu 751 . A B . . . . -0.68 . .
. -0.32 0.31 Ala 752 . A B . . . . -1.27 * . . -0.60 0.23 Ile 753 .
A B . . . . -0.92 . . . -0.60 0.27 Lys 754 A A . . . . . -0.97 . .
. 0.30 0.55 Ala 755 A A . . . . . -0.58 . . . 0.30 0.54 Ala 756 A A
. . . . . -0.01 . . F 0.60 1.12 Asp 757 A . . . . T . -0.31 . . F
0.85 0.87 Gly 758 . . B . . T . -0.23 . * F 0.25 0.61 Thr 759 . . B
. . T . -0.28 . . F -0.05 0.50 Tyr 760 . . B . . T . -0.03 . * .
-0.20 0.48 Ile 761 . . B . . . . 0.56 . * . -0.40 0.48 Leu 762 . .
B . . . . 0.31 . * . -0.40 0.55 Asn 763 . . B . . T . 0.34 . * F
-0.50 0.55 Gly 764 . . . . T T . -0.16 . * F 0.50 1.14 Asp 765 . .
. . T T . -0.21 . * F 0.50 1.14 Tyr 766 . . . . . T C 0.37 . * F
0.45 0.95 Thr 767 . . B B . . . 0.37 . * . -0.15 1.38 Leu 768 . . B
B . . . 0.37 * * . -0.60 0.68 Ser 769 . . B B . . . 0.71 * . F
-0.45 0.75 Thr 770 . . B B . . . 0.71 * * F -0.15 0.90 Leu 771 A .
. B . . . 0.07 * . F 0.60 1.83 Glu 772 A . . B . . . -0.22 * . F
0.45 0.96 Gln 773 A . . B . . . 0.34 * * F 0.45 0.66 Asp 774 A . .
B . . . 0.69 . * F 0.00 1.25 Ile 775 A . . B . . . 0.66 . * . 0.75
1.44 Met 776 A . . B . . . 0.61 . * . 0.30 0.82 Tyr 777 . . B B . .
. -0.24 . * . -0.30 0.37 Lys 778 . . B B . . . -1.06 . * . -0.60
0.39 Gly 779 . . B B . . . -0.94 . * . -0.60 0.32 Val 780 . . B B .
. . -0.30 . * . -0.30 0.40 Val 781 . . B B . . . 0.00 . * . -0.30
0.32 Leu 782 . . B B . . . -0.10 . * . -0.60 0.43 Arg 783 . . B B .
. . -0.44 . * . -0.60 0.57 Tyr 784 . . B . . T . -0.40 * * . 0.25
1.03 Ser 785 . . . . T T . -0.13 . * F 0.80 1.68 Gly 786 . . . . .
T C 0.13 * * F 1.05 0.86 Ser 787 . . . . . T C 0.13 . * F 0.45 0.56
Ser 788 . A . . . . C 0.02 . * F 0.05 0.34 Ala 789 A A . . . . .
0.38 * . F 0.45 0.60 Ala 790 A A . . . . . -0.21 * * . 0.60 0.88
Leu 791 A A . . . . . 0.24 * * . 0.30 0.46 Glu 792 A A . . . . .
0.24 * * . 0.60 0.89 Arg 793 . A B B . . . -0.16 * * F 0.90 1.18
Ile 794 A A . B . . . 0.13 * * F 0.60 1.24 Arg 795 A A . B . . .
0.51 * * F 0.75 0.96 Ser 796 . A . . T . . 0.51 . * F 1.13 0.76 Phe
797 . . . . . . C 0.56 . * F 0.81 0.89 Ser 798 . . . . . T C 0.44 .
* F 1.89 0.91 Pro 799 . . . . . T C 1.12 * * F 2.32 1.17 Leu 800 .
. . . T T . 0.20 * * F 2.80 2.10 Lys 801 . . . . . T C 0.19 * * F
2.32 1.29 Glu 802 . . . . . . C 0.00 . * F 1.84 1.20 Pro 803 A . .
B . . . 0.30 . * F 1.16 1.02 Leu 804 A . . B . . . -0.34 . * F 0.73
0.89 Thr 805 . . B B . . . -0.34 . * . -0.30 0.38 Ile 806 . . B B .
. . -0.70 . * . -0.60 0.20 Gln 807 . . B B . . . -1.56 . . . -0.60
0.35 Val 808 . . B B . . . -1.69 . * . -0.60 0.18 Leu 809 . . B B .
. . -0.88 . * . -0.60 0.26 Thr 810 . . B B . . . -1.16 . . . -0.60
0.24 Val 811 . . B B . . . -1.08 . * . -0.60 0.33 Gly 812 . . B B .
. . -0.97 * * . -0.60 0.33 Asn 813 A . . . . . . -0.32 * * . 0.12
0.44 Ala 814 A . . . . . . 0.53 * * . 0.34 0.92 Leu 815 A . . . . .
. -0.04 * * F 1.76 1.86 Arg 816 . . B . . . . 0.86 * * F 1.53 0.81
Pro 817 . . B . . . . 0.96 * * F 2.20 1.61 Lys 818 . . B B . . .
0.64 * * F 1.48 3.05 Ile 819 . . B B . . . 0.99 . * F 1.56 2.25 Lys
820 . . B B . . . 1.10 * * F 0.44 2.28 Tyr 821 . . B B . . . 0.13 *
* . -0.38 0.99 Thr 822 . . B B . . . 0.39 . * . -0.45 1.04 Tyr 823
A . . B . . . 0.39 . * . -0.45 1.04 Phe 824 A . . B . . . 1.32 . *
. -0.45 1.33 Val 825 A . . B . . . 1.32 . . . 0.45 1.85 Lys 826 A .
. B . . . 1.57 . . F 0.90 2.36 Lys 827 A A . . . . . 1.58 * . F
0.90 4.71 Lys 828 A A . . . . . 1.12 * . F 0.90 8.51 Lys 829 A A .
. . . . 1.82 * . F 0.90 3.68 Glu 830 A A . . . . . 2.09 * . F 0.90
2.96 Ser 831 A A . . . . . 1.16 * . F 0.90 1.50 Phe 832 A A . . . .
. 0.90 . . . 0.30 0.52 Asn 833 . A B . . . . 0.54 * . . -0.30 0.47
Ala 834 . . B . . . . -0.20 * * . -0.40 0.50 Ile 835 . . . . . . C
-0.50 * . . -0.20 0.50 Pro 836 . . . . . T C -0.79 . . . 0.00 0.42
Thr 837 . . . . T T . -0.38 * * . 0.20 0.42 Phe 838 A . . . . T .
-1.23 * . . -0.20 0.63 Ser 839 . . . . . T C -1.53 * . . 0.00 0.30
Ala 840 . A B B . . . -0.64 * . . -0.60 0.15 Trp 841 . A B B . . .
-0.43 . . . -0.60 0.29 Val 842 A A . B . . . -0.41 . . . -0.30 0.38
Ile 843 A A . B . . . -0.06 * . . -0.60 0.40 Glu 844 A A . B . . .
0.24 * . . -0.60 0.37 Glu 845 A A . . . . . 0.17 * . . 0.30 0.87
Trp 846 A A . . . . . 0.16 * . . 0.61 0.66 Gly 847 A A . . . . .
1.06 * . F 1.37 0.51 Glu 848 . A . . T . . 1.64 * . F 2.08 0.59 Cys
849 . A . . T . . 0.98 * . F 2.09 0.76 Ser 850 . . . . T T . 0.98 .
. F 3.10 0.41 Lys 851 . . . . T T . 0.46 . . F 2.79 0.41 Ser 852 .
. . . T T . 0.46 . . F 2.18 0.63 Cys 853 . . . . T T . 0.17 * * .
2.02 0.47 Glu 854 A A . . . . . 0.83 * . . 0.61 0.24 Leu 855 A A .
. . . . 1.24 . . . -0.30 0.32 Gly 856 . A . . . . . 1.31 . * . 0.85
1.16 Trp 857 A A . . . . . 0.80 * * . 0.75 1.31 Gln 858 A A . . . .
. 0.61 * * . -0.15 1.31 Arg 859 A A . . . . . 0.61 * * . -0.30 0.98
Arg 860 . A B . . . . 0.76 . * . 0.45 1.61 Leu 861 . A B . . . .
1.21 * . . 0.60 0.50 Val 862 . A B . . . . 1.50 * . . 0.60 0.50 Glu
863 . A B . . . . 0.61 . . . 0.94 0.43 Cys 864 . A B . . . . 0.50 .
. . 0.98 0.36 Arg 865 . A . . T . . 0.04 . . F 2.17 0.78 Asp 866 .
. . . T T . 0.86 . . F 2.91 0.45 Ile 867 . . . . T T . 1.50 . . F
3.40 1.45 Asn 868 . . . . T T . 0.91 . . F 3.06 1.14 Gly 869 . . .
. . T C 1.28 . . F 2.07 0.69 Gln 870 . . . . . T C 1.17 . * F 1.28
1.32 Pro 871 . . . . . T C 0.50 . * F 1.54 1.42 Ala 872 . . . . . T
C 0.80 . * F 1.05 0.77 Ser 873 A . . . . T . 0.84 * . F 0.85 0.45
Glu 874 A A . . . . . 1.19 * . F 0.75 0.58 Cys 875 A A . . . . .
0.33 * . . 0.60 1.00 Ala 876 A A . . . . . 0.59 * . . 0.60 0.55 Lys
877 A A . . . . . 0.97 * . F 0.75 0.64 Glu 878 A A . . . . . 0.68 *
. F 0.90 1.84 Val 879 A A . . . . . 0.38 * . F 0.90 1.84 Lys 880 A
A . . . . . 0.73 * . F 0.90 1.23 Pro 881 A . . . . T . 1.43 * . F
1.30 1.03 Ala 882 . . . . T T . 1.18 * . F 2.01 2.71 Ser 883 . . .
. T T . 0.51 . * F 2.32 2.10 Thr 884 . . . . T T . 0.78 . * F 2.18
0.73 Arg 885 . . B . . T . 0.73 . * F 2.09 0.73 Pro 886 . . . . T T
. 0.91 . * F 3.10 0.91 Cys 887 . . . . T T . 1.29 . * . 2.64 0.85
Ala 888 . . . . T T . 0.92 . * . 2.43 0.67 Asp 889 . . . . T . .
1.02 . * . 1.72 0.23 His 890 . . . . . T C 0.91 . * . 1.51 0.67 Pro
891 . . . . T T . 0.83 . . . 1.65 1.16 Cys 892 . . . . T T . 1.50 .
* . 1.00 0.73 Pro 893 . . . . T T . 1.28 . * . 0.60 0.93 Gln 894 .
A . . T . . 0.93 . . . 0.10 0.49 Trp 895 . A B . . . . 0.97 . . .
-0.40 0.91 Gln 896 . A B . . . . 0.89 . . . -0.05 1.02 Leu 897 . A
B . . . . 1.26 . . . -0.60 0.62 Gly 898 . . . . T . . 1.17 . . .
0.00 0.79 Glu 899 . . . . T . . 0.50 . . F 0.45 0.61 Trp 900 . . .
. T . . 0.49 . . F 0.45 0.40 Ser 901 . . . . T T . 0.53 . . F 0.65
0.54 Ser 902 . . . . T T . 1.03 . . F 1.25 0.62 Cys 903 . . . . T T
. 0.71 . * F 0.65 0.85 Ser 904 . . . . T T . 0.37 * * F 1.25 0.34
Lys 905 . . . . T . . 0.70 * * F 1.05 0.25 Thr 906 . . . . T . .
0.66 * * F 1.69 0.94 Cys 907 . . . . T . . 0.71 * . F 2.03 0.69 Gly
908 . . . . T T . 1.42 * * F 2.27 0.54 Lys 909 . . . . T T . 1.77 *
* F 2.61 0.75 Gly 910 . . . . T T . 1.83 * * F 3.40 2.81 Tyr 911 .
. . . T T . 1.84 . . F 3.06 5.57 Lys 912 . A B . . . . 1.70 * . F
1.92 3.73 Lys 913 . A B . . . . 2.09 * . F 1.58 3.11 Arg 914 . A B
. . . . 1.38 * . F 1.24 3.97 Ser 915 . A B . . . . 0.91 * . F 0.90
1.06 Leu 916 . A B . . . . 0.86 * . F 0.75 0.44 Lys 917 . A B . . .
. 0.78 * . . 0.30 0.30 Cys 918 . A B . . . . 0.73 * . . -0.30 0.30
Leu 919 . A B . . . . 0.28 * . . 0.30 0.62 Ser 920 . . B . . . .
0.23 . . . 0.50 0.31 His 921 . . B . . T . 0.19 * . F 0.85 0.56 Asp
922 . . . . T T . -0.67 * . F 0.65 0.51 Gly 923 . . . . T T . -0.30
. . F 0.65 0.31 Gly 924 . . . . T T . 0.48 . . F 0.65 0.31 Val 925
. . B . . . . 0.78 . . . -0.10 0.25 Leu 926 . . B . . . . 0.51 . .
. -0.10 0.44 Ser 927 . . B . . . . -0.16 . . . -0.10 0.59 His 928 .
. B . . T . 0.19 . . . 0.10 0.43 Glu 929 . . B . . T . 0.32 . . F
0.85 0.87 Ser 930 A . . . . T . 0.37 * . F 1.30 1.00 Cys 931 A . .
. . T . 1.22 * . F 0.85 0.61 Asp 932 A . . . . T . 1.57 * . F 1.15
0.70 Pro 933 A . . . . T . 1.39 * . F 1.30 1.05 Leu 934 A . . . . T
. 1.43 * . F 1.30 3.02 Lys 935 A . . . . T . 1.70 * . F 1.30 3.62
Lys 936 A A . . . . . 1.67 * . F 0.90 3.18 Pro 937 A A . . . . .
0.78 * . F 0.90 3.34 Lys 938 A A . . . . . 0.99 * * F 0.90 1.17 His
939 A A . . . . . 1.10 * * . 0.60 0.98 Phe 940 . A B . . . . 0.39 *
* . -0.30 0.55 Ile 941 . A B . . . . 0.03 * * . -0.30 0.15 Asp 942
A A . . . . . -0.36 * * . -0.60 0.16 Phe 943 A A . . . . . -0.99 *
* . -0.60 0.18 Cys 944 A A . . . . . -0.96 . . . -0.60 0.26 Thr 945
A A . . . . . -0.92 . * . 0.30 0.27 Met 946 A A . . . . . -0.33 . *
. -0.60 0.16 Ala 947 A A . . . . . -0.72 . . . -0.30 0.41 Glu 948 A
A . . . . . -0.41 . . . 0.30 0.36 Cys 949 A A . . . . . -0.13 . . .
0.30 0.47 Ser 950 A A . . . . . -0.21 . . . 0.30 0.60
TABLE-US-00002 TABLE 2 Chou- . . . Chou- . . . Chou- . . . Kyte- .
. . Eisen . . . James . . . Emini Res Pos. Garni . . . Alpha Alpha
Garni . . . Beta Beta Garni . . . Turn Turn Garni . . . Coil Hydro
. . . Eisen . . . Alpha Beta Karpl . . . Flexi . . . Antig . . .
Surfa . . . Met 1 . . B . . . . -0.37 . . . -0.40 0.50 Phe 2 . . B
. . . . -0.57 . . . -0.40 0.61 Pro 3 . . B . . . . -0.77 . . .
-0.40 0.48 Ala 4 . . . . . . C -0.59 . * . -0.20 0.49 Pro 5 . . . .
. . C -0.09 . * . -0.20 0.87 Ala 6 . . . . . . C 0.22 * * . 0.85
1.11 Ala 7 . . . . . T C 0.11 * * . 0.45 1.15 Pro 8 A . . . . T .
0.11 * . . -0.20 0.61 Arg 9 . . . . T T . 0.00 * . . 0.20 0.94 Trp
10 . . B . . T . -0.60 * . . -0.20 0.81 Leu 11 . A B . . . . -0.82
* . . -0.60 0.43 Pro 12 . A B . . . . -1.04 * . . -0.60 0.18 Phe 13
. A B . . . . -1.64 * . . -0.60 0.14 Leu 14 A A . . . . . -2.57 * .
. -0.60 0.14 Leu 15 A A . . . . . -3.09 . . . -0.60 0.08 Leu 16 A A
. . . . . -3.09 . . . -0.60 0.07 Leu 17 A A . . . . . -3.69 . . .
-0.60 0.07 Leu 18 A A . . . . . -3.80 . . . -0.60 0.07 Leu 19 A A .
. . . . -3.20 . . . -0.60 0.07 Leu 20 A A . . . . . -3.20 . . .
-0.60 0.14 Leu 21 A A . . . . . -2.98 * . . -0.60 0.14 Leu 22 . A B
. . . . -2.06 * . . -0.60 0.17 Pro 23 . A B . . . . -1.59 * . .
-0.60 0.39 Leu 24 A A . . . . . -1.37 * . . -0.60 0.47 Ala 25 A A .
. . . . -0.77 * . . -0.04 0.58 Arg 26 . A B . . . . -0.54 * . .
0.82 0.58 Gly 27 . A B . . . . 0.38 . . F 0.63 0.71 Ala 28 . . B .
. . . 0.38 . . .F 2.14 1.37 Pro 29 . . . . . . C 0.60 . . F 2.60
1.08 Ala 30 . . B . . . . 0.60 . . F 1.84 1.11 Arg 31 . . B . . . .
0.14 . . F 1.58 1.11 Pro 32 . . B . . . . 0.14 . * F 1.17 0.71 Ala
33 . . B . . T . 0.73 . * F 1.11 0.69 Ala 34 A . . . . T . 0.36 . *
F 0.85 0.61 Gly 35 . . . . . T C 0.64 . * F 0.45 0.40 Gly 36 . . .
. . T C 0.53 . * F 0.45 0.53 Gln 37 A . . . . . . -0.07 . . F 0.65
0.91 Ala 38 . . B . . . . -0.33 . . F 0.65 0.76 Ser 39 . . B B B .
. -0.60 . . F -0.15 0.57 Glu 40 . . B B B . . -0.47 . . F -0.15
0.24 Leu 41 . . B B B . . -0.43 . * . -0.30 0.37 Val 42 . . B B B .
. -0.32 . * . -0.30 0.40 Val 43 . . B B B . . -0.54 . * . 0.30 0.46
Pro 44 . . B B B . . -0.46 . * F -0.24 0.46 Thr 45 . . B B B . .
-0.80 . * F 0.27 0.95 Arg 46 . . B B B . . -0.29 . * F 0.63 1.26
Leu 47 . . . . . T C -0.02 . * F 2.04 1.10 Pro 48 . . . . . T C
0.49 * * F 2.10 0.77 Gly 49 . . . . . T C 0.70 * * F 1.89 0.39 Ser
50 . . . . . T C 0.20 * * F 1.68 0.81 Ala 51 A A . . . . . -0.50 *
* F 0.87 0.43 Gly 52 A A . . . . . -0.50 . . F 0.66 0.44 Glu 53 A A
. . . . . -0.32 . * . -0.30 0.27 Leu 54 A A . . . . . -0.79 . * .
-0.30 0.37 Ala 55 A A . . . . . -0.79 . * . -0.60 0.31 Leu 56 A A .
. . . . -0.79 . * . -0.60 0.24 His 57 A A . . . . . -1.14 . * .
-0.60 0.29 Leu 58 A A . . . . . -1.49 * * . -0.60 0.25 Ser 59 A A .
. . . . -0.63 * * . -0.60 0.30 Ala 60 A A . . . . . -0.39 * * .
-0.30 0.44 Phe 61 A A . . . . . -0.28 * * . -0.30 0.53 Gly 62 . . .
. T T . -1.10 * . . 0.50 0.34 Lys 63 A . . . . T . -1.10 . * F
-0.05 0.25 Gly 64 . . B . . T . -0.69 . * . -0.20 0.24 Phe 65 . . B
. . T . -0.91 . * . 0.70 0.47 Val 66 . . B B . . . -0.80 * * .
-0.30 0.19 Leu 67 . . B B . . . -0.67 * * . -0.30 0.20 Arg 68 . . B
B . . . -0.71 . * . 0.00 0.35 Leu 69 . . B B . . . -0.37 * * . 1.20
0.80 Ala 70 . . . . . T C 0.03 . * . 2.55 1.61 Pro 71 . . . . . T C
0.19 * * F 3.00 1.10 Asp 72 . . . . T T . 0.19 . * F 2.60 1.16 Asp
73 A . . . . T . -0.51 . * F 1.75 0.95 Ser 74 A A . . . . . 0.09 .
. . 0.90 0.62 Phe 75 A A . . . . . 0.68 . . . 0.60 0.57 Leu 76 A A
. . . . . 0.19 . * . 0.30 0.59 Ala 77 A A . . . . . 0.23 . * .
-0.60 0.38 Pro 78 A A . . . . . -0.66 . * . -0.30 0.89 Glu 79 A A .
. . . . -0.36 * * F -0.15 0.75 Phe 80 A A . . . . . 0.46 * . F 0.90
1.29 Lys 81 A A . . . . . 0.46 * * F 0.90 1.63 Ile 82 A A . . . . .
0.70 * . F 0.75 0.78 Glu 83 A A . . . . . 0.57 * * F 0.45 0.89 Arg
84 A A . . . . . 0.27 * * F 0.75 0.44 Leu 85 . A . . T . . 0.62 * *
F 0.85 0.84 Gly 86 . A . . T . . 0.69 * * F 1.15 0.48 Gly 87 . . .
. . T C 0.99 * * F 1.35 0.48 Ser 88 . . . . . T C 0.68 * * F 1.05
0.59 Gly 89 . . . . . T C 0.22 * * F 1.05 0.86 Arg 90 . . B . . T .
0.69 . * F 1.19 0.86 Ala 91 . . . . . T C 1.03 . * F 1.73 0.63 Thr
92 . . B . . T . 1.49 . * F 2.32 1.11 Gly 93 . . B . . T . 1.44 . *
F 2.66 1.11 Gly 94 . . . . T T . 0.98 * * F 3.40 1.09 Glu 95 . . B
. . . . 0.98 * * F 2.31 0.62 Arg 96 . . B . . . . 1.22 * . F 2.12
1.23 Gly 97 . . . . T . . 0.87 * * F 2.18 1.23 Leu 98 . . B . . T .
0.51 * . F 1.49 0.38 Arg 99 . . B . . T . 0.16 * . . 0.70 0.17 Gly
100 . . B . . T . -0.14 * . . -0.20 0.15 Cys 101 . . B . . T .
-0.60 * . . -0.20 0.24 Phe 102 . . B . . . . -0.57 . * . -0.10 0.12
Phe 103 . . B . . T . -0.61 . * . -0.20 0.18 Ser 104 . . B . . T .
-0.72 . * F -0.05 0.24 Gly 105 . . . . . T C -0.72 . * F 0.15 0.45
Thr 106 . . . . . T C -0.06 * * F 0.45 0.52 Val 107 . . . B . . C
0.43 . * F 1.25 0.67 Asn 108 . . . B . . C 1.13 . * F 1.70 1.05 Gly
109 . . . B . . C 1.13 . * F 2.30 1.26 Glu 110 . . . . . T C 0.67 .
* F 3.00 2.27 Pro 111 A . . . . T . 0.39 . * F 2.50 1.16 Glu 112 A
. . . . T . 0.66 . * F 2.20 1.19 Ser 113 A . . . . T . -0.20 . . F
1.75 0.69 Leu 114 A A . B . . . -0.16 . . . 0.00 0.33 Ala 115 A A .
B . . . -0.97 . . . -0.30 0.26 Ala 116 A A . B . . . -1.42 . . .
-0.60 0.16 Val 117 A A . B . . . -1.31 . . . -0.60 0.10 Ser 118 . A
B B . . . -1.36 * . . -0.30 0.20 Leu 119 . . B B . . . -1.36 * . .
-0.30 0.20 Cys 120 . . B . . T . -1.07 * . . 0.10 0.22 Arg 121 . .
B . . T . -0.82 * . . 0.10 0.22 Gly 122 . . . . T T . -0.27 * . F
0.65 0.26 Leu 123 . . . . T T . -0.67 * . F 1.25 0.65 Ser 124 . . .
. . T C -0.67 * . F 0.45 0.29 Gly 125 . . B . . T . -0.81 . * F
-0.05 0.24 Ser 126 . . B . . T . -0.92 . * F -0.05 0.24 Phe 127 . .
B . . T . -0.92 . * . 0.10 0.30 Leu 128 . A B . . . C -0.11 . * .
-0.30 0.30 Leu 129 . A . . . . . 0.19 . * F 0.65 0.39 Asp 130 A A .
. . . . -0.17 . . F 0.45 0.77 Gly 131 A A . . . . . -0.18 . . F
0.45 0.81 Glu 132 A A . . . . . -0.37 . * F 0.90 1.42 Glu 133 A A .
. . . . 0.44 . * F 0.75 0.60 Phe 134 A A . . . . . 1.04 . * . 0.45
1.04 Thr 135 . A B . . . . 1.04 . * . 0.30 0.93 Ile 136 . . B . . .
. 1.04 . * F 0.05 0.93 Gln 137 . . B . . . . 0.46 . * F -0.10 1.06
Pro 138 . . . . . . C 0.11 . * F 0.25 0.75 Gln 139 . . . . T . .
0.47 . * F 0.60 1.05 Gly 140 . . . . . T C 0.48 . * F 0.45 0.60 Ala
141 . . . . T T . 0.56 . * F 1.25 0.52 Gly 142 . . . . . T C -0.03
. . F 0.45 0.25 Gly 143 . . . . . T C 0.18 . . F 0.65 0.25 Ser 144
. . . . . . C -0.03 . . F 0.65 0.43 Leu 145 . . B . . . . 0.28 * .
F 0.65 0.68 Ala 146 . . B . . . . 0.98 * . F 0.85 0.93 Gln 147 . .
B . . T . 0.51 . * F 2.00 1.36 Pro 148 . . B . . T . 0.86 * . .
1.05 1.36 His 149 . . B . . T . 1.27 * . . 1.45 2.34 Arg 150 . . B
. . T . 1.79 * . . 1.55 2.64 Leu 151 . . B . . . . 2.03 * . . 0.85
1.80 Gln 152 . . B . . . . 1.82 * . . 0.65 1.31 Arg 153 . . . . T .
. 1.44 * . . 1.05 1.03 Trp 154 . . . . T . . 1.13 * . F 0.84 1.26
Gly 155 . . . . . T C 0.43 * . F 0.93 0.72 Pro 156 . . . . . T C
1.36 * . F 1.17 0.37 Ala 157 . . . . T T . 1.14 * . F 1.61 0.69 Gly
158 . . . . . T C 0.22 * . F 2.40 1.08 Ala 159 . . . . . . C 0.30 *
* F 1.81 0.58 Arg 160 . . .B . . . . 0.76 * * F 1.37 0.89 Pro 161 .
. B . . . . 0.62 * * F 1.58 1.75 Leu 162 . . . . . . C 1.00 * * F
1.84 1.72 Pro 163 . . . . . . C 1.34 * * F 1.90 1.35 Arg 164 . . .
. . . C 1.64 * * F 2.20 1.52 Gly 165 . . . . . T C 1.53 * * F 2.40
1.93 Pro 166 . . . . . T C 0.89 * . F 3.00 2.17 Glu 167 . . . . . T
C 1.70 * . F 2.55 0.82 Trp 168 A . . . . T . 1.60 * . . 2.05 1.44
Glu 169 A . . . . . . 1.14 * . . 1.85 1.34 Val 170 A . . . . . .
1.49 . F 1.85 0.77 Glu 171 A . . . . . . 1.36 * F 2.00 1.26 Thr 172
A . . . . . . 1.36 * F 2.15 0.72 Gly 173 . . . . . T C 1.76 . F
3.00 1.68 Glu 174 A . . . . T . 1.76 . F 2.50 1.90 Gly 175 A . . .
. T . 2.61 . F 2.20 2.28 Gln 176 A . . . . T . 2.72 . F 1.90 4.00
Arg 177 A A . . . . . 2.69 * F 1.54 4.52 Gln 178 A A . . . . . 3.03
* F 1.58 4.52 Glu 179 . A . . T . . 3.00 * * F 2.32 4.36 Arg 180 .
A . . T . . 3.34 * . F 2.66 3.03 Gly 181 . . . . T T . 3.34 . * F
3.40 3.03 Asp 182 . . . . . T C 3.23 . . F 2.86 3.03 His 183 . . .
. . T C 2.93 . * F 2.52 2.58 Gln 184 . . . . . T C 2.93 . * F 2.18
3.50 Glu 185 . A . . . . C 2.82 . * F 1.44 3.63 Asp 186 A A . . . .
. 3.17 . . F 0.90 4.61 Ser 187 A A . . . . . 2.87 . . F 0.90 4.61
Glu 188 A A . . . . . 2.90 . . F 0.90 3.57 Glu 189 A A . . . . .
2.90 . . F 0.90 3.70 Glu 190 A A . . . . . 2.90 . . F 0.90 4.79 Ser
191 A A . . . . . 2.90 . . F 0.90 4.79 Gln 192 A A . . . . . 2.61 .
. F 0.90 4.79 Glu 193 A A . . . . . 2.61 . . F 0.90 2.79 Glu 194 A
A . . . . . 2.27 . . F 0.90 3.61 Glu 195 A A . . . . . 1.68 . . F
0.90 2.06 Ala 196 A A . . . . . 1.68 . . F 1.16 1.20 Glu 197 A A .
. . . . 1.68 . . F 1.27 0.93 Gly 198 A A . . . . . 1.47 . . F 1.53
0.93 Ala 199 . A . . T . . 1.26 . . F 2.34 1.42 Ser 200 . . . . . .
C 1.04 . . F 2.60 1.27 Glu 201 . . . . . . C 1.42 * . F 2.04 1.99
Pro 202 . . . . . . C 0.61 * . F 1.78 3.04 Pro 203 . . . . . . C
0.61 . . F 1.52 1.87 Pro 204 . . . . . T C 0.61 . . F 1.46 1.07 Pro
205 . . . . . T C 0.60 . . F 0.45 0.70 Leu 206 . . . . . T C 0.30 *
* F 0.45 0.65 Gly 207 . . B . . T . 0.62 . * F 0.51 0.57 Ala 208 .
. B . . . . 0.52 . * F 1.17 0.72 Thr 209 . . B . . . . 0.78 * * F
1.58 1.25 Ser 210 . . B . . T . 1.10 * . F 2.34 2.53 Arg 211 . . B
. . T . 1.21 * . F 2.60 4.91 Thr 212 . . B . . T . 0.70 * . F 2.34
2.95 Lys 213 . . B . . T . 0.99 * . F 2.08 1.63 Arg 214 . . B B . .
. 1.30 * . F 1.42 1.12 Phe 215 . . B B . . . 1.01 * * . 1.01 1.34
Val 216 . . B B . . . 1.01 * * . 0.60 0.68 Ser 217 A . . B . . .
0.62 * * . 0.60 0.68 Glu 218 A A . . . . . -0.28 * * . -0.30 0.68
Ala 219 A A . B . . . -0.39 * * . 0.30 0.68 Arg 220 A A . B . . .
0.00 * * . 0.60 0.87 Phe 221 A A . B . . . 0.04 * . . 0.60 0.73 Val
222 A A . B . . . -0.47 * * . -0.30 0.59 Glu 223 A A . B . . .
-1.32 * * . -0.30 0.25 Thr 224 A A . B . . . -1.32 * * . -0.60 0.21
Leu 225 A A . B . . . -1.43 * * . -0.60 0.29 Leu 226 A A . B . . .
-1.32 . . . 0.30 0.28 Val 227 A A . B . . . -0.77 . . . -0.60 0.20
Ala 228 A A . B . . . -1.37 . . . -0.30 0.32 Asp 229 A A . B . . .
-1.64 . . . -0.30 0.38 Ala 230 A A . . . . . -1.42 . . . -0.30 0.52
Ser 231 A A . . . . . -1.31 . . . 0.30 0.52 Met 232 A A . . . . .
-0.70 . . . -0.30 0.27 Ala 233 A A . . . . . -0.46 . . . -0.60 0.42
Ala 234 A A . . . . . -1.04 . . . -0.60 0.31 Phe 235 A A . . . . .
-0.46 . . . -0.60 0.32 Tyr 236 A A . . . . . -0.97 . . . -0.60 0.52
Gly 237 A A . . . . . -0.37 . . . -0.60 0.43 Ala 238 A A . . . . .
0.22 . . . -0.60 0.86 Asp 239 A A . . . . . 0.78 * * . -0.30 0.88
Leu 240 A A . . . . . 0.59 * . . 0.45 1.21 Gln 241 A A . B . . .
0.02 * * . -0.30 0.84
Asn 242 A A . B . . . 0.06 * . . -0.30 0.41 His 243 . A B B . . .
-0.17 * * . -0.60 0.72 Ile 244 . A B B . . . -0.77 * . . -0.60 0.35
Leu 245 . A B B . . . -0.26 * . . -0.60 0.21 Thr 246 . A B B . . .
-1.11 * . . -0.60 0.21 Leu 247 . A B B . . . -1.70 * . . -0.60 0.22
Met 248 A A . B . . . -2.26 * * . -0.60 0.27 Ser 249 A A . B . . .
-1.26 * * . -0.60 0.19 Val 250 A A . B . . . -1.33 * * . -0.30 0.45
Ala 251 A A . B . . . -1.27 * * . -0.30 0.32 Ala 252 A A . B . . .
-0.41 * * . -0.60 0.37 Arg 253 A A . B . . . 0.16 * * . -0.15 1.01
Ile 254 A A . B . . . 0.24 * * . 0.45 1.36 Tyr 255 A . . . . . .
0.80 * * . 0.99 2.08 Lys 256 . . B . . . . 0.50 * * . 1.33 1.42 His
257 . . B . . T . 1.13 . * F 1.12 1.42 Pro 258 . . . . T C 1.02 . *
F 2.56 1.81 Ser 259 . . . . T T . 1.61 . * F 3.40 1.46 Ile 260 . .
. . T T . 0.97 . * F 2.76 1.44 Lys 261 . . B . . . . 0.92 . * F
1.67 0.65 Asn 262 . . . . T . . 0.14 * * F 1.73 0.78 Ser 263 . . B
B . . . -0.24 * * F 0.19 0.92 Ile 264 . . B B . . . -0.80 * * .
-0.30 0.45 Asn 265 . . B B . . . -0.77 * * . -0.60 0.21 Leu 266 . .
B B . . . -0.77 . * . -0.60 0.12 Met 267 A . . B . . . -1.62 * . .
-0.60 0.33 Val 268 . . B B . . . -2.13 . * . -0.60 0.15 Val 269 . .
B B . . . -2.13 . . . -0.60 0.15 Lys 270 A . . B . . . -2.99 . . .
-0.60 0.11 Val 271 . . B B . . . -2.18 . . . -0.60 0.11 Leu 272 . .
B B . . . -1.58 . . . -0.30 0.25 Ile 273 A . . B . . . -0.72 . . .
0.30 0.21 Val 274 A . . B . . . 0.18 . * . 0.30 0.49 Glu 275 A . .
B . . . -0.16 . . . 0.75 1.19 Asp 276 A A . . . . . 0.36 . . F 0.90
1.79 Glu 277 A A . . . . . 0.96 * . F 0.90 2.39 Lys 278 . A . . T .
. 1.84 * * F 1.30 2.13 Trp 279 . A . . . . C 1.84 . * F 1.10 2.21
Gly 280 . . . . . T C 1.54 * . F 1.35 0.95 Pro 281 . . . . . T C
1.54 * * F 1.36 0.64 Glu 282 . . B . . T . 1.54 * * F 1.62 1.01 Val
283 . . B . . T . 1.16 * * F 2.23 1.64 Ser 284 . . . . . T C 1.10 .
* F 2.74 1.05 Asp 285 . . . . T T . 0.63 . . F 3.10 0.60 Asn 286 .
. . . T T . 0.53 . * F 2.49 0.67 Gly 287 . . . . T T . -0.28 * * F
2.18 0.72 Gly 288 . . . . T . . 0.69 * * F 1.07 0.35 Leu 289 . . B
. . . . 0.99 * * F 0.36 0.43 Thr 290 . . B . . . . 0.29 * * . -0.10
0.70 Leu 291 . . B . . . . -0.38 * * . -0.40 0.61 Arg 292 . . B . .
. . -0.03 * * . -0.40 0.40 Asn 293 . . B . . . . 0.02 * * . -0.10
0.44 Phe 294 . . . . T T . 0.83 * * . 0.20 0.57 Cys 295 . . . . T T
. 1.26 * * . 0.20 0.50 Asn 296 . . . . T T . 2.18 * * . 0.20 0.61
Trp 297 . . . . T T . 1.37 * * . 0.65 1.38 Gln 298 . . . . T . .
1.37 * . . 0.45 2.23 Arg 299 . . . . T . . 2.07 * . . 1.05 2.23 Arg
300 . . . . T . . 2.52 * * F 1.20 3.67 Phe 301 . . . . T . . 2.22 *
* F 1.84 3.28 Asn 302 . . . . T . . 2.51 * * F 2.18 2.24 Gln 303 .
. . . . T C 2.62 * . F 2.52 1.91 Pro 304 . . . . . T C 2.48 * . F
2.86 4.33 Ser 305 . . . . T T . 2.16 * * F 3.40 3.66 Asp 306 . . .
. T T . 2.86 * . F 3.06 3.27 Arg 307 . . . . . . C 2.82 * . F 2.32
3.66 His 308 . . . . . . C 2.58 * . F 1.98 3.72 Pro 309 . . . . . .
C 2.79 * . F 1.64 3.49 Glu 310 . . . . T . . 2.78 * . F 1.50 2.97
His 311 A . . . . T . 2.19 * . F 1.00 3.15 Tyr 312 A . . . . T .
1.19 * . F 1.00 2.06 Asp 313 A . . . . T . 0.41 . . F 0.85 0.83 Thr
314 A . . . . T . -0.19 . . . -0.20 0.51 Ala 315 A . . B . . .
-0.50 * . . -0.60 0.27 Ile 316 . . B B . . . -0.36 * . . -0.60 0.23
Leu 317 . . B B . . . -0.11 . . . -0.60 0.31 Leu 318 . . B B . . .
-0.11 . * . -0.60 0.53 Thr 319 . . B B . . . -0.50 . . F 0.00 1.23
Arg 320 . . B B . . . -0.58 . * F -0.08 1.29 Gln 321 . . . B T . .
-0.03 . * F 0.69 0.84 Asn 322 . . . . T T . 0.78 . * F 1.31 0.57
Phe 323 . . . . T T . 1.59 . . . 1.98 0.51 Cys 324 . . . . T T .
1.56 . * . 2.20 0.51 Gly 325 . . . . T T . 0.63 . * F 1.53 0.31 Gln
326 . . . . T . . -0.03 . . F 1.11 0.30 Glu 327 . . . . T . . -0.03
. . F 0.89 0.30 Gly 328 . . . . T . . 0.36 . . F 1.27 0.50 Leu 329
. . B . . . . 0.21 . . F 0.65 0.42 Cys 330 . . B . . . . 0.21 . . .
0.50 0.20 Asp 331 . . B . . T . -0.64 . . . 0.10 0.20 Thr 332 . . B
. . T . -1.23 * . . -0.20 0.18 Leu 333 . . B . . T . -0.89 . . .
0.10 0.34 Gly 334 . . B . . T . -0.97 . . . 0.70 0.34 Val 335 . . B
. . . . -0.64 . . . -0.40 0.16 Ala 336 . . B . . . . -0.96 . . .
-0.10 0.20 Asp 337 . . B . . T . -1.53 . . . 0.10 0.29 Ile 338 . .
B . . T . -1.39 . . . -0.20 0.27 Gly 339 . . B . . T . -1.04 * . .
0.10 0.14 Thr 340 . . B . . T . -0.40 . . . 0.70 0.14 Ile 341 . . B
. . . . 0.19 . . . 0.24 0.32 Cys 342 . . B . . . . 0.23 . . . 1.18
0.52 Asp 343 . . B . . T . 0.82 * . F 1.87 0.72 Pro 344 . . . . T T
. 0.50 . . F 3.06 1.37 Asn 345 . . . . T T . 0.51 . . F 3.40 1.37
Lys 346 . . . . T T . 0.54 * . F 3.06 1.10 Ser 347 . . . B T . .
0.32 . . F 1.87 0.53 Cys 348 . . B B . . . 0.32 * . . 0.38 0.23 Ser
349 . . B B . . . 0.53 * . . 0.64 0.20 Val 350 . . B B . . . 0.53 *
. . 0.30 0.25 Ile 351 . . B B . . . 0.14 * . . 0.60 0.80 Glu 352 A
. . B . . . -0.37 . . . 0.60 0.59 Asp 353 A A . . . . . 0.30 . . F
0.75 0.66 Glu 354 A A . . . . . 0.01 * . F 0.90 1.62 Gly 355 A A .
. . . . 0.28 * . F 0.75 0.95 Leu 356 A A . . . . . 1.13 * . . 0.30
0.57 Gln 357 A A . . . . . 0.82 * . . -0.30 0.45 Ala 358 A A . . .
. . 0.01 * . . -0.60 0.66 Ala 359 A A . . . . . -0.58 * . . -0.60
0.66 His 360 A A . . . . . -0.27 * . . -0.60 0.38 Thr 361 A A . . .
. . 0.54 * . . -0.60 0.52 Leu 362 A A . . . . . -0.27 * . . -0.30
0.88 Ala 363 A A . . . . . -0.02 * . . -0.30 0.54 His 364 A A . . .
. . 0.53 * . . -0.30 0.37 Glu 365 A A . . . . . -0.29 * . . -0.30
0.61 Leu 366 A A . B . . . -0.79 * . . -0.30 0.45 Gly 367 A A . B .
. . -0.28 * . . -0.60 0.27 His 368 A A . B . . . -0.29 * . . -0.30
0.21 Val 369 A A . B . . . -0.47 * . . -0.60 0.25 Leu 370 . A B B .
. . -0.50 * . . -0.26 0.39 Ser 371 . A B B . . . 0.31 * . . 0.08
0.39 Met 372 . . B . . . . 0.66 . . . 0.92 0.88 Pro 373 . . . . T .
. 0.39 * . . 2.41 1.78 His 374 . . . . T T . 1.29 * . F 3.40 1.78
Asp 375 . . . . T T . 1.89 . . F 3.06 3.61 Asp 376 . . . . T T .
1.52 . . F 2.89 3.61 Ser 377 . . . . T T . 1.81 * * F 2.72 1.42 Lys
378 . . B . . T . 2.13 * * F 2.15 1.23 Pro 379 . . . . T T . 1.36 *
* F 2.38 1.44 Cys 380 . . B . . T . 0.66 * * F 1.70 0.89 Thr 381 .
. B . . T . 0.31 * * F 1.53 0.38 Arg 382 . . B B . . . 0.40 * * F
0.36 0.25 Leu 383 . . B B . . . -0.24 * * . 0.04 0.71 Phe 384 . . B
B . . . -0.38 * . . -0.43 0.49 Gly 385 . . . B . . C 0.33 * . F
0.05 0.25 Pro 386 . . . . . T C 0.61 * * F 0.45 0.59 Met 387 . . .
. T T . 0.47 * * F 0.65 0.93 Gly 388 A . . . . T . 0.42 . . F 1.00
1.29 Lys 389 A . . . . T . 0.52 . . . 0.10 0.62 His 390 A A . . . .
. 0.28 . . . -0.30 0.62 His 391 A A . . . . . 0.28 . * . -0.30 0.63
Val 392 . A B . . . . 0.07 . . . -0.30 0.49 Met 393 A A . . . . .
-0.29 . * . -0.60 0.30 Ala 394 A A . . . . . -1.19 . * . -0.60 0.19
Pro 395 A A . . . . . -1.19 . * . -0.60 0.19 Leu 396 A A . . . . .
-1.97 . * . -0.60 0.26 Phe 397 A A . . . . . -1.11 * * . -0.60 0.21
Val 398 A A . . . . . -0.51 * . . -0.60 0.22 His 399 . A B . . . .
-0.23 * * . -0.60 0.46 Leu 400 . A B . . . . -0.83 * * . -0.60 0.77
Asn 401 . A . . T . . -0.23 * * F -0.05 0.85 Gln 402 . A . . T . .
0.18 . * F -0.05 0.97 Thr 403 . A . . T . . 0.73 . * F 0.10 1.24
Leu 404 . A . . . . C 0.56 . * F -0.10 1.03 Pro 405 . . . . T . .
0.70 . . . 0.00 0.92 Trp 406 . . . . T . . 0.40 . . . 0.00 0.34 Ser
407 . . . . . T C -0.19 . . . 0.00 0.55 Pro 408 . . . . T T . -0.48
. . . 0.20 0.36 Cys 409 . . . . T T . 0.09 . . . 0.20 0.34 Ser 410
. . B . . T . -0.51 . . . -0.20 0.40 Ala 411 . A B . . . . -0.53 .
. . -0.60 0.21 Met 412 . A B . . . . -0.23 . . . -0.60 0.57 Tyr 413
. A B . . . . -0.83 . . . -0.60 0.74 Leu 414 . A B . . . . -0.98 *
. . -0.60 0.60 Thr 415 . A B . . . . -0.68 * . . -0.60 0.50 Glu 416
A A . . . . . -0.43 * . . -0.30 0.54 Leu 417 A A . . . . . -0.18 *
. F 0.76 0.64 Leu 418 A . . . . T . 0.03 * . F 1.47 0.44 Asp 419 .
. . . T T . 0.50 * . F 2.18 0.35 Gly 420 . . . . T T . 0.81 . . F
1.89 0.42 Gly 421 . . . . T T . 0.14 . . F 3.10 0.84 His 422 . . .
. T T . 0.14 . . F 2.79 0.27 Gly 423 . . . . T T . 0.14 . . F 1.58
0.23 Asp 424 . . B . . T . 0.14 . * . 0.72 0.19 Cys 425 . . B . . T
. -0.10 . * . 1.01 0.23 Leu 426 . . B . . . . 0.03 . * . 0.50 0.24
Leu 427 . . B . . . . -0.28 . * . 0.50 0.22 Asp 428 . . B . . . .
-0.52 * * . -0.10 0.40 Ala 429 . . B . . T . -1.11 * . F 0.25 0.49
Pro 430 A . . . . T . -1.26 . . F 0.25 0.60 Gly 431 . . . . T T .
-0.66 . . F 0.65 0.30 Ala 432 . . B . . T . -0.66 . . . -0.20 0.46
Ala 433 . . B . . . . -0.87 . . . -0.40 0.24 Leu 434 . . B . . . .
-0.59 . . . -0.40 0.38 Pro 435 . . B . . . . -0.72 . . . -0.40 0.54
Leu 436 . . B . . T . -1.19 . . . -0.20 0.53 Pro 437 . . B . . T .
-0.81 . . F 0.00 0.53 Thr 438 . . . . T T . -0.57 . * F 0.45 0.53
Gly 439 . . . . . T C 0.36 . * F 0.30 0.64 Leu 440 . . . . . T C
-0.03 . * F 1.25 0.81 Pro 441 . . B . . T . 0.19 . * F 0.50 0.55
Gly 442 . . B . . T . -0.41 . * F 0.45 0.57 Arg 443 . . B . . T .
-0.34 . * . 0.25 0.57 Met 444 . A B . . . . 0.00 . * . -0.50 0.57
Ala 445 . A B . . . . 0.00 * * . -0.10 1.00 Leu 446 . A B . . . .
0.21 * . . -0.60 0.42 Tyr 447 . A B . . . . 0.56 * * . -0.60 0.71
Gln 448 . A B . . . . 0.44 * * . -0.45 1.22 Leu 449 A A . . . . .
0.38 * * . -0.15 2.57 Asp 450 A A . . . . . 1.08 * * F -0.15 0.88
Gln 451 . A B . . . . 1.89 * * F 0.75 0.99 Gln 452 . A B . . . .
1.24 * * F 0.90 2.09 Cys 453 . A B . . . . 0.54 * * F 0.75 0.88 Arg
454 . A B . . . . 1.01 * * . -0.30 0.44 Gln 455 . A B . . . . 0.80
* * . -0.30 0.25 Ile 456 . A B . . . . 0.80 * . . -0.30 0.72 Phe
457 . A . . T . . 0.10 * * . 0.70 0.62 Gly 458 . . . . . T C 0.88 *
* . 0.00 0.31 Pro 459 . . . . T T . 0.73 * * F 0.65 0.86 Asp 460 .
. . . T T . 0.07 * * F 1.40 1.35 Phe 461 . . . . T T . 0.74 * * .
1.35 0.73 Arg 462 . . . . T . . 1.44 * * . 1.40 0.73 His 463 . . .
. T . . 1.48 * * . 1.65 0.71 Cys 464 . . . . . T C 1.39 * * . 1.45
1.18 Pro 465 . . . . T T . 0.80 * . F 2.50 0.80 Asn 466 . . . . T T
. 1.50 * * F 1.65 0.60 Thr 467 . . . . T T . 1.39 * * F 1.55 1.93
Ser 468 . A . . T . . 0.57 * . F 1.50 2.08 Ala 469 . A . . T . .
0.57 . . F 1.10 0.96 Gln 470 . A B . . . . 0.19 . . F 0.45 0.36 Asp
471 . A B . . . . 0.19 * * F 0.45 0.27 Val 472 . A B . . . . -0.31
* . . -0.30 0.46 Cys 473 . A B . . . . -0.30 * . . -0.30 0.22 Ala
474 . A B . . . . -0.38 * * . -0.60 0.14 Gln 475 . A B . . . .
-0.41 . * . -0.60 0.10 Leu 476 . A B . . . . -0.72 * * . -0.60 0.25
Trp 477 . A B . . . . 0.13 . * . -0.60 0.36 Cys 478 . A B . . . .
0.46 . . . -0.26 0.35 His 479 . . . . T T . 0.46 . . . 0.88 0.42
Thr 480 . . . . T T . 0.46 . * . 1.52 0.40 Asp 481 . . . . T T .
1.06 . . F 3.06 1.30 Gly 482 . . . . T T . 0.53 . . F 3.40 1.48 Ala
483 . . . . T . C 0.53 * . F 2.41 0.85 Glu 484 A . . . . . . 0.53 *
. F 1.67 0.27 Pro 485 A . . . . . . 0.53 . . F 0.73 0.37 Leu 486 A
. . . . . . 0.58 * . . 0.24 0.53 Cys 487 A . . . . . . 0.92 . . .
0.78 0.62 His 488 . . B . . . . 1.17 . . F 0.61 0.64 Thr 489 . . .
. T T . 0.87 . . F 1.49 0.77 Lys 490 . . . . T T . 0.27 . . F 2.52
1.92 Asn 491 . . . . T T . 0.87 . . F 2.80 1.16 Gly 492 . . . . T T
. 1.24 . . F 2.52 1.25
Ser 493 . . . . . . C 0.69 . . F 1.09 0.66 Leu 494 . . . . . . C
1.00 . . . 0.36 0.41 Pro 495 . . B . . . . 0.61 . . . 0.18 0.69 Trp
496 . . . . T T . 0.30 . . . 0.50 0.51 Ala 497 . . B . . T . 0.43 .
. . 0.05 0.90 Asp 498 . . . . T T . 0.07 . . F 1.15 0.90 Gly 499 .
. . . T T . 0.53 . . F 1.40 0.46 Thr 500 . . . . . T C 0.53 . . F
2.05 0.45 Pro 501 . . . . T T . 0.48 . . F 2.50 0.42 Cys 502 . . .
. T T . 1.03 . * F 1.65 0.42 Gly 503 . . . . . T C 0.22 . . F 1.20
0.39 Pro 504 . . . . T . . -0.10 . . F 0.65 0.21 Gly 505 . . . . T
. . -0.09 . . . 0.25 0.21 His 506 . . B . . . . 0.12 . . . -0.40
0.28 Leu 507 . . B . . . . 0.44 . . . 0.50 0.32 Cys 508 . . B . . T
. 0.49 . * . 0.91 0.32 Ser 509 . . . . T T . 0.03 . . F 1.67 0.31
Glu 510 . . . . T T . -0.43 . . F 1.28 0.20 Gly 511 . . . . T T .
-0.61 * . F 1.49 0.31 Ser 512 . . . . T . . 0.20 * . F 2.10 0.36
Cys 513 . A . . . . C 0.87 . . F 1.79 0.36 Leu 514 . A . . . . C
1.17 . . F 1.58 0.63 Pro 515 A A . . . . . 0.31 . . F 1.17 0.81 Glu
516 A A . . . . . 0.66 * . F 1.11 1.13 Glu 517 A A . . . . . 1.07 *
. F 0.90 2.37 Glu 518 A A . . . . . 1.52 . . F 0.90 3.00 Val 519 A
A . . . . . 2.38 . . F 0.90 2.68 Glu 520 A A . . . . . 2.38 * . F
0.90 3.09 Arg 521 A . . . . T . 1.52 * . F 1.30 2.76 Pro 522 A . .
. . T . 0.67 * * F 1.30 2.76 Lys 523 A . . . . T . 0.67 * * F 1.30
1.18 Pro 524 . . B . . T . 1.18 * * F 1.30 1.01 Val 525 . . B . . .
. 0.83 * * F 0.65 0.65 Val 526 . . B . . . . 0.43 . * F 0.65 0.32
Asp 527 . . B . . T . 0.06 * . F -0.05 0.22 Gly 528 . . B . . T .
-0.20 * . F -0.05 0.30 Gly 529 . . . . T T . -0.28 . . F 0.65 0.62
Trp 530 . . . . . T C 0.23 . . . 0.00 0.39 Ala 531 . . . . . . C
0.88 . . . -0.20 0.39 Pro 532 . . . . T . . 0.59 . . . 0.00 0.61
Trp 533 . . . . T . . 0.59 . . . 0.00 0.61 Gly 534 . . . . . T C
0.93 . . . 0.00 0.59 Pro 535 . . . . T T . 0.56 . . F 0.35 0.66 Trp
536 . . . . T T . 0.84 * . F 0.66 0.34 Gly 537 . . . . . T C 1.17 *
. F 1.07 0.46 Glu 538 . . . . T . . 1.14 * . F 1.98 0.58 Cys 539 .
. . . T T . 0.82 * . F 2.49 0.80 Ser 540 . . . . T T . 0.69 * . F
3.10 0.43 Arg 541 . . . . T T . 0.63 * . F 2.79 0.25 Thr 542 . . .
. T T . 0.63 * . F 2.18 0.46 Cys 543 . . . . T T . -0.22 * . F 1.87
0.34 Gly 544 . . . . T T . 0.44 * . F 1.56 0.13 Gly 545 . . . . T T
. 0.04 * * F 0.65 0.15 Gly 546 . . . . T T . -0.37 * * F 0.35 0.25
Val 547 . . B B . . . -0.09 * * . -0.60 0.33 Gln 548 . . B B . . .
0.69 * * . -0.60 0.46 Phe 549 . . B B . . . 1.03 * * . -0.30 0.91
Ser 550 . . B B . . . 0.71 * * . 0.79 2.13 His 551 . . B . . . .
1.10 * * . 1.18 0.66 Arg 552 . . . . T . . 1.96 * * . 2.37 1.52 Glu
553 . . . . T . . 1.74 * * F 2.86 1.89 Cys 554 . . . . T T . 2.44 *
. F 3.40 2.15 Lys 555 . . . . T T . 2.53 * . F 3.06 1.90 Asp 556 .
. . . . T C 2.57 * . F 2.52 1.70 Pro 557 . . . . . T C 2.46 * . F
2.52 5.49 Glu 558 . . . . . . C 2.11 . . F 2.32 4.41 Pro 559 . . .
. T T . 2.43 . * F 2.72 2.62 Gln 560 . . . . T T . 2.50 . * F 2.76
1.67 Asn 561 . . . . T T . 2.26 * * F 3.40 1.89 Gly 562 . . . . T T
. 1.80 * * F 2.76 1.92 Gly 563 . . . . T T . 0.99 * * F 2.27 0.59
Arg 564 . . B . . T . 0.86 * . F 0.93 0.30 Tyr 565 . . B . . T .
0.97 . . . 0.44 0.30 Cys 566 . . B . . T . 1.08 . . . 1.00 0.60 Leu
567 . . B . . . . 0.83 . * . 1.40 0.60 Gly 568 . . B . . . . 1.22 .
* F 1.55 0.39 Arg 569 . . B . . . . 0.87 . * F 2.30 1.45 Arg 570 .
. . . T . . 1.11 * * F 3.00 2.75 Ala 571 . . . . T . . 1.48 * * F
2.70 4.82 Lys 572 . . . . T . . 1.62 * * F 2.40 3.30 Tyr 573 . . .
. T T . 1.93 * . F 1.85 0.90 Gln 574 . . . . T T . 1.51 . . F 1.10
1.22 Ser 575 . . . . T T . 1.40 . * . 0.50 0.88 Cys 576 . . . . T T
. 1.99 . . . 0.50 0.97 His 577 . A B . . . . 1.28 . . . 0.60 0.97
Thr 578 . A . . T . . 1.31 . . F 0.85 0.39 Glu 579 . A . . T . .
1.10 . . F 1.00 1.12 Glu 580 . A . . T . . 1.40 . . F 1.64 1.27 Cys
581 . A B . . . . 1.72 . * F 1.58 1.47 Pro 582 . . . . . T C 1.80 .
* F 2.37 0.84 Pro 583 . . . . T T . 1.81 * . F 2.91 0.97 Asp 584 .
. . . T T . 1.11 * . F 3.40 2.43 Gly 585 . . . . T T . 1.22 * . F
3.06 1.36 Lys 586 . A . . T . . 1.89 * . F 2.32 1.72 Ser 587 A A .
. . . . 2.10 . . F 1.58 1.79 Phe 588 A A . . . . . 2.31 . . F 1.24
3.13 Arg 589 A A . . . . . 1.64 . . F 0.90 2.71 Glu 590 A A . . . .
. 1.99 . . F 0.60 1.08 Gln 591 A A . . . . . 1.99 . . F 0.90 2.17
Gln 592 A A . . . . . 2.04 . * F 0.90 2.21 Cys 593 A A . . . . .
2.74 . * F 1.15 2.00 Glu 594 . A . . T . . 2.04 . . F 1.50 1.86 Lys
595 . A . . T . . 1.80 . . F 1.75 1.08 Tyr 596 . . . . T . . 1.80 .
. . 2.05 3.17 Asn 597 . . . . T T . 1.56 . . . 2.50 2.94 Ala 598 .
. . . T T . 1.91 . . . 1.35 2.30 Tyr 599 . . B . . T . 1.91 . . .
0.70 2.12 Asn 600 . . B . . T . 1.27 . * . 0.75 2.20 Tyr 601 . . B
. . . . 1.51 . . . 0.25 2.16 Thr 602 . . B . . . . 1.17 . * F 0.70
2.30 Asp 603 . . B . . T . 1.76 . * F 1.75 1.42 Met 604 . . B . . T
. 1.19 . * F 2.00 1.45 Asp 605 . . . . T T . 0.38 * . F 2.50 0.83
Gly 606 . . B . . T . 0.62 * * F 1.85 0.41 Asn 607 . . B B . . .
0.64 * * F 0.60 0.72 Leu 608 A . . B . . . -0.21 * * . -0.10 0.45
Leu 609 . . B B . . . 0.18 * * . -0.35 0.34 Gln 610 . . B B . . .
0.22 * . . -0.60 0.33 Trp 611 . . B B . . . 0.32 * . . -0.60 0.79
Val 612 . . B B . . . -0.27 * . . -0.45 1.50 Pro 613 . . B . . T .
0.20 * . . -0.20 0.88 Lys 614 . . B . . T . 0.16 * * . -0.20 0.82
Tyr 615 . . B . . T . -0.14 . . . 0.10 0.82 Ala 616 . . . . T T .
-0.07 * * . 0.50 0.71 Gly 617 . . . . T . . 0.90 * . . 0.64 0.55
Val 618 . . B . . . . 1.11 . * . 0.58 0.69 Ser 619 . . B . . T .
1.18 . * F 2.32 1.14 Pro 620 . . B . . T . 0.76 . * F 2.66 2.26 Arg
621 . . . . T T . 1.39 . * F 3.40 1.63 Asp 622 . . . . T T . 0.92 .
* F 3.06 2.43 Arg 623 . A . . T . . 1.08 . * F 2.32 1.30 Cys 624 .
A B . . . . 0.71 * * F 1.43 0.57 Lys 625 . A B . . . . 1.03 * * .
0.64 0.18 Leu 626 . A B . . . . 0.33 * * . 0.30 0.18 Phe 627 . A B
. . . . 0.44 . * . 0.04 0.35 Cys 628 . A B . . . . -0.01 . * . 0.98
0.34 Arg 629 . A B . . . . 0.77 * * . 1.32 0.41 Ala 630 A . . . . T
. 0.42 * * . 2.36 0.92 Arg 631 . . . . T T . 1.23 . * F 3.40 2.31
Gly 632 . . . . T T . 1.23 . * F 3.06 2.04 Arg 633 . . . . T T .
1.94 . * F 2.72 1.75 Ser 634 A A . . . . . 0.98 * * F 1.58 1.79 Glu
635 A A . . . . . 0.87 * * F 1.24 1.34 Phe 636 A A . . . . . 0.76 *
* F 0.45 0.59 Lys 637 A A . . . . . 0.51 * * . 0.30 0.77 Val 638 A
A . . . . . 0.44 * * . 0.30 0.45 Phe 639 A A . . . . . -0.11 . . .
0.45 1.03 Glu 640 A A . . . . . -1.00 * . . 0.30 0.38 Ala 641 A . .
B . . . -0.30 * . . -0.30 0.36 Lys 642 A . . B . . . -0.69 . . .
0.30 0.70 Val 643 A . . B . . . -0.14 . . . 0.60 0.40 Ile 644 A . .
B . . . -0.26 . * F 0.45 0.57 Asp 645 . . B B . . . -0.92 . . F
0.45 0.23 Gly 646 . . B B . . . -0.68 * . F -0.45 0.17 Thr 647 . .
B B . . . -0.93 * . F -0.15 0.24 Leu 648 . . . B . . C -0.08 . . F
0.05 0.22 Cys 649 . . . B T . . 0.50 * * . 0.10 0.39 Gly 650 . . .
. . T C -0.31 . . F 0.45 0.39 Pro 651 . . . . T T . -0.56 . . F
0.65 0.39 Glu 652 A . . . . T . -1.13 . . F 0.25 0.73 Thr 653 A . .
. . T . -0.99 . . F 0.25 0.52 Leu 654 A . . B . . . -1.18 * * .
-0.30 0.18 Ala 655 . . B B . . . -0.72 * * . -0.60 0.08 Ile 656 . .
B B . . . -0.86 . * . -0.60 0.10 Cys 657 . . B B . . . -0.86 . * .
-0.60 0.13 Val 658 A . . B . . . -1.21 . * . -0.30 0.21 Arg 659 . .
B B . . . -1.26 . * . -0.30 0.16 Gly 660 . . . B T T . -0.62 . * F
0.25 0.23 Gln 661 . . B B . . . -0.32 . * F 0.45 0.61 Cys 662 . . B
B . . . 0.00 . * . 0.30 0.32 Val 663 . . B B . . . 0.19 . * . 0.30
0.32 Lys 664 . . B . . T . 0.08 . * . 0.10 0.10 Ala 665 . . B . . T
. 0.39 * . . 0.70 0.30 Gly 666 . . B . . T . -0.47 * . . 0.70 0.56
Cys 667 . . B . . T . -0.66 * * . 0.70 0.21 Asp 668 . . B B . . .
0.20 * * . -0.30 0.15 His 669 . . B B . . . -0.14 * . . 0.30 0.26
Val 670 . . B B . . . 0.23 * . . 0.30 0.64 Val 671 . . B B . . .
0.69 * . . 0.64 0.59 Asp 672 . . B B . . . 1.40 * . F 1.13 0.86 Ser
673 . . B . . T . 0.59 * . F 2.32 2.31 Pro 674 A . . . . T . 0.62 *
. F 2.66 2.56 Arg 675 . . . . T T . 1.52 * . F 3.40 2.56 Lys 676 .
. . . T T . 1.71 * . F 3.06 3.82 Leu 677 . . . . T . . 1.37 * . F
2.52 1.33 Asp 678 . . . . T T . 0.81 * . F 2.23 0.67 Lys 679 . . B
. . T . 0.36 * . F 1.49 0.25 Cys 680 . . B . . T . -0.10 * . . 0.70
0.16 Gly 681 . . B . . T . -0.49 * . . 0.70 0.10 Val 682 . . B . .
. . 0.37 * . . -0.10 0.05 Cys 683 . . B . . T . 0.02 . . . 0.10
0.18 Gly 684 . . . . T T . -0.02 . . F 1.59 0.18 Gly 685 . . . . T
T . 0.34 . . F 1.93 0.38 Lys 686 . . . . T T . 0.02 . . F 2.27 0.96
Gly 687 . . . . T . . 0.99 . . F 2.41 0.52 Asn 688 . . . . T T .
1.70 . . F 3.40 1.03 Ser 689 . . B . . T . 1.19 . . F 2.66 1.03 Cys
690 . . B . . T . 1.23 . . F 2.34 0.77 Arg 691 . . B . . T . 0.84 .
. F 2.17 0.64 Lys 692 . . B . . . . 0.89 * . F 1.80 0.47 Val 693 .
. B . . T . 0.08 * . F 1.98 1.18 Ser 694 . . B . . T . 0.07 * . F
1.70 0.50 Gly 695 . . B . . T . 0.52 * . F 0.93 0.36 Ser 696 . . B
. . T . 0.10 * . F 0.46 0.75 Leu 697 . . B . . . . 0.06 . * F 0.39
0.81 Thr 698 . . B . . . . 0.67 . . F 0.37 1.31 Pro 699 . . B . . T
. 0.62 . . F 0.10 1.53 Thr 700 . . . . T T . 0.72 . . F 0.50 1.84
Asn 701 . . B . . T . 1.02 . . F 0.10 2.00 Tyr 702 . . . . T T .
1.83 * . . 0.35 2.08 Gly 703 . . . . T T . 1.26 * . . 0.65 2.41 Tyr
704 . . . . T T . 0.61 * . . 0.35 1.05 Asn 705 . . B . . T . 0.61 *
. . -0.20 0.50 Asp 706 . . B . . T . -0.28 * . . 0.10 0.72 Ile 707
. . B B . . . -0.24 * . . -0.60 0.32 Val 708 . . B B . . . -0.49 .
. . -0.30 0.31 Thr 709 . . B B . . . -0.59 * . . -0.60 0.19 Ile 710
. . B B . . . -1.18 . . . -0.60 0.27 Pro 711 . . B . . T . -1.49 *
. . -0.20 0.36 Ala 712 . . B . . T . -0.60 * . . -0.20 0.36 Gly 713
. . . . . T C -0.63 . * . 0.00 0.83 Ala 714 . . . . . T C -0.32 . *
F 0.15 0.38 Thr 715 . . B B . . . -0.29 . * F 0.45 0.62 Asn 716 . .
B B . . . -0.03 . * F -0.15 0.47 Ile 717 . . B B . . . 0.56 . * F
0.45 0.92 Asp 718 . . B B . . . 1.01 . * F 0.60 1.11 Val 719 . . B
B . . . 1.30 . * F 0.90 1.35 Lys 720 . . B B . . . 1.58 . * F 0.90
2.58 Gln 721 . . B . . . . 1.37 . * F 1.10 2.10 Arg 722 . . B . . .
. 1.91 . * F 1.10 4.38 Ser 723 . . . . . . C 1.06 * * F 1.30 2.17
His 724 . . . . . T C 1.91 * * F 1.05 0.93 Pro 725 . . . . . T C
1.87 . * F 1.33 0.82 Gly 726 . . . . T T . 1.87 * * F 1.21 0.99 Val
727 . . B . . T . 1.41 * * F 1.84 1.21 Gln 728 . . B . . . . 1.71 .
* F 1.77 0.77 Asn 729 . . B . T T . 1.50 * . F 2.80 1.26 Asp 730 .
. . . T T . 0.90 * . F 1.92 2.66 Gly 731 . . . . T T . 0.66 . . F
1.64 1.27 Asn 732 . . B . . T . 0.70 . * F 0.81 0.80 Tyr 733 . A B
. . . . 0.74 . . . -0.32 0.39 Leu 734 . A B . . . . 0.43 * . .
-0.60 0.79 Ala 735 . A B . . . . -0.16 * . . -0.60 0.71 Leu 736 . A
B . . . . 0.19 . . . -0.40 0.46 Lys 737 . A B . . . . -0.16 . . F
0.85 0.93 Thr 738 . . B . . T . 0.09 . . F 1.45 0.91 Ala 739 A . .
. . T . 0.66 . . F 2.10 1.91 Asp 740 . . B . . T . 0.43 . . F 2.00
1.50 Gly 741 . . B . . T . 0.43 . * F 1.05 0.86 Gln 742 . . B . . .
. 0.39 . * F 0.35 0.70 Tyr 743 . . B . . . . 0.36 . * . 0.30
0.67
Leu 744 . . B . . . . 0.94 . * . -0.20 0.67 Leu 745 . . B . . . .
0.13 . * . -0.40 0.63 Asn 746 . . B . . T . -0.11 . * F -0.05 0.33
Gly 747 . . . . T T . -1.00 . * F 0.35 0.40 Asn 748 . . . . . T C
-1.06 . * . 0.00 0.34 Leu 749 . . . . . T C -0.83 . * . 0.00 0.29
Ala 750 A A B . . . . -0.91 . * . -0.60 0.29 Ile 751 . A B . . . .
-0.91 * * . -0.60 0.13 Ser 752 . A B . . . . -0.57 * . . -0.60 0.27
Ala 753 A A . . . . . -0.57 * * . -0.30 0.46 Ile 754 A A . . . . .
-0.64 * . . 0.45 1.09 Glu 755 A A . . . . . -0.87 * . F 0.45 0.57
Gln 756 A . . B . . . -0.83 . * F 0.45 0.47 Asp 757 A . . B . . .
-0.49 . * F -0.15 0.49 Ile 758 A . . B . . . -0.24 . * . 0.60 0.57
Leu 759 A . . B . . . 0.33 . * . 0.30 0.33 Val 760 A . . B . . .
-0.56 . * . 0.30 0.28 Lys 761 A . . B . . . -1.37 . * F -0.45 0.28
Gly 762 . . B B . . . -1.32 . * F -0.45 0.28 Thr 763 . . B B . . .
-0.68 . * F 0.45 0.76 Ile 764 . . B B . . . -0.17 . . F -0.15 0.59
Leu 765 . . B B . . . 0.34 . * . -0.60 0.80 Lys 766 . . B B . . .
0.00 . * F -0.45 0.55 Tyr 767 . . B . . T . -0.54 * * F 0.40 1.05
Ser 768 . . . . . T C -0.82 * * F 0.45 0.89 Gly 769 . . . . . T C
-0.24 * . F 0.45 0.45 Ser 770 . . . . . T C -0.24 . * F 0.15 0.42
Ile 771 . A B . . . . -0.29 * * . -0.60 0.26 Ala 772 . A B . . . .
0.07 * . . -0.30 0.45 Thr 773 . A B . . . . -0.44 * * . 0.30 0.66
Leu 774 . A B . . . . -0.10 * . . -0.30 0.77 Glu 775 A A . . . . .
-0.10 * . 0.45 1.32 Arg 776 . A B . . . . 0.09 . . F 0.60 1.23 Leu
777 . A . . T . . 0.79 . . F 1.00 1.29 Gln 778 . A . . T . . 0.89 .
. F 1.30 1.46 Ser 779 . A . . T . . 0.89 . . F 1.00 1.15 Phe 780 .
. B . . . . 0.68 * . F 0.41 1.15 Arg 781 . . . . . . C 0.57 . * F
0.82 1.03 Pro 782 . . . . . . C 1.17 * . F 1.63 1.33 Leu 783 . . .
. . T C 0.36 * . F 2.04 2.37 Pro 784 . . . . . T C 0.34 * * F 2.10
1.00 Glu 785 . . . . . T C 0.19 * * F 1.29 0.93 Pro 786 . . B . . T
. 0.08 * * F 0.88 0.84 Leu 787 . . B B . . . -0.52 . * F 0.27 0.94
Thr 788 . . B B . . . -0.52 . * . -0.09 0.45 Val 789 . . B B . . .
-0.62 . . . -0.60 0.24 Gln 790 . . B B . . . -1.48 . . . -0.60 0.42
Leu 791 . . B B . . . -1.48 . . . -0.60 0.21 Leu 792 . . B B . . .
-1.01 . * . -0.60 0.45 Thr 793 . . B B . . . -0.70 . * . -0.60 0.26
Val 794 . . B . . T . -0.70 * . F 0.25 0.54 Pro 795 . . B . . T .
-1.40 * . F 0.25 0.48 Gly 796 . . B . . T . -0.80 * . F -0.05 0.29
Glu 797 . . B . . T . -0.20 . * F 0.25 0.60 Val 798 . . B . . . .
0.16 . * F 0.05 0.60 Phe 799 . . B . . . . 0.16 . * F 1.00 1.22 Pro
800 . . B . . T . 0.41 * * F 1.25 0.52 Pro 801 . . . . T T . 0.51 *
* F 2.00 1.41 Lys 802 . . . . T T . 0.20 * * F 1.60 2.55 Val 803 .
. B . . T . 0.36 . * F 2.00 2.38 Lys 804 . . B B . . . 0.36 . * F
0.80 1.33 Tyr 805 . . B B . . . -0.29 . * . 0.00 0.58 Thr 806 . . B
B . . . -0.29 . * . -0.20 0.58 Phe 807 . . B B . . . -0.33 . * .
-0.40 0.45 Phe 808 . . B B . . . 0.52 * * . -0.60 0.46 Val 809 . .
B . . T . -0.38 * * . -0.20 0.53 Pro 810 . . B . . T . -0.13 . * F
-0.05 0.45 Asn 811 . . . . T T . -0.52 . * F 1.25 0.88 Asp 812 . .
. . T T . -0.12 . * F 1.40 1.02 Val 813 A . . . . . . -0.02 * * F
0.65 0.89 Asp 814 A . . . . . . 0.83 * * . 0.50 0.54 Phe 815 A . .
. . . . 0.74 . * . 0.80 0.57 Ser 816 A . . . . . . 0.44 . * . 0.65
1.02 Met 817 A . . . . . . 0.49 . * . 1.40 0.82 Gln 818 A . . . . T
. 1.34 . * F 2.20 1.89 Ser 819 . . . . . T C 1.46 . * F 3.00 2.44
Ser 820 . . . . . T C 1.57 . * F 2.70 4.84 Lys 821 A . . . . T .
1.56 . * F 2.20 2.82 Glu 822 A . . . . . . 1.84 . * F 1.70 3.04 Arg
823 A . . B . . . 1.84 * * F 1.20 3.27 Ala 824 A . . B . . . 1.26 *
* F 0.90 2.63 Thr 825 . . B B . . . 0.67 * * F 0.60 1.06 Thr 826 .
. B B . . . 0.62 * * F -0.15 0.38 Asn 827 . . B B . . . 0.41 * * .
-0.60 0.65 Ile 828 . . B B . . . -0.51 * * . -0.60 0.70 Ile 829 . .
B B . . . -0.73 * . . -0.60 0.40 Gln 830 . A B . . . . -0.46 * . .
-0.60 0.21 Pro 831 . A B . . . . -0.73 * . . -0.60 0.40 Leu 832 . A
B . . . . -0.73 * * . -0.60 0.57 Leu 833 . A B . . . . -0.13 . . .
-0.60 0.57 His 834 . A B . . . . -0.10 . * . -0.60 0.39 Ala 835 . A
B B . . . -0.91 . * . -0.60 0.35 Gln 836 . A B B . . . -1.04 . . .
-0.60 0.35 Trp 837 . A B B . . . -0.23 . . . -0.60 0.26 Val 838 . A
B B . . . 0.29 . * . -0.60 0.42 Leu 839 . . B . . T . 0.02 * . .
-0.20 0.26 Gly 840 . . . . T T . 0.61 * . . 0.45 0.33 Asp 841 . . .
. T T . -0.06 . . F 1.15 0.76 Trp 842 . . . . T T . -0.07 . . F
2.00 0.50 Ser 843 . . . . . T C 0.49 * . F 2.05 0.67 Glu 844 . . .
. T T . 0.99 . . F 2.50 0.54 Cys 845 . . . . T T . 0.67 . . F 1.65
0.74 Ser 846 . . . . T T . 0.32 . . F 2.00 0.30 Ser 847 . . . . T .
. 0.02 . . F 1.55 0.17 Thr 848 . . . . T . . -0.02 . . F 0.70 0.32
Cys 849 . . . . T . . -0.31 . . F 0.45 0.24 Gly 850 . . . . T T .
0.36 * . . 0.20 0.18 Ala 851 . . . . T T . 0.77 . . . 0.20 0.22 Gly
852 . . . . T T . 1.18 . . . 0.50 0.81 Trp 853 . . . . T T . 1.18 *
. . 1.25 1.60 Gln 854 . . B B . . . 0.99 * . F 0.60 2.29 Arg 855 .
. B B . . . 1.33 * . F 0.60 1.72 Arg 856 . . B B . . . 1.26 . * F
0.90 2.83 Thr 857 . . B B . . . 1.71 . . F 1.05 0.87 Val 858 . . B
B . . . 2.00 . . . 1.20 0.87 Glu 859 . . B B . . . 1.79 . . . 1.50
0.75 Cys 860 . . . . T . . 1.38 . * . 2.40 0.80 Arg 861 . . . . T .
. 0.92 . . F 3.00 1.44 Asp 862 . . . . . T C 1.23 . * F 2.55 0.82
Pro 863 . . . . T T . 1.50 . * F 2.60 2.66 Ser 864 . . . . T T .
1.20 . * F 2.30 1.37 Gly 865 . . . . T T . 1.28 . . F 1.70 1.10 Gln
866 A . . . . . . 0.86 . * F 0.05 0.72 Ala 867 . . B . . . . 0.19 .
* F 0.05 0.78 Ser 868 . . B . . . . 0.40 . * . -0.10 0.42 Ala 869 A
. . . . . . 0.74 . * . -0.10 0.39 Thr 870 A . . . . T . 0.50 * . .
0.70 0.77 Cys 871 A . . . . T . -0.31 * . . 0.70 0.58 Asn 872 A . .
. . T . 0.32 * . . 0.10 0.48 Lys 873 A . . . . T . 0.41 . . F 0.85
0.66 Ala 874 A . . . . . . 1.00 * . F 0.80 1.90 Leu 875 A . . . . .
. 1.31 * . F 1.10 2.05 Lys 876 A . . . . T . 1.39 . . F 1.30 1.71
Pro 877 A . . . . T . 1.43 . . F 1.30 1.71 Glu 878 A . . . . T .
1.18 . . F 1.30 4.14 Asp 879 A . . . . T . 1.10 . . F 1.30 3.20 Ala
880 A . . . . . . 1.91 . . F 1.10 1.11 Lys 881 A . . . . T . 1.57 .
. F 1.30 1.11 Pro 882 A . . . . T . 1.78 * . F 1.15 0.89 Cys 883 A
. . . . T . 0.97 * . F 1.30 1.53 Glu 884 A . . . . T . 0.30 . . F
1.15 0.63 Ser 885 A A . . . . . 0.68 * . F -0.15 0.22 Gln 886 . A B
. . . . -0.18 * . F -0.15 0.63 Leu 887 . A B . . . . -0.36 . . .
-0.30 0.30 Cys 888 . A B . . . . -0.08 . . . -0.60 0.29 Pro 889 . A
B . . . . -0.47 . . . -0.60 0.21 Leu 890 . . B . . . . -0.56 . . .
-0.40 0.33
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] By screening cDNA libraries with cDNA encoding the
anti-angiogenic domain of TSP-1, the present inventors have
identified two novel proteins, METH1 and METH2 (also called VEGA-1
and VEGA-2, respectively, for vascular endothelial growth
antagonist) which contain the anti-angiogenic domain of TSP-1, a
metalloproteinase domain, and a disintegrin-like domain. The
present inventors have demonstrated that both METH1 and METH2 have
anti-angiogenic activity.
[0028] Thus, the present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a METH1 polypeptide
having the amino acid sequence shown in SEQ ID NO:2, which was
determined by sequencing a cloned cDNA. The METH1 protein of the
present invention shares sequence homology with thrombospondin-1
and pNPI. The nucleotide sequence shown in SEQ ID NO:1 was obtained
by sequencing a cDNA clone, which was deposited on Jan. 15, 1998 at
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, and given accession number 209581. The
cDNA clone contained in ATCC.TM. Deposit No. 209581 contains a
METH1 sequence, encoding amino acids 1 to 950 of SEQ ID NO:2.
[0029] The present invention also provides isolated nucleic acid
molecules comprising a polynucleotide encoding a METH2 polypeptide
having the amino acid sequence shown in SEQ ID NO:4, which was
partially determined by sequencing a cloned cDNA. The METH2 protein
of the present invention shares sequence homology with
thrombospondin-1 and pNPI. The nucleotide sequence shown in SEQ ID
NO: 3 was partially obtained by sequencing a cDNA clone, which was
deposited on Jan. 15, 1998 at the American Type Culture Collection,
University Boulevard, Manassas, Va. 20110-2209, and given accession
number 209582. The cDNA clone contained in ATCC.TM. Deposit No.
209582 contains a partial METH2 sequence, encoding amino acids
112-890 of SEQ ID NO:4.
[0030] Nucleic Acid Molecules
[0031] Some of the nucleotide sequences determined by sequencing a
DNA molecule herein were determined using an automated DNA
sequencer (such as the Model 373 from Applied Biosystems, Inc.),
and all amino acid sequences of polypeptides encoded by DNA
molecules determined herein were predicted by translation of a DNA
sequence determined as above. Therefore, as is known in the art for
any DNA sequence determined by this automated approach, any
nucleotide sequence determined herein may contain some errors.
Nucleotide sequences determined by automation are typically at
least about 90% identical, more typically at least about 95% to at
least about 99.9% identical to the actual nucleotide sequence of
the sequenced DNA molecule. The actual sequence can be more
precisely determined by other approaches including manual DNA
sequencing methods well known in the art. As is also known in the
art, a single insertion or deletion in a determined nucleotide
sequence compared to the actual sequence will cause a frame shift
in translation of the nucleotide sequence such that the predicted
amino acid sequence encoded by a determined nucleotide sequence
will be completely different from the amino acid sequence actually
encoded by the sequenced DNA molecule, beginning at the point of
such an insertion or deletion.
[0032] Using the information provided herein, such as the
nucleotide sequence in SEQ ID NO: 1 or SEQ ID NO:3, a nucleic acid
molecule of the present invention encoding a METH1 or METH2
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in SEQ ID NO:1 was discovered in a cDNA library derived
from human heart and the nucleic acid molecule described in SEQ ID
NO:3 was discovered in a cDNA library derived from human lung. The
determined nucleotide sequence of the METH1 cDNA of SEQ ID NO:1
contains an open reading frame encoding a protein of about 950
amino acid residues, including a predicted leader sequence of about
28 amino acid residues. The present inventors have determined that
the nucleotide sequence of the METH2 cDNA of SEQ ID NO:3 contains
an open reading frame encoding a protein of about 890 amino acid
residues, including a predicted leader sequence of about 23 amino
acid residues.
[0033] The present invention also provides the mature form(s) of
the METH1 and METH2 proteins of the present invention. According to
the signal hypothesis, proteins secreted by mammalian cells have a
signal or secretory leader sequence which is cleaved from the
mature protein once export of the growing protein chain across the
rough endoplasmic reticulum has been initiated. Most mammalian
cells and even insect cells cleave secreted proteins with the same
specificity. However, in some cases, cleavage of a secreted protein
is not entirely uniform, which results in two or more mature
species on the protein. Further, it has long been known that the
cleavage specificity of a secreted protein is ultimately determined
by the primary structure of the complete protein, that is, it is
inherent in the amino acid sequence of the polypeptide. Therefore,
the present invention provides a nucleotide sequence encoding the
mature METH1 polypeptide having the amino acid sequence encoded by
the cDNA clone contained in the host identified as ATCC.TM. Deposit
No. 209581 and as shown in SEQ ID NO:2. The present invention also
provides a nucleotide sequence encoding the mature METH2
polypeptide having the amino acid sequence as shown in SEQ ID NO:4.
By the mature METH1 protein having the amino acid sequence encoded
by the cDNA clone contained in the host identified as ATCC.TM.
Deposit No. 209581 is meant the mature form(s) of the METH1 protein
produced by expression in a mammalian cell (e.g., COS cells, as
described below) of the complete open reading frame encoded by the
human DNA sequence of the clone contained in the vector in the
deposited host. As indicated below, the mature METH1 having the
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209581 may or may not differ from the predicted
"mature" METH1 protein shown in SEQ ID NO:2 (amino acids from about
29 to about 950) depending on the accuracy of the predicted
cleavage site based on computer analysis; and the mature METH2 may
or may not differ from the predicted "mature" METH2 protein shown
in SEQ ID NO: 4 (amino acids from about 24 to about 890) depending
on the accuracy of the predicted cleavage site based on computer
analysis. Additionally, the mature form of the protein may then
undergo even more processing after the prodomain has been cleaved
(e.g., a second cleavage distal to the prodomain, located in the
metalloprotease domain/cysteine-rich region). Thus, "mature" forms
of the proteins encompass not only those forms produced by cleavage
of the prodomain, but also other processed forms of the
protein.
[0034] Methods for predicting whether a protein has a secretory
leader as well as the cleavage point for that leader sequence are
available. For instance, the methods of McGeoch (Virus Res.
3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690
(1986)) can be used. The accuracy of predicting the cleavage points
of known mammalian secretory proteins for each of these methods is
in the range of 75-80%. von Heinje, supra. However, the two methods
do not always produce the same predicted cleavage point(s) for a
given protein.
[0035] In the present case, the predicted amino acid sequence of
the complete METH1 and METH2 polypeptides of the present invention
were analyzed by a computer program ("PSORT") (K. Nakai and M.
Kanehisa, Genomics 14:897-911 (1992)), which is an expert system
for predicting the cellular location of a protein based on the
amino acid sequence. As part of this computational prediction of
localization, the methods of McGeoch and von Heinje are
incorporated. The analysis by the PSORT program predicted the
cleavage site between amino acids 28 and 29 in SEQ ID NO:2 and
amino acids 23 and 24 in SEQ ID NO:4. Thereafter, the complete
amino acid sequences were further analyzed by visual inspection,
applying a simple form of the (-1, -3) rule of von Heinje. von
Heinje, supra. Thus, the leader sequence for the METH1 protein is
predicted to consist of amino acid residues from about 1 to about
28 in SEQ ID NO:2, while the mature METH1 protein is predicted to
consist of residues from about 29 to about 950; and the leader
sequence for the METH2 protein is predicted to consist of amino
acid residues from about 1 to about 23 in SEQ ID NO:4, while the
mature METH2 protein is predicted to consist of residues from about
24 to about 890. An alternative predicted mature METH1 protein
consists of residues 30 to 950 in SEQ ID NO:2. Another alternative
predicted mature METH1 protein consists of residues 35 to 950 of
SEQ ID NO:2. An alternative predicted mature METH2 protein consists
of residues 31 to 890 of SEQ ID NO:4.
[0036] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors, as well as the variability of
cleavage sites for leaders in different known proteins, the
predicted METH1 polypeptide encoded by the deposited cDNA comprises
about 950 amino acids, but may be anywhere in the range of 910-990
amino acids; and the predicted leader sequence of this protein is
about 28 amino acids, but may be anywhere in the range of about 18
to about 38 amino acids. An alternative predicted METH1 polypeptide
is shown in SEQ ID NO:126, encoded by SEQ ID NO:125, and comprises
an additional 18 amino acid residues on the N-terminus. Also, the
predicted METH2 polypeptide comprises about 890 amino acids, but
may be anywhere in the range of 850 to about 930 amino acids; and
the predicted leader sequence of this protein is about 23 amino
acids, but may be anywhere in the range of about 13 to about 33
amino acids.
[0037] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0038] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0039] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) shown
in SEQ ID NO:1; DNA molecules comprising the coding sequence for
the mature METH1 protein; and DNA molecules which comprise a
sequence substantially different from those described above but
which, due to the degeneracy of the genetic code, still encode the
METH1 protein. Also included are DNA molecules comprising an open
reading frame (ORF) shown in SEQ ID NO: 3; DNA molecules comprising
the coding sequence for the mature METH2 protein; and DNA molecules
which comprise a sequence substantially different from those
described above but which, due to the degeneracy of the genetic
code, still encode the METH2 protein. Of course, the genetic code
is well known in the art. Thus, it would be routine for one skilled
in the art to generate such degenerate variants.
[0040] Polynucleotides of the present invention encompass not only
polynucleotides encoding the full length sequence, but
polynucleotides encoding the mature, proprotein, processed forms of
the protein, deletion mutants, substitution variants, allelic
variants, analogs, derivatives, etc.
[0041] In another aspect, the invention provides isolated nucleic
acid molecules encoding the METH1 or METH2 polypeptides having an
amino acid sequence as encoded by the cDNA clones contained in the
plasmids deposited as ATCC.TM. Deposit No. 209581 on Jan. 15, 1998
or ATCC.TM. Deposit No. 209582 on Jan. 15, 1998, respectively. In a
further embodiment, nucleic acid molecules are provided encoding
the mature METH1 or METH2 polypeptide or the full-length METH1 or
METH2 polypeptide lacking the N-terminal methionine. The invention
also provides an isolated nucleic acid molecule having the
nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or the
nucleotide sequence of the METH1 or METH2 cDNA contained in the
above-described deposited clones, or a nucleic acid molecule having
a sequence complementary to one of the above sequences. Such
isolated molecules, particularly DNA molecules, are useful as
probes for gene mapping, by in situ hybridization with chromosomes,
and for detecting expression of the METH1 or METH2 gene in human
tissue, for instance, by Northern blot analysis.
[0042] The present invention is further directed to fragments of
the isolated nucleic acid molecules described herein. By a fragment
of an isolated nucleic acid molecule having the nucleotide sequence
of the deposited cDNA or the nucleotide sequence shown in SEQ ID
NO:1 or SEQ ID NO: 3 is intended fragments at least about 15 nt,
and more preferably at least about 20 nt, still more preferably at
least about 30 nt, and even more preferably, at least about 40 nt
in length which are useful as diagnostic probes and primers as
discussed herein. Of course, larger fragments 50, 100, 150, 200,
250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700,
1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800,
2900, or 3000 nt in length are also useful according to the present
invention as are fragments corresponding to most, if not all, of
the nucleotide sequence of the deposited cDNA or as shown in SEQ ID
NO:1 or SEQ ID NO:3. By a fragment at least 20 nt in length, for
example, is intended fragments which include 20 or more contiguous
bases from the nucleotide sequence of the deposited cDNA or the
nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:3.
[0043] Preferred nucleic acid fragments of the present invention
include nucleic acid molecules encoding epitope-bearing portions of
the METH1 or METH2 protein. Methods for determining epitope-bearing
portions of the METH1 and METH2 proteins are described in detail
below.
[0044] Other preferred nucleic acid fragments of the present
invention include nucleic acid molecules encoding: the
metalloprotease domain of METH1, amino acids 235 to 459 in SEQ ID
NO:2; the disintegrin domain of METH1, amino acids 460 to 544 in
SEQ ID NO:2; the first TSP-like domain of METH1, amino acids 545 to
598 in SEQ ID NO:2; the second TSP-like domain of METH1, amino
acids 841 to 894 in SEQ ID NO:2; the third TSP-like domain of
METH1, amino acids 895 to 934 in SEQ ID NO:2; amino acids 536 to
613 in SEQ ID NO:2; amino acids 549 to 563 in SEQ ID NO:2; the
metalloprotease domain of METH2, amino acids 214 to 439 in SEQ ID
NO:4; the disintegrin domain of METH2, amino acids 440 to 529 in
SEQ ID NO:4; the first TSP-like domain of METH2, amino acids 530 to
583 in SEQ ID NO:4; the second TSP-like domain of METH2, amino
acids 837 to 890 in SEQ ID NO:4; amino acids 280 to 606 in SEQ ID
NO:4; and amino acids 529 to 548 in SEQ ID NO:4; and nucleic acid
molecules encoding combinations of these domains.
[0045] Thus, preferred embodiments include a nucleic acid molecule
encoding a METH1 or METH2 protein lacking the signal sequence
(cleavage occurs for METH1 somewhere about 1-24 to about 1-34 and
about 1-23 to about 1-30 for METH2); a METH1 or METH2 protein
lacking the signal sequence and the prodomain (cleavage for the
prodomain can occur in METH1 between amino acids about 232 to 236
and in METH2 between amino acids about 211 to 215); a METH1 or
METH2 protein lacking the signal sequence, the prodomain, and the
metalloprotease domain; a METH1 or METH2 protein lacking the signal
sequence, the prodomain, the metalloprotease domain, and the
cysteine rich domain; a METH1 or METH2 protein lacking the signal
sequence, the prodomain, the metalloprotease domain, cysteine rich
domain and TSP1; a METH1 or METH2 protein lacking the signal
sequence, the prodomain, the metalloprotease domain, cysteine rich
domain, TSP1 and TSP2. Also preferred are polypeptides encoded by
such nucleic acids.
[0046] Similarly, preferred embodiments include a nucleic acid
encoding a METH1 protein lacking TSP3; a METH1 protein lacking TSP2
and TSP3; a METH1 protein lacking TSP3, TSP2, and TSP1; a METH1
protein lacking the cysteine-rich domain, TSP1, TSP2, and TSP3; a
METH1 protein lacking the metalloprotease domain, the cysteine-rich
domain, TSP1, TSP2 and TSP3; and a METH1 protein lacking the
prodomain, the metalloprotease domain, the cysteine-rich domain,
TSP1, TSP2, and TSP3; a METH2 protein lacking TSP2; a METH2 protein
lacking TSP1 and TSP2; a METH2 protein lacking the cysteine-rich
domain, TSP1 and TSP2; a METH2 protein lacking the metalloprotease
domain, the cysteine-rich domain, TSP1 and TSP2; and a METH2
protein lacking the prodomain, the metalloprotease domain, the
cysteine-rich domain, TSP1 and TSP2. Also preferred are polypeptide
encoded by such nucleic acids.
[0047] Also preferred are nucleic acids encoding any combination of
METH1 domains. For example, nucleic acid molecule encoding
polypeptides comprising the following domains of METH1 are
preferred: signal sequence and prodomain; signal sequence,
prodomain and metalloprotease domain; signal sequence and
metalloprotease domain; signal sequence, prodomain, metalloprotease
domain, and cysteine rich domain; signal sequence and cysteine rich
domain; signal sequence, metalloprotease domain and cysteine rich
domain; signal sequence, prodomain, and cysteine rich domain;
signal sequence, prodomain, metalloprotease domain, cysteine rich
domain, and TSP1; signal sequence and TSP1; signal sequence,
prodomain and TSP1; signal sequence, prodomain, metalloprotease
domain and TSP1; signal sequence, metalloprotease domain, and TSP1;
signal sequence, prodomain, cysteine rich domain and TSP1; signal
sequence, cysteine rich domain and TSP1; signal sequence,
metalloprotease domain, cysteine rich domain and TSP1; signal
sequence, prodomain, metalloprotease domain, cysteine rich domain,
TSP1 and TSP2; signal sequence and TSP2; signal sequence, prodomain
and TSP2; signal sequence, metalloprotease domain and TSP2; signal
sequence, cysteine rich domain and TSP2; signal sequence, TSP1 and
TSP2; signal sequence, prodomain, metalloprotease domain and TSP2;
signal sequence, prodomain, cysteine rich domain and TSP2; signal
sequence, prodomain, TSP1 and TSP2; signal sequence,
metalloprotease domain, cysteine rich domain and TSP2; signal
sequence, metalloprotease domain, TSP1 and TSP2; signal sequence,
metalloprotease domain, cysteine rich domain, TSP1 and TSP2; signal
sequence, prodomain, cysteine rich domain, TSP1 and TSP2; signal
sequence and TSP3; signal sequence, prodomain and TSP3; signal
sequence, prodomain, metalloprotease domain and TSP3; signal
sequence, metalloprotease domain and TSP3; signal sequence,
prodomain, metalloprotease domain, cysteine rich domain and TSP3;
signal sequence, cysteine rich domain and TSP3; signal sequence,
prodomain, cysteine rich domain and TSP3; signal sequence,
prodomain, metalloprotease domain, cysteine rich domain, TSP1 and
TSP3; signal sequence, TSP1 and TSP3; signal sequence, prodomain,
TSP1 and TSP3; signal sequence, prodomain, metalloprotease domain,
TSP1 and TSP3; signal sequence, prodomain, cysteine rich domain,
TSP1 and TSP3; signal sequence, TSP2 and TSP3; signal sequence,
prodomain, cysteine rich domain, TSP1, TSP2 and TSP3; signal
sequence, prodomain, metalloprotease domain, TSP1, TSP2 and TSP3;
signal sequence, metalloprotease domain, TSP1, TSP2 and TSP3;
signal sequence, TSP1, TSP2 and TSP3;
[0048] signal sequence, metalloprotease domain, cysteine rich
domain, TSP1, TSP2 and TSP3; signal sequence, prodomain,
metalloprotease domain, TSP1 and TSP2; signal sequence, prodomain,
metalloprotease domain, cysteine rich domain, and TSP2; signal
sequence, prodomain, metalloprotease domain, cysteine rich domain,
TSP2 and TSP3; signal sequence, TSP1, TSP2 and TSP3; signal
sequence, cysteine rich domain, TSP1 and TSP2; signal sequence,
cysteine rich domain, TSP1 and TSP3; signal sequence, cysteine rich
domain, TSP2 and TSP3; signal sequence, cysteine rich domain, TSP1,
TSP2, and TSP3; signal sequence, metalloprotease domain, cysteine
rich domain, and TSP3; signal sequence, metalloprotease domain,
cysteine rich domain, TSP1 and TSP3; signal sequence,
metalloprotease domain, cysteine rich domain, TSP2 and TSP3; signal
sequence, metalloprotease domain, TSP1 and TSP3; signal sequence,
metalloprotease domain, TSP2 and TSP3; signal sequence, prodomain,
metalloprotease domain, TSP1 and TSP3; signal sequence, prodomain,
metalloprotease domain, TSP2 and TSP3; prodomain and
metalloprotease domain; prodomain and cysteine rich domain;
prodomain and TSP1; prodomain and TSP2; prodomain and TSP3;
prodomain, metalloprotease domain and cysteine rich domain;
prodomain, metalloprotease domain and TSP1; prodomain,
metalloprotease domain and TSP2; prodomain, metalloprotease domain
and TSP3; prodomain, metalloprotease domain, cysteine rich domain
and TSP1; prodomain, metalloprotease domain, cysteine rich domain
and TSP2; prodomain, metalloprotease domain, cysteine rich domain
and TSP3; prodomain, cysteine rich domain and TSP1; prodomain,
cysteine rich domain and TSP2; prodomain, cysteine rich domain and
TSP3; prodomain, metalloprotease domain, cysteine rich domain, TSP1
and TSP2; prodomain, metalloprotease domain, cysteine rich domain,
TSP1, TSP2 and TSP3; prodomain, cysteine rich domain, TSP1 and
TSP2; prodomain, metalloprotease domain, TSP1 and TSP2; prodomain,
metalloprotease domain, cysteine rich domain, TSP2 and TSP3;
prodomain, cysteine rich domain, TSP1 and TSP3; prodomain, cysteine
rich domain, TSP2 and TSP3; prodomain, TSP1 and TSP2; prodomain,
TSP1 and TSP3; prodomain, TSP2 and TSP3; prodomain, metalloprotease
domain, TSP1 and TSP2; prodomain, metalloprotease domain, TSP1 and
TSP3; prodomain, metalloprotease domain, TSP2 and TSP3; prodomain,
metalloprotease domain, cysteine rich domain, TSP2 and TSP3;
prodomain, TSP1 and TSP2; prodomain, TSP1 and TSP3; prodomain, TSP2
and TSP3; prodomain, metalloprotease domain, TSP1 and TSP2;
prodomain, metalloprotease domain, TSP1 and TSP3; prodomain,
metalloprotease domain, TSP2 and TSP3; prodomain, metalloprotease
domain, cysteine domain, TSP1 and TSP3; prodomain, cysteine rich
domain, TSP1, TSP2 and TSP3; prodomain, metalloprotease domain,
TSP1, TSP2, and TSP3; metalloprotease domain and cysteine rich
domain; metalloprotease domain and TSP1; metalloprotease domain and
TSP2; metalloprotease domain and TSP3; metalloprotease domain,
cysteine rich domain and TSP1; metalloprotease domain, cysteine
rich domain and TSP2; metalloprotease domain, cysteine rich domain
and TSP3; metalloprotease domain, cysteine rich domain, TSP1 and
TSP2; metalloprotease domain, cysteine rich domain, TSP1, TSP2 and
TSP3; metalloprotease domain, cysteine rich domain, TSP1 and TSP3;
metalloprotease domain, cysteine rich domain, TSP2 and TSP3;
metalloprotease domain, TSP1 and TSP2; metalloprotease domain, TSP1
and TSP3; metalloprotease domain, TSP2 and TSP3; metalloprotease
domain, TSP1, TSP2 and TSP3; cysteine rich domain and TSP1;
cysteine rich domain and TSP2; cysteine rich domain and TSP3;
cysteine rich domain, TSP1 and TSP2; cysteine rich domain, TSP1 and
TSP3; cysteine rich domain, TSP2 and TSP3; cysteine rich domain,
TSP1, TSP2 and TSP3; TSP1 and TSP2; TSP1 and TSP3; TSP2 and TSP3;
and/or TSP1, TSP2 and TSP3. These domains may be present in the
METH1 molecule in the same order or a different order than in the
naturally occurring molecule. Also preferred are polypeptides
encoded by such nucleic acids.
[0049] Also preferred are nucleic acids encoding any combination of
METH2 domains. For example, nucleic acid molecule encoding
polypeptides comprising the following domains of METH2 are
preferred: signal sequence and prodomain; signal sequence,
prodomain and metalloprotease domain; signal sequence and
metalloprotease domain; signal sequence, prodomain, metalloprotease
domain, and cysteine rich domain; signal sequence and cysteine rich
domain; signal sequence, metalloprotease domain and cysteine rich
domain; signal sequence, prodomain, and cysteine rich domain;
signal sequence, prodomain, metalloprotease domain, cysteine rich
domain, and TSP1; signal sequence and TSP1; signal sequence,
prodomain and TSP1; signal sequence, prodomain, metalloprotease
domain and TSP1; signal sequence, metalloprotease domain, and TSP1;
signal sequence, prodomain, cysteine rich domain and TSP1; signal
sequence, cysteine rich domain and TSP1; signal sequence,
metalloprotease domain, cysteine rich domain and TSP1; signal
sequence, prodomain, metalloprotease domain, cysteine rich domain,
TSP1 and TSP2; signal sequence and TSP2; signal sequence, prodomain
and TSP2; signal sequence, metalloprotease domain and TSP2; signal
sequence, cysteine rich domain and TSP2; signal sequence, TSP1 and
TSP2; signal sequence, prodomain, metalloprotease domain and TSP2;
signal sequence, prodomain, cysteine rich domain and TSP2; signal
sequence, prodomain, TSP1 and TSP2; signal sequence,
metalloprotease domain, cysteine rich domain and TSP2; signal
sequence, metalloprotease domain, TSP1 and TSP2; signal sequence,
metalloprotease domain, cysteine rich domain, TSP1 and TSP2; signal
sequence, prodomain, cysteine rich domain, TSP1 and TSP2; signal
sequence, prodomain, metalloprotease domain, TSP1 and TSP2; signal
sequence, prodomain, metalloprotease domain, cysteine rich domain,
and TSP2; signal sequence, cysteine rich domain, TSP1 and TSP2;
prodomain and metalloprotease domain; prodomain and cysteine rich
domain; prodomain and TSP1; prodomain and TSP2; prodomain,
metalloprotease domain and cysteine rich domain; prodomain,
metalloprotease domain and TSP1; prodomain, metalloprotease domain
and TSP2; prodomain, metalloprotease domain, cysteine rich domain
and TSP1; prodomain, metalloprotease domain, cysteine rich domain
and TSP2; prodomain, cysteine rich domain and TSP1; prodomain,
cysteine rich domain and TSP2; prodomain, metalloprotease domain,
cysteine rich domain, TSP1 and TSP2; prodomain, cysteine rich
domain, TSP1 and TSP2; prodomain, metalloprotease domain, TSP1 and
TSP2; prodomain, metalloprotease domain, TSP1 and TSP2; prodomain,
TSP1 and TSP2; prodomain, metalloprotease domain, TSP1 and TSP2;
metalloprotease domain and cysteine rich domain; metalloprotease
domain and TSP1; metalloprotease domain and TSP2; metalloprotease
domain, cysteine rich domain and TSP1; metalloprotease domain,
cysteine rich domain and TSP2; metalloprotease domain, cysteine
rich domain, TSP1 and TSP2; metalloprotease domain, TSP1 and TSP2;
cysteine rich domain and TSP1; cysteine rich domain and TSP2;
cysteine rich domain, TSP1 and TSP2. These domains may be present
in the METH2 molecule in the same order or a different order than
in the naturally occurring molecule. Also preferred are
polypeptides encoded by such nucleic acids.
[0050] Additionally, METH1 and METH2 domains may be combined to
form hybrid molecules. Any domain of METH1 may be combined with any
domain of METH2 to form a hybrid molecule. For example, the TSP1
domain of METH1 may be replaced with the TSP1 domain of METH2 to
form a hybrid molecule, leaving the remainder of the METH1 molecule
intact. Also, the TSP1 domain of METH1 may be replaced with the
TSP2 domain of METH2 to form a hybrid molecule, leaving the
remainder of the METH1 molecule intact. Additionally, the TSP1
domain of METH1 may be combined with the TSP2 domain of METH2 to
form a hybrid molecule, without any additional METH1 and/or METH2
sequences. These domains may be present in the same or a different
order as occurs in the naturally occurring molecules. Also
preferred are polypeptides encoded by such nucleic acids.
[0051] Further embodiments include nucleic acids encoding a METH1
or METH2 polypeptide in which: one or more TSP domains have been
replaced with other known TSP domains; the metalloprotease domain
has been replaced with another known metalloprotease domain; the
disintegrin domain has been replaced with another known disintegrin
domain. One or more domains may be replaced in this manner. For
example, the both the metalloprotease and disintegrin domains may
be replaced. Alternatively, all three TSP domains may be replaced.
Also preferred are polypeptides encoded by such nucleic acids.
[0052] Preferred embodiments are polynucleotides encoding the amino
acid sequence of SEQ ID NO:2 or SEQ ID NO:4 except for several,
5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted,
deleted or added, in any combination.
[0053] In addition, the present inventors have identified the
following cDNA clones related to portions of the sequence shown in
SEQ ID NO:1: HOUCQ17RA (SEQ ID NO:14), HPLBM11R (SEQ ID NO:15),
HGBI07R (SEQ ID NO:16), HNTMA49R (SEQ ID NO:17), HNALE27R (SEQ ID
NO:18), and HIBDB45R (SEQ ID NO:19).
[0054] The following public ESTs, which relate to portions of SEQ
ID NO:1, have also been identified: D67076 (SEQ ID NO:20), AB001735
(SEQ ID NO:21), X14787 (SEQ ID NO:22), U64857 (SEQ ID NO:23),
X04665 (SEQ ID NO:24), M64866 (SEQ ID NO:25), L07803 (SEQ ID
NO:26), U08006 (SEQ ID NO:27), M16974 (SEQ ID NO:28), L13855 (SEQ
ID NO:29), AL021529 (SEQ ID NO:30), D86074 (SEQ ID NO:31), L05390
(SEQ ID NO:32), Z69361 (SEQ ID NO:33), X99599 (SEQ ID NO:34),
AF018073 (SEQ ID NO:35), L23760 (SEQ ID NO:36), Z46970 (SEQ ID
NO:37), AC004449 (SEQ ID NO:38), Z69589 (SEQ ID NO:39), Z22279 (SEQ
ID NO:40), X17524 (SEQ ID NO:41), AI126019 (SEQ ID NO:103),
AI571069 (SEQ ID NO:104), AI148739 (SEQ ID NO:105), AI335849 (SEQ
ID NO:106), AA677116 (SEQ ID NO:107), H27128 (SEQ ID NO:108),
AA368429 (SEQ ID NO:109), AA345812 (SEQ ID NO:110), AA373718 (SEQ
ID NO:111), AI537518 (SEQ ID NO:112), N88341 (SEQ ID NO:113),
C03600 (SEQ ID NO:114), AA066142 (SEQ ID NO:115), AI40095 (SEQ ID
NO:94), AA288689 (SEQ ID NO:116), AI464076 (SEQ ID NO:97), R13547
(SEQ ID NO:117), R19976 (SEQ ID NO:118), Z43925 (SEQ ID NO:19),
AA670987 (SEQ ID NO:120), AA635657 (SEQ ID NO:96), W24878 (SEQ ID
NO:121), W47316 (SEQ ID NO:122), W35345 (SEQ ID NO:123), and N27243
(SEQ ID NO:124).
[0055] The present inventors have also identified the following
cDNA clones related to portions of SEQ ID NO:3: HCE4D69FP02 (SEQ ID
NO:42), HIBDB45F (SEQ ID NO:43), HKIXH64R (SEQ ID NO:44), HIBDB45R
(SEQ ID NO:19), HCE3Z95R (SEQ ID NO:45), HTLEQ90R (SEQ ID NO:46),
HMWEF45R (SEQ ID NO:47), HTOFC34RA (SEQ ID NO:48), HHFDI20R (SEQ ID
NO:49), HMSHY47R (SEQ ID NO:50), HCESF90R (SEQ ID NO:51), HMCAO46R
(SEQ ID NO:52), HTTAQ67R (SEQ ID NO:53), HFKCF19F (SEQ ID NO:54),
HMCAS31R (SEQ ID NO:55), HMWGP26R (SEQ ID NO:56), HLHTP36R (SEQ ID
NO:57), HE8AN11R (SEQ ID NO:58), HEONN73R (SEQ ID NO:59), HBNBG53R
(SEQ ID NO:60), and HMSCH94R (SEQ ID NO:61).
[0056] The following public ESTs, which relate to portions of the
sequence shown in SEQ ID NO:3, have also been identified: D67076
(SEQ ID NO:20), AB001735 (SEQ ID NO:21), AB005287 (SEQ ID NO:62),
X87619 (SEQ ID NO:63), X14787 (SEQ ID NO:22), X04665 (SEQ ID
NO:24), M87276 (SEQ ID NO:64), M62458 (SEQ ID NO:65), AB002364 (SEQ
ID NO:66), AB005297 (SEQ ID NO:67), X69161 (SEQ ID NO:68), X16619
(SEQ ID NO:69), 136448 (SEQ ID NO:70), L12260 (SEQ ID NO:71),
136352 (SEQ ID NO:72), X15898 (SEQ ID NO:73), 107789 (SEQ ID
NO:74), 108144 (SEQ ID NO:75) U31814 (SEQ ID NO:76), AF001444 (SEQ
ID NO:77), AI400905 (SEQ ID NO:94), AI378857 (SEQ ID NO:95),
AA635657 (SEQ ID NO:96), AI464076 (SEQ ID NO:97), CO6578 (SEQ ID
NO:98), AA855532 (SEQ ID NO:99), H11881 (SEQ ID NO:100), AA350801
(SEQ ID NO:101), and AA350802 (SEQ ID NO:102).
[0057] In specific embodiments, the polynucleotides of the
invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10
kb, or 7.5 kb in length. In a further embodiment, polynucleotides
of the invention comprise at least 15 contiguous nucleotides of
METH1 or METH2 coding sequence, but do not comprise all or a
portion of any METH1 or METH2 intron. In another embodiment, the
nucleic acid comprising METH1 or METH2 coding sequence does not
contain coding sequences of a genomic flanking gene (i.e., 5' or 3'
to the METH1 or METH2 gene in the genome).
[0058] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clones contained in
ATCC.TM. Deposit No. 209581 or ATCC.TM. Deposit No. 209582. By
"stringent hybridization conditions" is intended overnight
incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C.
[0059] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30, 40, 50, 60 or 70 nt
of the reference polynucleotide. These are useful as diagnostic
probes and primers as discussed above and in more detail below.
[0060] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNAs or the nucleotide sequence as shown in SEQ ID
NO:1 or SEQ ID NO:3). Of course, a polynucleotide which hybridizes
only to a poly A sequence (such as the 3' terminal poly(A) tract of
the METH1 or METH2 cDNA shown in SEQ ID NO:1 and SEQ ID NO:3,
respectively) or to a complementary stretch of T (or U) resides,
would not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0061] Also contemplated are nucleic acid molecules that hybridize
to the METH1 or METH2 polynucleotides at moderately high stringency
hybridization conditions. Changes in the stringency of
hybridization and signal detection are primarily accomplished
through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt
conditions, or temperature. For example, moderately high stringency
conditions include an overnight incubation at 3.degree. C. in a
solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl; 0.2M
NaH.sub.2PO.sub.4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide,
100 .mu.g/ml salmon sperm blocking DNA; followed by washes at
50.degree. C. with 1.times.SSPE, 0.1% SDS. In addition, to achieve
even lower stringency, washes performed following stringent
hybridization can be done at higher salt concentrations (e.g.
5.times.SSC).
[0062] Note that variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. Typical blocking reagents include Denhardt's reagent,
BLOTTO, heparin, denatured salmon sperm DNA, and commercially
available proprietary formulations. The inclusion of specific
blocking reagents may require modification of the hybridization
conditions described above, due to problems with compatibility.
[0063] Of course, a polynucleotide which hybridizes only to polyA+
sequences (such as any 3' terminal polyA+ tract of a cDNA shown in
the sequence listing), or to a complementary stretch of T (or U)
residues, would not be included in the definition of
"polynucleotide," since such a polynucleotide would hybridize to
any nucleic acid molecule containing a poly (A) stretch or the
complement thereof (e.g., practically any double-stranded cDNA
clone).
[0064] The METH1 or METH2 polynucleotide can be composed of any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. For example, METH1 or
METH2 polynucleotides can be composed of single- and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, the METH1 or METH2
polynucleotides can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. METH1 or METH2
polynucleotides may also contain one or more modified bases or DNA
or RNA backbones modified for stability or for other reasons.
"Modified" bases include, for example, tritylated bases and unusual
bases such as inosine. A variety of modifications can be made to
DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically, or metabolically modified forms.
[0065] "SEQ ID NO:1" refers to a METH1 polynucleotide sequence
while "SEQ ID NO:2" refers to a METH1 polypeptide sequence. "SEQ ID
NO:3" refers to a METH2 polynucleotide sequence while "SEQ ID NO:4"
refers to a METH2 polypeptide sequence.
[0066] As indicated, nucleic acid molecules of the present
invention which encode a METH1 or METH2 polypeptide may include,
but are not limited to, those encoding the amino acid sequence of
the mature polypeptide, by itself; the coding sequence for the
mature polypeptide and additional sequences, such as those encoding
the leader or secretory sequence, such as a pre-, or pro- or
prepro-protein sequence; the coding sequence of the mature
polypeptide, with or without the aforementioned additional coding
sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (Qiagen, Inc.), among others, many of
which are commercially available. As described in Gentz et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson et al.,
Cell 37:767-778 (1984). As discussed below, other such fusion
proteins include the METH1 or METH2 fused to Fc at the N- or
C-terminus. Other fusion proteins include METH1 or METH2 fused to
Flag at the N- or C-terminus. Other fusion proteins include METH1
fragments or METH2 fragments fused to Flag or Fc at the N- or
C-terminus. Particularly preferred are fragments of METH1 or METH2,
such as H541-Q894, M1-P799, F236-E614, or K801-Q950 of SEQ ID NO:2,
fused to Fc or Flag at the N- or C-terminus.
[0067] As stated above, METH1 or METH2 may be fused with the FLAG
polypeptide sequence (see U.S. Pat. No. 4,851,341; see also Hopp et
al., Bio/Technology 6:1204, 1988). The FLAG polypeptide sequence is
highly antigenic and provides an epitope for binding by a specific
monoclonal antibody, enabling rapid purification of the expressed
recombinant protein. This sequence is also specifically cleaved by
bovine mucosal enterokinase at the residue immediately following
the Asp-Lys pairing.
[0068] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the METH1 or METH2 protein.
Variants may occur naturally, such as a natural allelic variant. By
an "allelic variant" is intended one of several alternate forms of
a gene occupying a given locus on a chromosome of an organism.
Lewin, B., ed., Genes II, John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0069] Such variants include those produced by nucleotide
substitutions, deletions or additions, which may involve one or
more nucleotides. The variants may be altered in coding regions,
non-coding regions, or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions. Especially preferred among these are silent
substitutions, additions and deletions, which do not alter the
properties and activities of the METH1 or METH2 protein or portions
thereof. Also especially preferred in this regard are conservative
substitutions.
[0070] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to: a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-terminal
methionine; a nucleotide sequence encoding the polypeptide having
the amino acid sequence at positions from about 29 to about 950 in
SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having
the amino acid sequence at position from about 30 to about 950 in
SEQ ID NO:2; a nucleotide sequence encoding the polypeptide having
the amino acid sequence encoded by the cDNA clone contained in
ATCC.TM. Deposit No. 209581; a nucleotide sequence encoding the
mature METH1 polypeptide having the amino acid sequence encoded by
the cDNA clone contained in ATCC.TM. Deposit No. 209581; a
nucleotide sequence encoding amino acids 235 to 459 in SEQ ID NO:2
(the metalloprotease domain of METH1); a nucleotide sequence
encoding amino acids 460 to 544 in SEQ ID NO:2 (the disintegrin
domain of METH1); a nucleotide sequence encoding amino acids 545 to
598 in SEQ ID NO:2 (the first TSP-like domain of METH1); a
nucleotide sequence encoding amino acids 841 to 894 in SEQ ID NO:2
(the second TSP-like domain of METH1); a nucleotide sequence
encoding amino acids 895 to 934 in SEQ ID NO:2 (the third TSP-like
domain of METH1); a nucleotide sequence encoding amino acids 536 to
613 in SEQ ID NO:2; a nucleotide sequence encoding amino acids 549
to 563 in SEQ ID NO:2; a nucleotide sequence encoding the
polypeptide having the amino acid sequence in SEQ ID NO:4; a
nucleotide sequence encoding the polypeptide having the amino acid
sequence in SEQ ID NO:4, but lacking the N-terminal methionine; a
nucleotide sequence encoding the polypeptide having the amino acid
sequence at positions from about 24 to about 890 in SEQ ID NO:4; a
nucleotide sequence encoding the polypeptide having the amino acid
sequence at positions from about 112 to about 890 in SEQ ID NO:4; a
nucleotide sequence encoding the polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209582; a nucleotide sequence encoding the mature METH2
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 209582; a nucleotide
sequence encoding amino acids 214 to 439 in SEQ ID NO:4 (the
metalloprotease domain of METH2); a nucleotide sequence encoding
amino acids 440 to 529 in SEQ ID NO:4 (the disintegrin domain of
METH2); a nucleotide sequence encoding amino acids 530 to 583 in
SEQ ID NO:4 (the first TSP-like domain of METH2); a nucleotide
sequence encoding amino acids 837 to 890 in SEQ ID NO:4 (the second
TSP-like domain of METH2); a nucleotide sequence encoding amino
acids 280 to 606 in SEQ ID NO:4; a nucleotide sequence encoding
amino acids 529 to 548 in SEQ ID NO:4; or a nucleotide sequence
complementary to any of the above nucleotide sequences. By a
polynucleotide having a nucleotide sequence at least, for example,
95% "identical" to a reference nucleotide sequence encoding a METH1
or METH2 polypeptide is intended that the nucleotide sequence of
the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence encoding the METH1 or METH2 polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0071] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID
NO: 3 or to the nucleotide sequence of the deposited cDNA clones
can be determined conventionally using known computer programs such
as the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0072] A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al., Comp. Appl. Biosci.
6:237-245 (1990). In a sequence alignment, the query and subject
sequences are both DNA sequences. An RNA sequence can be compared
by converting U's to T's. The result of said global sequence
alignment is in percent identity. Preferred parameters used in a
FASTDB alignment of DNA sequences to calculate percent identity
are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject nucleotide sequence, whichever is shorter.
[0073] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by the results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence are calculated for the
purposes of manually adjusting the percent identity score.
[0074] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and, therefore, the
FASTDB alignment does not show a match/alignment of the first 10
bases at the 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence), so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal, so that there are no bases on the 5' or 3'
ends of the subject sequence which are not matched/aligned with the
query. In this case, the percent identity calculated by FASTDB is
not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to be made for the purposes of the present invention.
[0075] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or to the
nucleic acid sequence of the deposited cDNAs, irrespective of
whether they encode a polypeptide having METH1 or METH2 activity.
This is because even where a particular nucleic acid molecule does
not encode a polypeptide having METH1 or METH2 activity, one of
skill in the art would still know how to use the nucleic acid
molecule, for instance, as a hybridization probe or a polymerase
chain reaction (PCR) primer. Uses of the nucleic acid molecules of
the present invention that do not encode a polypeptide having METH1
or METH2 activity include, inter alia, (1) isolating the METH1 or
METH2 gene or allelic variants thereof in a cDNA library; (2) in
situ hybridization (e.g., "FISH") to metaphase chromosomal spreads
to provide precise chromosomal location of the METH1 or METH2 gene,
as described in Verma et al., Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York (1988); and (3) Northern Blot
analysis for detecting METH1 or METH2 mRNA expression in specific
tissues.
[0076] Preferred, however, are nucleic acid molecules having
sequences at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 or to a
nucleic acid sequence of the deposited cDNAs which do, in fact,
encode a polypeptide having METH1 or METH2 protein activity. By "a
polypeptide having METH1 activity" is intended polypeptides
exhibiting METH1 activity in a particular biological assay. For
example, METH1 protein activity can be measured using the
chorioallantoic membrane assay (Iruela-Arispe et al., Thrombosis
and Haemostasis 78(1):672-677 (1997)) or the cornea pocket assay
(Tolsma et al., J. Cell. Biol. 122:497-511 (1993)), both described
in Example 4, below. By "a polypeptide having METH2 activity" is
intended polypeptides exhibiting METH2 activity in a particular
biological assay. For example, METH2 protein activity can also be
measured using the chorioallantoic membrane assay (Iruela-Arispe et
al., Thrombosis and Haemostasis 78(1):672-677 (1997)) or the cornea
pocket assay (Tolsma et al., J. Cell. Biol. 122:497-5111 (1993)),
both described in Example 4, below.
[0077] Briefly, in the chorioallantoic assay, the potentially
anti-angiogenic compound of interest is added to type I collagen
pellets (Vitrogen), along with an angiogenic growth factor, such as
bFGF. The samples are mixed and placed onto nylon meshes, and
allowed to polymerize. After polymerization is complete, the meshes
are placed onto the chorioallantoic membrane of 12 day old chick
embryos and placed at 37.degree. C. for 24 hours. The embryos are
then injected with a fluorescent agent, such as FITC-dextran, and
the meshes are fixed and mounted for observation under a
fluorescent microscope.
[0078] In the cornea pocket assay, hydron pellets containing the
compound of interest and an angiogenic growth factor, such as bFGF,
are implanted 1 to 2 mm from the limbus of the cornea of rats or
mice. Response is examined after a period of time, for example 5
days. The extent of angiogenesis is evaluated by measuring the
capillaries migrating from the limb of the cornea.
[0079] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence of
the deposited cDNAs or a nucleic acid sequence shown in SEQ ID NO:1
or SEQ ID NO:3 will encode a polypeptide "having METH1 or METH2
protein activity." In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing the above
described comparison assay. It will be further recognized in the
art that, for such nucleic acid molecules that are not degenerate
variants, a reasonable number will also encode a polypeptide having
METH1 or METH2 protein activity. This is because the skilled
artisan is fully aware of amino acid substitutions that are either
less likely or not likely to significantly effect protein function
(e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid).
[0080] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that proteins are surprisingly tolerant of amino
acid substitutions.
[0081] Vectors and Host Cells
[0082] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of METH1 or METH2 polypeptides or fragments thereof
by recombinant techniques. Cell-free translation systems can also
be employed to produce such proteins using RNAs derived from the
DNA constructs of the present invention.
[0083] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0084] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0085] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0086] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0087] In addition to the use of expression vectors in the practice
of the present invention, the present invention further includes
novel expression vectors comprising operator and promoter elements
operatively linked to nucleotide sequences encoding a protein of
interest. One example of such a vector is pHE4-5 which is described
in detail below.
[0088] As summarized in FIGS. 8 and 9, components of the pHE4-5
vector (SEQ ID NO:12) include: 1) a neomycinphosphotransferase gene
as a selection marker, 2) an E. coli origin of replication, 3) a T5
phage promoter sequence, 4) two lac operator sequences, 5) a
Shine-Delgarno sequence, 6) the lactose operon repressor gene
(lacIq). The origin of replication (oriC) is derived from pUC19
(LTI, Gaithersburg, Md.). The promoter sequence and operator
sequences were made synthetically. Synthetic production of nucleic
acid sequences is well known in the art. Clontech 95/96 Catalog,
pages 215-216, Clontech, 1020 East Meadow Circle, Palo Alto, Calif.
94303. A nucleotide sequence encoding METH1 (SEQ ID NO:2) or METH2
(SEQ ID NO:4), is operatively linked to the promoter and operator
by inserting the nucleotide sequence between the NdeI and Asp718
sites of the pHE4-5 vector.
[0089] As noted above, the pHE4-5 vector contains a lacIq gene.
LacIq is an allele of the lacI gene which confers tight regulation
of the lac operator. Amann, E. et al., Gene 69:301-315 (1988);
Stark, M., Gene 51:255-267 (1987). The lacIq gene encodes a
repressor protein which binds to lac operator sequences and blocks
transcription of down-stream (i.e., 3') sequences. However, the
lacIq gene product dissociates from the lac operator in the
presence of either lactose or certain lactose analogs, e.g.,
isopropyl B-D-thiogalactopyranoside (IPTG). METH1 or METH2 thus is
not produced in appreciable quantities in uninduced host cells
containing the pHE4-5 vector. Induction of these host cells by the
addition of an agent such as IPTG, however, results in the
expression of the METH1 or METH2 coding sequence.
[0090] The promoter/operator sequences of the pHE4-5 vector (SEQ ID
NO:13) comprise a T5 phage promoter and two lac operator sequences.
One operator is located 5' to the transcriptional start site and
the other is located 3' to the same site. These operators, when
present in combination with the lacIq gene product, confer tight
repression of down-stream sequences in the absence of a lac operon
inducer, e.g., IPTG. Expression of operatively linked sequences
located down-stream from the lac operators may be induced by the
addition of a lac operon inducer, such as IPTG. Binding of a lac
inducer to the lacIq proteins results in their release from the lac
operator sequences and the initiation of transcription of
operatively linked sequences. Lac operon regulation of gene
expression is reviewed in Devlin, T., Textbook of Biochemistry with
Clinical Correlations, 4th Edition (1997), pages 802-807.
[0091] The pHE4 series of vectors contain all of the components of
the pHE4-5 vector except for the METH1 or METH2 coding sequence.
Features of the pHE4 vectors include optimized synthetic T5 phage
promoter, lac operator, and Shine-Delgarno sequences. Further,
these sequences are also optimally spaced so that expression of an
inserted gene may be tightly regulated and high level of expression
occurs upon induction.
[0092] Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL promoters and the trp promoter. Suitable
eukaryotic promoters include the CMV immediate early promoter, the
HSV thymidine kinase promoter, the early and late SV40 promoters,
the promoters of retroviral LTRs, such as those of the Rous Sarcoma
Virus (RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0093] The pHE4-5 vector also contains a Shine-Delgarno sequence 5'
to the AUG initiation codon. Shine-Delgarno sequences are short
sequences generally located about 10 nucleotides up-stream (i.e.,
5') from the AUG initiation codon. These sequences essentially
direct prokaryotic ribosomes to the AUG initiation codon.
[0094] Thus, the present invention is also directed to expression
vectors useful for the production of the proteins of the present
invention. This aspect of the invention is exemplified by the
pHE4-5 vector (SEQ ID NO:12).
[0095] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0096] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when the Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as an antigen for
immunizations. In drug discovery, for example, human proteins, such
as the hIL5-receptor, have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
of hIL-5. See, D. Bennett et al., J. Mol. Recognition 8:52-58
(1995) and K. Johanson et al., J. of Biol. Chem. 270(16):9459-9471
(1995).
[0097] The METH1 or METH2 protein can be recovered and purified
from recombinant cell cultures by well-known methods including
ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
[0098] In addition, polypeptides of the invention can be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller, M., et al., 1984, Nature 310:
105-111). For example, a peptide corresponding to a fragment of the
METH1 and/or METH2 polypeptides of the invention can be synthesized
by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino acids or chemical amino acid analogs can be
introduced as a substitution or addition into the METH1 and/or
METH2 polynucleotide sequence. Non-classical amino acids include,
but are not limited to, to the D-isomers of the common amino acids,
2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric
acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine,
t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,
fluoro-amino acids, designer amino acids such as b-methyl amino
acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid
analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or L (levorotary).
[0099] The invention encompasses METH1 and/or METH2 polypeptides
which are differentially modified during or after translation,
e.g., by glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc. Any of numerous chemical modifications may be carried out by
known techniques, including but not limited, to specific chemical
cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8
protease, NaBH.sub.4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin;
etc.
[0100] Additional post-translational modifications encompassed by
the invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of procaryotic host cell expression. The polypeptides may
also be modified with a detectable label, such as an enzymatic,
fluorescent, isotopic or affinity label to allow for detection and
isolation of the protein.
[0101] Also provided by the invention are chemically modified
derivatives of METH1 and/or METH2 which may provide additional
advantages such as increased solubility, stability and circulating
time of the polypeptide, or decreased immunogenicity (see U.S. Pat.
No. 4,179,337). The chemical moieties for derivitization may be
selected from water soluble polymers such as polyethylene glycol,
ethylene glycol/propylene glycol copolymers,
carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
The polypeptides may be modified at random positions within the
molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0102] The polymer may be of any molecular weight, and may be
branched or unbranched. For polyethylene glycol, the preferred
molecular weight is between about 1 kDa and about 100 kDa (the term
"about" indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the stated
molecular weight) for ease in handling and manufacturing. Other
sizes may be used, depending on the desired therapeutic profile
(e.g., the duration of sustained release desired, the effects, if
any on biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the polyethylene
glycol to a therapeutic protein or analog).
[0103] The polyethylene glycol molecules (or other chemical
moieties) should be attached to the protein with consideration of
effects on functional or antigenic domains of the protein. There
are a number of attachment methods available to those skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG to G-CSF), see also Malik et al., Exp. Hematol.
20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl
chloride). For example, polyethylene glycol may be covalently bound
through amino acid residues via a reactive group, such as, a free
amino or carboxyl group. Reactive groups are those to which an
activated polyethylene glycol molecule may be bound. The amino acid
residues having a free amino group may include lysine residues and
the N-terminal amino acid residues; those having a free carboxyl
group may include aspartic acid residues glutamic acid residues and
the C-terminal amino acid residue. Sulfhydryl groups may also be
used as a reactive group for attaching the polyethylene glycol
molecules. Preferred for therapeutic purposes is attachment at an
amino group, such as attachment at the N-terminus or lysine
group.
[0104] One may specifically desire proteins chemically modified at
the N-terminus. Using polyethylene glycol as an illustration of the
present composition, one may select from a variety of polyethylene
glycol molecules (by molecular weight, branching, etc.), the
proportion of polyethylene glycol molecules to protein (or peptide)
molecules in the reaction mix, the type of pegylation reaction to
be performed, and the method of obtaining the selected N-terminally
pegylated protein. The method of obtaining the N-terminally
pegylated preparation (i.e., separating this moiety from other
monopegylated moieties if necessary) may be by purification of the
N-terminally pegylated material from a population of pegylated
protein molecules. Selective proteins chemically modified at the
N-terminus modification may be accomplished by reductive alkylation
which exploits differential reactivity of different types of
primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate
reaction conditions, substantially selective derivatization of the
protein at the N-terminus with a carbonyl group containing polymer
is achieved.
[0105] The METH1 and/or METH2 polypeptides of the invention may be
in monomers or multimers (i.e., dimers, trimers, tetramers and
higher multimers). Accordingly, the present invention relates to
monomers and multimers of the METH1 and/or METH2 polypeptides of
the invention, their preparation, and compositions (preferably,
pharmaceutical compositions) containing them. In specific
embodiments, the polypeptides of the invention are monomers,
dimers, trimers or tetramers. In additional embodiments, the
multimers of the invention are at least dimers, at least trimers,
or at least tetramers.
[0106] Multimers encompassed by the invention may be homomers or
heteromers. As used herein, the term homomer, refers to a multimer
containing only METH1 and/or METH2 polypeptides of the invention
(including METH1 and/or METH2 fragments, variants, splice variants,
and fusion proteins, as described herein). These homomers may
contain METH1 and/or METH2 polypeptides having identical or
different amino acid sequences. In a specific embodiment, a homomer
of the invention is a multimer containing only METH1 and/or METH2
polypeptides having an identical amino acid sequence. In another
specific embodiment, a homomer of the invention is a multimer
containing METH1 and/or METH2 polypeptides having different amino
acid sequences. In specific embodiments, the multimer of the
invention is a homodimer (e.g., containing METH1 and/or METH2
polypeptides having identical or different amino acid sequences) or
a homotrimer (e.g., containing METH1 and/or METH2 polypeptides
having identical and/or different amino acid sequences). In
additional embodiments, the homomeric multimer of the invention is
at least a homodimer, at least a homotrimer, or at least a
homotetramer.
[0107] As used herein, the term heteromer refers to a multimer
containing one or more heterologous polypeptides (i.e.,
polypeptides of different proteins) in addition to the METH1 and/or
METH2 polypeptides of the invention. In a specific embodiment, the
multimer of the invention is a heterodimer, a heterotrimer, or a
heterotetramer. In additional embodiments, the homomeric multimer
of the invention is at least a homodimer, at least a homotrimer, or
at least a homotetramer.
[0108] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic and/or covalent associations and/or may be
indirectly linked by, for example, liposome formation. Thus, in one
embodiment, multimers of the invention, such as, for example,
homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of the invention, such as, for example,
heterotrimers or heterotetramers, are formed when polypeptides of
the invention contact antibodies to the polypeptides of the
invention (including antibodies to the heterologous polypeptide
sequence in a fusion protein of the invention) in solution. In
other embodiments, multimers of the invention are formed by
covalent associations with and/or between the METH1 and/or METH2
polypeptides of the invention. Such covalent associations may
involve one or more amino acid residues contained in the
polypeptide sequence (e.g., that recited in SEQ ID NO:2 or 4, or
contained in the polypeptide encoded by the clone HATCK89). In one
instance, the covalent associations are cross-linking between
cysteine residues located within the polypeptide sequences which
interact in the native (i.e., naturally occurring) polypeptide. In
another instance, the covalent associations are the consequence of
chemical or recombinant manipulation. Alternatively, such covalent
associations may involve one or more amino acid residues contained
in the heterologous polypeptide sequence in a METH1 and/or METH2
fusion protein. In one example, covalent associations are between
the heterologous sequence contained in a fusion protein of the
invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific
example, the covalent associations are between the heterologous
sequence contained in a METH1 and/or METH2-Fc fusion protein of the
invention (as described herein). In another specific example,
covalent associations of fusion proteins of the invention are
between heterologous polypeptide sequence from another Fibroblast
Growth Factor family member that is capable of forming covalently
associated multimers, such as for example, osteoprotegerin (see,
e.g., International Publication No. WO 98/49305, the contents of
which are herein incorporated by reference in its entirety).
[0109] The multimers of the invention may be generated using
chemical techniques known in the art. For example, polypeptides
desired to be contained in the multimers of the invention may be
chemically cross-linked using linker molecules and linker molecule
length optimization techniques known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). Additionally, multimers of the invention may be
generated using techniques known in the art to form one or more
inter-molecule cross-links between the cysteine residues located
within the sequence of the polypeptides desired to be contained in
the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Further, polypeptides
of the invention may be routinely modified by the addition of
cysteine or biotin to the C terminus or N-terminus of the
polypeptide and techniques known in the art may be applied to
generate multimers containing one or more of these modified
polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety). Additionally,
techniques known in the art may be applied to generate liposomes
containing the polypeptide components desired to be contained in
the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925,
which is herein incorporated by reference in its entirety).
[0110] Alternatively, multimers of the invention may be generated
using genetic engineering techniques known in the art. In one
embodiment, polypeptides contained in multimers of the invention
are produced recombinantly using fusion protein technology
described herein or otherwise known in the art (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In a specific embodiment, polynucleotides coding for
a homodimer of the invention are generated by ligating a
polynucleotide sequence encoding a polypeptide of the invention to
a sequence encoding a linker polypeptide and then further to a
synthetic polynucleotide encoding the translated product of the
polypeptide in the reverse orientation from the original C-terminus
to the N-terminus (lacking the leader sequence) (see, e.g., U.S.
Pat. No. 5,478,925, which is herein incorporated by reference in
its entirety). In another embodiment, recombinant techniques
described herein or otherwise known in the art are applied to
generate recombinant polypeptides of the invention which contain a
transmembrane domain (or hydrophobic or signal peptide) and which
can be incorporated by membrane reconstitution techniques into
liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein
incorporated by reference in its entirety).
[0111] METH1 and METH2 Polypeptides and Fragments
[0112] The invention further provides an isolated METH1 polypeptide
having the amino acid sequence encoded by the deposited cDNA, or
the amino acid sequence in SEQ ID NO:2, or a peptide or polypeptide
comprising a portion of the above polypeptides. The invention also
provides an isolated METH2 polypeptide having the amino acid
sequence encoded by the deposited cDNA, or the amino acid sequence
in SEQ ID NO:4, or a peptide or polypeptide comprising a portion of
the above polypeptides.
[0113] Polypeptides of the present invention encompass not only
full length polypeptides, but the mature, proprotein, processed
forms of the protein, deletion mutants, substitution variants,
allelic variants, analogs, derivatives, etc.
[0114] METH1 or METH2 polypeptides can be composed of amino acids
joined to each other by peptide bonds or modified peptide bonds,
i.e., peptide isosteres, and may contain amino acids other than the
20 gene-encoded amino acids. The METH1 or METH2 polypeptides may be
modified by either natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in the METH1
or METH2 polypeptide, including the peptide backbone, the amino
acid side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given METH1 or
METH2 polypeptide. Also, a given METH1 or METH2 polypeptide may
contain many types of modifications. METH1 or METH2 polypeptides
may be branched, for example, as a result of ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched,
and branched cyclic METH1 or METH2 polypeptides may result from
posttranslation natural processes or may be made by synthetic
methods. Modifications include acetylation, acylation,
ADP-ribosylation, amidation, covalent attachment of flavin,
covalent attachment of a heme moiety, covalent attachment of a
nucleotide or nucleotide derivative, covalent attachment of a lipid
or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation,
demethylation, formation of covalent cross-links, formation of
cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination. (See, for instance, PROTEINS--STRUCTURE AND
MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W.H. Freeman and
Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION
OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);
Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
[0115] It will be recognized in the art that some amino acid
sequences of the METH1 and METH2 polypeptides can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity.
[0116] The present inventors have shown that METH1 and METH2
inhibit angiogenesis in vitro and in vivo. METH1 and METH2 each
contain a metalloprotease domain, a disintegrin domain, and
TSP-like domains. The metalloprotease domain may be catalytically
active. The disintegrin domain may play a role in inhibiting
angiogenesis by interacting with integrins, since integrins are
essential for the mediation of both proliferative and migratory
signals. The present inventors have shown that peptides derived
from the TSP-like domains of METH1 and METH2 inhibit angiogenesis
in vitro and in vivo.
[0117] Thus, the invention further includes variations of the METH1
polypeptide which show substantial METH1 polypeptide activity or
which include regions of METH1 protein such as the protein portions
discussed below; and variations of the METH2 polypeptide which show
substantial METH2 polypeptide activity or which include regions of
METH2 protein such as the protein portions discussed below. Such
mutants include deletions, insertions, inversions, repeats, and
type substitutions. As indicated above, guidance concerning which
amino acid changes are likely to be phenotypically silent can be
found in Bowie, J. U., et al., "Deciphering the Message in Protein
Sequences: Tolerance to Amino Acid Substitutions," Science
247:1306-1310 (1990).
[0118] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2 or SEQ ID NO:4, or that encoded by the deposited
cDNA, may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as an IgG Fc fusion
region peptide or leader or secretory sequence or a sequence which
is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0119] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the METH1
or METH2 proteins. The prevention of aggregation is highly
desirable. Aggregation of proteins not only results in a loss of
activity but can also be problematic when preparing pharmaceutical
formulations, because they can be immunogenic. (Pinckard et al.,
Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes
36:838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug
Carrier Systems 10:307-377 (1993)).
[0120] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 3).
TABLE-US-00003 TABLE 3 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Serine
Threonine Methionine Glycine
[0121] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions for any given METH1 or METH2 polypeptide will not be
more than 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.
[0122] In particular, preferred METH1 molecules contain one or more
of the following conservative substitutions: M1 replaced with A, G,
I, L, S, T, or V; G2 replaced with A, I, L, S, T, M, or V; N3
replaced with Q; A4 replaced with G, I, L, S, T, M, or V; E5
replaced with D; R6 replaced with H, or K; A7 replaced with G, I,
L, S, T, M, or V; G9 replaced with A, I, L, S, T, M, or V; S10
replaced with A, G, I, L, T, M, or V; R11 replaced with H, or K;
S12 replaced with A, G, I, L, T, M, or V; F13 replaced with W, or
Y; G14 replaced with A, I, L, S, T, M, or V; V16 replaced with A,
G, I, L, S, T, or M; T18 replaced with A, G, I, L, S, M, or V; L19
replaced with A, G, I, S, T, M, or V; L20 replaced with A, G, I, S,
T, M, or V; L21 replaced with A, G, I, S, T, M, or V; L22 replaced
with A, G, I, S, T, M, or V; A23 replaced with G, I, L, S, T, M, or
V; A24 replaced with G, I, L, S, T, M, or V; A25 replaced with G,
I, L, S, T, M, or V; L26 replaced with A, G, I, S, T, M, or V; L27
replaced with A, G, I, S, T, M, or V; A28 replaced with G, I, L, S,
T, M, or V; V29 replaced with A, G, I, L, S, T, or M; S30 replaced
with A, G, I, L, T, M, or V; D31 replaced with E; A32 replaced with
G, I, L, S, T, M, or V; L33 replaced with A, G, I, S, T, M, or V;
G34 replaced with A, I, L, S, T, M, or V; R35 replaced with H, or
K; S37 replaced with A, G, I, L, T, M, or V; E38 replaced with D;
E39 replaced with D; D40 replaced with E; E41 replaced with D; E42
replaced with D; L43 replaced with A, G, I, S, T, M, or V; V44
replaced with A, G, I, L, S, T, or M; V45 replaced with A, G, I, L,
S, T, or M; E47 replaced with D; L48 replaced with A, G, I, S, T,
M, or V; E49 replaced with D; R50 replaced with H, or K; A51
replaced with G, I, L, S, T, M, or V; G53 replaced with A, I, L, S,
T, M, or V; H54 replaced with K, or R; G55 replaced with A, I, L,
S, T, M, or V; T56 replaced with A, G, I, L, S, M, or V; T57
replaced with A, G, I, L, S, M, or V; R58 replaced with H, or K;
L59 replaced with A, G, I, S, T, M, or V; R60 replaced with H, or
K; L61 replaced with A, G, I, S, T, M, or V; H62 replaced with K,
or R; A63 replaced with G, I, L, S, T, M, or V; F64 replaced with
W, or Y; D65 replaced with E; Q66 replaced with N; Q67 replaced
with N; L68 replaced with A, G, I, S, T, M, or V; D69 replaced with
E; L70 replaced with A, G, I, S, T, M, or V; E71 replaced with D;
L72 replaced with A, G, I, S, T, M, or V; R73 replaced with H, or
K; D75 replaced with E; S76 replaced with A, G, I, L, T, M, or V;
S77 replaced with A, G, I, L, T, M, or V; F78 replaced with W, or
Y; L79 replaced with A, G, I, S, T, M, or V; A80 replaced with G,
I, L, S, T, M, or V; G82 replaced with A, I, L, S, T, M, or V; F83
replaced with W, or Y; T84 replaced with A, G, I, L, S, M, or V;
L85 replaced with A, G, I, S, T, M, or V; Q86 replaced with N; N87
replaced with Q; V88 replaced with A, G, I, L, S, T, or M; G89
replaced with A, I, L, S, T, M, or V; R90 replaced with H, or K;
K91 replaced with H, or R; S92 replaced with A, G, I, L, T, M, or
V; G93 replaced with A, I, L, S, T, M, or V; S94 replaced with A,
G, I, L, T, M, or V; E95 replaced with D; T96 replaced with A, G,
I, L, S, M, or V; L98 replaced with A, G, I, S, T, M, or V; E100
replaced with D; T101 replaced with A, G, I, L, S, M, or V; D102
replaced with E; L103 replaced with A, G, I, S, T, M, or V; A104
replaced with G, I, L, S, T, M, or V; H105 replaced with K, or R;
F107 replaced with W, or Y; Y108 replaced with F, or W; S109
replaced with A, G, I, L, T, M, or V; G10 replaced with A, I, L, S,
T, M, or V; T111 replaced with A, G, I, L, S, M, or V; V112
replaced with A, G, I, L, S, T, or M; N113 replaced with Q; G114
replaced with A, I, L, S, T, M, or V; D115 replaced with E; S117
replaced with A, G, I, L, T, M, or V; S118 replaced with A, G, I,
L, T, M, or V; A119 replaced with G, I, L, S, T, M, or V; A120
replaced with G, I, L, S, T, M, or V; A121 replaced with G, I, L,
S, T, M, or V; L122 replaced with A, G, I, S, T, M, or V; S123
replaced with A, G, I, L, T, M, or V; L124 replaced with A, G, I,
S, T, M, or V; E126 replaced with D; G127 replaced with A, I, L, S,
T, M, or V; V128 replaced with A, G, I, L, S, T, or M; R129
replaced with H, or K; G130 replaced with A, I, L, S, T, M, or V;
A131 replaced with G, I, L, S, T, M, or V; F132 replaced with W, or
Y; Y133 replaced with F, or W; L134 replaced with A, G, I, S, T, M,
or V; L135 replaced with A, G, I, S, T, M, or V; G136 replaced with
A, I, L, S, T, M, or V; E137 replaced with D; A138 replaced with G,
I, L, S, T, M, or V; Y139 replaced with F, or W; F140 replaced with
W, or Y; I141 replaced with A, G, L, S, T, M, or V; Q142 replaced
with N; L144 replaced with A, G, I, S, T, M, or V; A146 replaced
with G, I, L, S, T, M, or V; A147 replaced with G, I, L, S, T, M,
or V; S148 replaced with A, G, I, L, T, M, or V; E149 replaced with
D; R150 replaced with H, or K; L151 replaced with A, G, I, S, T, M,
or V; A152 replaced with G, I, L, S, T, M, or V; T153 replaced with
A, G, I, L, S, M, or V; A154 replaced with G, I, L, S, T, M, or V;
A155 replaced with G, I, L, S, T, M, or V; G157 replaced with A, I,
L, S, T, M, or V; E158 replaced with D; K159 replaced with H, or R;
A162 replaced with G, I, L, S, T, M, or V; L164 replaced with A, G,
I, S, T, M, or V; Q165 replaced with N; F166 replaced with W, or Y;
H167 replaced with K, or R; L168 replaced with A, G, I, S, T, M, or
V; L169 replaced with A, G, I, S, T, M, or V; R170 replaced with H,
or K; R171 replaced with H, or K; N172 replaced with Q; R173
replaced with H, or K; Q174 replaced with N; G175 replaced with A,
I, L, S, T, M, or V; D176 replaced with E; V177 replaced with A, G,
I, L, S, T, or M; G178 replaced with A, I, L, S, T, M, or V; G179
replaced with A, I, L, S, T, M, or V; T180 replaced with A, G, I,
L, S, M, or V; G182 replaced with A, I, L, S, T, M, or V; V183
replaced with A, G, I, L, S, T, or M; V184 replaced with A, G, I,
L, S, T, or M; D185 replaced with E; D186 replaced with E; E187
replaced with D; R189 replaced with H, or K; T191 replaced with A,
G, I, L, S, M, or V; G192 replaced with A, I, L, S, T, M, or V;
K193 replaced with H, or R; A194 replaced with G, I, L, S, T, M, or
V; E195 replaced with D; T196 replaced with A, G, I, L, S, M, or V;
E197 replaced with D; D198 replaced with E; E199 replaced with D;
D200 replaced with E; E201 replaced with D; G202 replaced with A,
I, L, S, T, M, or V; T203 replaced with A, G, I, L, S, M, or V;
E204 replaced with D; G205 replaced with A, I, L, S, T, M, or V;
E206 replaced with D; D207 replaced with E; E208 replaced with D;
G209 replaced with A, I, L, S, T, M, or V; Q211 replaced with N;
W212 replaced with F, or Y; S213 replaced with A, G, I, L, T, M, or
V; Q215 replaced with N; D216 replaced with E; A218 replaced with
G, I, L, S, T, M, or V; L219 replaced with A, G, I, S, T, M, or V;
Q220 replaced with N; G221 replaced with A, I, L, S, T, M, or V;
V222 replaced with A, G, I, L, S, T, or M; G223 replaced with A, I,
L, S, T, M, or V; Q224 replaced with N; T226 replaced with A, G, I,
L, S, M, or V; G227 replaced with A, I, L, S, T, M, or V; T228
replaced with A, G, I, L, S, M, or V; G229 replaced with A, I, L,
S, T, M, or V; S230 replaced with A, G, I, L, T, M, or V; I231
replaced with A, G, L, S, T, M, or V; R232 replaced with H, or K;
K233 replaced with H, or R; K234 replaced with H, or R; R235
replaced with H, or K; F236 replaced with W, or Y; V237 replaced
with A, G, I, L, S, T, or M; S238 replaced with A, G, I, L, T, M,
or V; S239 replaced with A, G, I, L, T, M, or V; H240 replaced with
K, or R; R241 replaced with H, or K; Y242 replaced with F, or W;
V243 replaced with A, G, I, L, S, T, or M; E244 replaced with D;
T245 replaced with A, G, I, L, S, M, or V; M246 replaced with A, G,
I, L, S, T, or V; L247 replaced with A, G, I, S, T, M, or V; V248
replaced with A, G, I, L, S, T, or M; A249 replaced with G, I, L,
S, T, M, or V; D250 replaced with E; Q251 replaced with N; S252
replaced with A, G, I, L, T, M, or V; M253 replaced with A, G, I,
L, S, T, or V; A254 replaced with G, I, L, S, T, M, or V; E255
replaced with D; F256 replaced with W, or Y; H257 replaced with K,
or R; G258 replaced with A, I, L, S, T, M, or V; S259 replaced with
A, G, I, L, T, M, or V; G260 replaced with A, I, L, S, T, M, or V;
L261 replaced with A, G, I, S, T, M, or V; K262 replaced with H, or
R; H263 replaced with K, or R; Y264 replaced with F, or W; L265
replaced with A, G, I, S, T, M, or V; L266 replaced with A, G, I,
S, T, M, or V; T267 replaced with A, G, I, L, S, M, or V; L268
replaced with A, G, I, S, T, M, or V; F269 replaced with W, or Y;
S270 replaced with A, G, I, L, T, M, or V; V271 replaced with A, G,
I, L, S, T, or M; A272 replaced with G, I, L, S, T, M, or V; A273
replaced with G, I, L, S, T, M, or V; R274 replaced with H, or K;
L275 replaced with A, G, I, S, T, M, or V; Y276 replaced with F, or
W; K277 replaced with H, or R; H278 replaced with K, or R; S280
replaced with A, G, I, L, T, M, or V; I281 replaced with A, G, L,
S, T, M, or V; R282 replaced with H, or K; N283 replaced with Q;
S284 replaced with A, G, I, L, T, M, or V; V285 replaced with A, G,
I, L, S, T, or M; S286 replaced with A, G, I, L, T, M, or V; L287
replaced with A, G, I, S, T, M, or V; V288 replaced with A, G, I,
L, S, T, or M; V289 replaced with A, G, I, L, S, T, or M; V290
replaced with A, G, I, L, S, T, or M; K291 replaced with H, or R;
I292 replaced with A, G, L, S, T, M, or V; L293 replaced with A, G,
I, S, T, M, or V; V294 replaced with A, G, I, L, S, T, or M; I295
replaced with A, G, L, S, T, M, or V; H296 replaced with K, or R;
D297 replaced with E; E298 replaced with D; Q299 replaced with N;
K300 replaced with H, or R; G301 replaced with A, I, L, S, T, M, or
V; E303 replaced with D; V304 replaced with A, G, I, L, S, T, or M;
T305 replaced with A, G, I, L, S, M, or V; S306 replaced with A, G,
I, L, T, M, or V; N.sub.3O.sub.7 replaced with Q; A308 replaced
with G, I, L, S, T, M, or V; A309 replaced with G, I, L, S, T, M,
or V; L310 replaced with A, G, I, S, T, M, or V; T311 replaced with
A, G, I, L, S, M, or V; L312 replaced with A, G, I, S, T, M, or V;
R313 replaced with H, or K; N314 replaced with Q; F315 replaced
with W, or Y; N317 replaced with Q; W318 replaced with F, or Y;
Q319 replaced with N; K320 replaced with H, or R; Q321 replaced
with N; H322 replaced with K, or R; N323 replaced with Q; S326
replaced with A, G, I, L, T, M, or V; D327 replaced with E; R328
replaced with H, or K; D329 replaced with E; A330 replaced with G,
I, L, S, T, M, or V; E331 replaced with D; H332 replaced with K, or
R; Y333 replaced with F, or W; D334 replaced with E; T335 replaced
with A, G, I, L, S, M, or V; A336 replaced with G, I, L, S, T, M,
or V; I337 replaced with A, G, L, S, T, M, or V; L338 replaced with
A, G, I, S, T, M, or V; F339 replaced with W, or Y; T340 replaced
with A, G, I, L, S, M, or V; R341 replaced with H, or K; Q342
replaced with N; D343 replaced with E; L344 replaced with A, G, I,
S, T, M, or V; G346 replaced with A, I, L, S, T, M, or V; S347
replaced with A, G, I, L, T, M, or V; Q348 replaced with N; T349
replaced with A, G, I, L, S, M, or V; D351 replaced with E; T352
replaced with A, G, I, L, S, M, or V; L353 replaced with A, G, I,
S, T, M, or V; G354 replaced with A, I, L, S, T, M, or V; M355
replaced with A, G, I, L, S, T, or V; A356 replaced with G, I, L,
S, T, M, or V; D357 replaced with E; V358 replaced with A, G, I, L,
S, T, or M; G359 replaced with A, I, L, S, T, M, or V; T360
replaced with A, G, I, L, S, M, or V; V361 replaced with A, G, I,
L, S, T, or M; D363 replaced with E; S365 replaced with A, G, I, L,
T, M, or V; R366 replaced with H, or K; S367 replaced with A, G, I,
L, T, M, or V; S369 replaced with A, G, I, L, T, M, or V; V370
replaced with A, G, I, L, S, T, or M; I371 replaced with A, G, L,
S, T, M, or V; E372 replaced with D; D373 replaced with E; D374
replaced with E; G375 replaced with A, I, L, S, T, M, or V; L376
replaced with A, G, I, S, T, M, or V; Q377 replaced with N; A378
replaced with G, I, L, S, T, M, or V; A379 replaced with G, I, L,
S, T, M, or V; F380 replaced with W, or Y; T381 replaced with A, G,
I, L, S, M, or V; T382 replaced with A, G, I, L, S, M, or V; A383
replaced with G, I, L, S, T, M, or V; H384 replaced with K, or R;
E385 replaced with D; L386 replaced with A, G, I, S, T, M, or V;
G387 replaced with A, I, L, S, T, M, or V; H388 replaced with K, or
R; V389 replaced with A, G, I, L, S, T, or M; F390 replaced with W,
or Y; N391 replaced with Q; M392 replaced with A, G, I, L, S, T, or
V; H394 replaced with K, or R; D395 replaced with E; D396 replaced
with E; A397 replaced with G, I, L, S, T, M, or V; K398 replaced
with H, or R; Q399 replaced with N; A401 replaced with G, I, L, S,
T, M, or V; S402 replaced with A, G, I, L, T, M, or V; L403
replaced with A, G, I, S, T, M, or V; N.sub.4O.sub.4 replaced with
Q; G405 replaced with A, I, L, S, T, M, or V; V406 replaced with A,
G, I, L, S, T, or M; N407 replaced with Q; Q408 replaced with N;
D409 replaced with E; S410 replaced with A, G, I, L, T, M, or V;
H411 replaced with K, or R; M412 replaced with A, G, I, L, S, T, or
V; M413 replaced with A, G, I, L, S, T, or V; A414 replaced with G,
I, L, S, T, M, or V; S415 replaced with A, G, I, L, T, M, or V;
M416 replaced with A, G, I, L, S, T, or V; L417 replaced with A, G,
I, S, T, M, or V; S418 replaced with A, G, I, L, T, M, or V; N419
replaced with Q; L420 replaced with A, G, I, S, T, M, or V; D421
replaced with E; H422 replaced with K, or R; S423 replaced with A,
G, I, L, T, M, or V; Q424 replaced with N; W426 replaced with F, or
Y; S427 replaced with A, G, I, L, T, M, or V; S430 replaced with A,
G, I, L, T, M, or V; A431 replaced with G, I, L, S, T, M, or V;
Y432 replaced with F, or W; M433 replaced with A, G, I, L, S, T, or
V; I434 replaced with A, G, L, S, T, M, or V; T435 replaced with A,
G, I, L, S, M, or V; S436 replaced with A, G, I, L, T, M, or V;
F437 replaced with W, or Y; L438 replaced with A, G, I, S, T, M, or
V; D439 replaced with E; N440 replaced with Q; G441 replaced with
A, I, L, S, T, M, or V; H442 replaced with K, or R; G443 replaced
with A, I, L, S, T, M, or V; E444 replaced with D; L446 replaced
with A, G, I, S, T, M, or V; M447 replaced with A, G, I, L, S, T,
or V; D448 replaced with E; K449 replaced with H, or R; Q451
replaced with N; N452 replaced with Q; I454 replaced with A, G, L,
S, T, M, or V; Q455 replaced with N; L456 replaced with A, G, I, S,
T, M, or V; G458 replaced with A, I, L, S, T, M, or V; D459
replaced with E; L460 replaced with A, G, I, S, T, M, or V; G462
replaced with A, I, L, S, T, M, or V; T463 replaced with A, G, I,
L, S, M, or V; S464 replaced with A, G, I, L, T, M, or V; Y465
replaced with F, or W; D466 replaced with E; A467 replaced with G,
I, L, S, T, M, or V; N468 replaced with Q; R469 replaced with H, or
K; Q470 replaced with N; Q472 replaced with N; F473 replaced with
W, or Y; T474 replaced with A, G, I, L, S, M, or V; F475 replaced
with W, or Y; G476 replaced with A, I, L, S, T, M, or V; E477
replaced with D; D478 replaced with E; S479 replaced with A, G, I,
L, T, M, or V; K480 replaced with H, or R; H481 replaced with K, or
R; D484 replaced with E; A485 replaced with G, I, L, S, T, M, or V;
A486 replaced with G, I, L, S, T, M, or V; S487 replaced with A, G,
I, L, T, M, or V; T488 replaced with A, G, I, L, S, M, or V; S490
replaced with A, G, I, L, T, M, or V; T491 replaced with A, G, I,
L, S, M, or V; L492 replaced with A, G, I, S, T, M, or V; W493
replaced with F, or Y; T495 replaced with A, G, I, L, S, M, or V;
G496 replaced with A, I, L, S, T, M, or V; T497 replaced with A, G,
I, L, S, M, or V; S498 replaced with A, G, I, L, T, M, or V; G499
replaced with A, I, L, S, T, M, or V; G500 replaced with A, I, L,
S, T, M, or V; V501 replaced with A, G, I, L, S, T, or M; L502
replaced with A, G, I, S, T, M, or V; V503 replaced with A, G, I,
L, S, T, or M; Q505 replaced with N; T506 replaced with A, G, I, L,
S, M, or V; K507 replaced with H, or R; H508 replaced with K, or R;
F509 replaced with W, or Y; W511 replaced with F, or Y; A512
replaced with G, I, L, S, T, M, or V; D513 replaced with E; G514
replaced with A, I, L, S, T, M, or V; T515 replaced with A, G, I,
L, S, M, or V; S516 replaced with A, G, I, L, T, M, or V; G518
replaced with A, I, L, S, T, M, or V; E519 replaced with D; G520
replaced with A, I, L, S, T, M, or V; K521 replaced with H, or R;
W522 replaced with F, or Y; I524 replaced with A, G, L, S, T, M, or
V; N525 replaced with Q; G526 replaced with A, I, L, S, T, M, or V;
K527 replaced with H, or R; V529 replaced with A, G, I, L, S, T, or
M; N530 replaced with Q; K531 replaced with H, or R; T532 replaced
with A, G, I, L, S, M, or V; D533 replaced with E; R534 replaced
with H, or K; K535 replaced with H, or R; H536 replaced with K, or
R; F537 replaced with W, or Y; D538 replaced with E; T539 replaced
with A, G, I, L, S, M, or V; F541 replaced with W, or Y; H542
replaced with K, or R; G543 replaced with A, I, L, S, T, M, or V;
S544 replaced with A, G, I, L, T, M, or V; W545 replaced with F, or
Y; G546 replaced with A, I, L, S, T, M, or V; M547 replaced with A,
G, I, L, S, T, or V; W548 replaced with F, or Y; G549 replaced with
A, I, L, S, T, M, or V; W551 replaced with F, or Y; G552 replaced
with A, I, L, S, T, M, or V; D553 replaced with E; S555 replaced
with A, G, I, L, T, M, or V; R556 replaced with H, or K; T557
replaced with A, G, I, L, S, M, or V; G559 replaced with A, I, L,
S, T, M, or V; G560 replaced with A, I, L, S, T, M, or V; G561
replaced with A, I, L, S, T, M, or V; V562 replaced with A, G, I,
L, S, T, or M; Q563 replaced with N; Y564 replaced with F, or W;
T565 replaced with A, G, I, L, S, M, or V; M566 replaced with A, G,
I, L, S, T, or V; R567 replaced with H, or K; E568 replaced with D;
D570 replaced with E; N571 replaced with Q; V573 replaced with A,
G, I, L, S, T, or M; K575 replaced with H, or R; N576 replaced with
Q; G577 replaced with A, I, L, S, T, M, or V; G578 replaced with A,
I, L, S, T, M, or V; K579 replaced with H, or R;
Y580 replaced with F, or W; E582 replaced with D; G583 replaced
with A, I, L, S, T, M, or V; K584 replaced with H, or R; R585
replaced with H, or K; V586 replaced with A, G, I, L, S, T, or M;
R587 replaced with H, or K; Y588 replaced with F, or W; R589
replaced with H, or K; S590 replaced with A, G, I, L, T, M, or V;
N592 replaced with Q; L593 replaced with A, G, I, S, T, M, or V;
E594 replaced with D; D595 replaced with E; D598 replaced with E;
N599 replaced with Q; N600 replaced with Q; G601 replaced with A,
I, L, S, T, M, or V; K602 replaced with H, or R; T603 replaced with
A, G, I, L, S, M, or V; F604 replaced with W, or Y; R605 replaced
with H, or K; E606 replaced with D; E607 replaced with D; Q608
replaced with N; E610 replaced with D; A611 replaced with G, I, L,
S, T, M, or V; H612 replaced with K, or R; N613 replaced with Q;
E614 replaced with D; F615 replaced with W, or Y; S616 replaced
with A, G, I, L, T, M, or V; K617 replaced with H, or R; A618
replaced with G, I, L, S, T, M, or V; S619 replaced with A, G, I,
L, T, M, or V; F620 replaced with W, or Y; G621 replaced with A, I,
L, S, T, M, or V; S622 replaced with A, G, I, L, T, M, or V; G623
replaced with A, I, L, S, T, M, or V; A625 replaced with G, I, L,
S, T, M, or V; V626 replaced with A, G, I, L, S, T, or M; E627
replaced with D; W628 replaced with F, or Y; I629 replaced with A,
G, L, S, T, M, or V; K631 replaced with H, or R; Y632 replaced with
F, or W; A633 replaced with G, I, L, S, T, M, or V; G634 replaced
with A, I, L, S, T, M, or V; V635 replaced with A, G, I, L, S, T,
or M; S636 replaced with A, G, I, L, T, M, or V; K638 replaced with
H, or R; D639 replaced with E; R640 replaced with H, or K; K642
replaced with H, or R; L643 replaced with A, G, I, S, T, M, or V;
1644 replaced with A, G, L, S, T, M, or V; Q646 replaced with N;
A647 replaced with G, I, L, S, T, M, or V; K648 replaced with H, or
R; G649 replaced with A, I, L, S, T, M, or V; I650 replaced with A,
G, L, S, T, M, or V; G651 replaced with A, I, L, S, T, M, or V;
Y652 replaced with F, or W; F653 replaced with W, or Y; F654
replaced with W, or Y; V655 replaced with A, G, I, L, S, T, or M;
L656 replaced with A, G, I, S, T, M, or V; Q657 replaced with N;
K659 replaced with H, or R; V660 replaced with A, G, I, L, S, T, or
M; V661 replaced with A, G, I, L, S, T, or M; D662 replaced with E;
G663 replaced with A, I, L, S, T, M, or V; T664 replaced with A, G,
I, L, S, M, or V; S667 replaced with A, G, I, L, T, M, or V; D669
replaced with E; S670 replaced with A, G, I, L, T, M, or V; T671
replaced with A, G, I, L, S, M, or V; S672 replaced with A, G, I,
L, T, M, or V; V673 replaced with A, G, I, L, S, T, or M; V675
replaced with A, G, I, L, S, T, or M; Q676 replaced with N; G677
replaced with A, I, L, S, T, M, or V; Q678 replaced with N; V680
replaced with A, G, I, L, S, T, or M; K681 replaced with H, or R;
A682 replaced with G, I, L, S, T, M, or V; G683 replaced with A, I,
L, S, T, M, or V; D685 replaced with E; R686 replaced with H, or K;
I687 replaced with A, G, L, S, T, M, or V; I688 replaced with A, G,
L, S, T, M, or V; D689 replaced with E; S690 replaced with A, G, I,
L, T, M, or V; K691 replaced with H, or R; K692 replaced with H, or
R; K693 replaced with H, or R; F694 replaced with W, or Y; D695
replaced with E; K696 replaced with H, or R; G698 replaced with A,
I, L, S, T, M, or V; V699 replaced with A, G, I, L, S, T, or M;
G701 replaced with A, I, L, S, T, M, or V; G702 replaced with A, I,
L, S, T, M, or V; N703 replaced with Q; G704 replaced with A, I, L,
S, T, M, or V; S705 replaced with A, G, I, L, T, M, or V; T706
replaced with A, G, I, L, S, M, or V; K708 replaced with H, or R;
K709 replaced with H, or R; I710 replaced with A, G, L, S, T, M, or
V; S711 replaced with A, G, I, L, T, M, or V; G712 replaced with A,
I, L, S, T, M, or V; S713 replaced with A, G, I, L, T, M, or V;
V714 replaced with A, G, I, L, S, T, or M; T715 replaced with A, G,
I, L, S, M, or V; S716 replaced with A, G, I, L, T, M, or V; A717
replaced with G, I, L, S, T, M, or V; K718 replaced with H, or R;
G720 replaced with A, I, L, S, T, M, or V; Y721 replaced with F, or
W; H722 replaced with K, or R; D723 replaced with E; I724 replaced
with A, G, L, S, T, M, or V; I725 replaced with A, G, L, S, T, M,
or V; T726 replaced with A, G, I, L, S, M, or V; I727 replaced with
A, G, L, S, T, M, or V; T729 replaced with A, G, I, L, S, M, or V;
G730 replaced with A, I, L, S, T, M, or V; A731 replaced with G, I,
L, S, T, M, or V; T732 replaced with A, G, I, L, S, M, or V; N733
replaced with Q; I734 replaced with A, G, L, S, T, M, or V; E735
replaced with D; V736 replaced with A, G, I, L, S, T, or M; K737
replaced with H, or R; Q738 replaced with N; R739 replaced with H,
or K; N740 replaced with Q; Q741 replaced with N; R742 replaced
with H, or K; G743 replaced with A, I, L, S, T, M, or V; S744
replaced with A, G, I, L, T, M, or V; R745 replaced with H, or K;
N746 replaced with Q; N747 replaced with Q; G748 replaced with A,
I, L, S, T, M, or V; S749 replaced with A, G, I, L, T, M, or V;
F750 replaced with W, or Y; L751 replaced with A, G, I, S, T, M, or
V; A752 replaced with G, I, L, S, T, M, or V; I753 replaced with A,
G, L, S, T, M, or V; K754 replaced with H, or R; A755 replaced with
G, I, L, S, T, M, or V; A756 replaced with G, I, L, S, T, M, or V;
D757 replaced with E; G758 replaced with A, I, L, S, T, M, or V;
T759 replaced with A, G, I, L, S, M, or V; Y760 replaced with F, or
W; I761 replaced with A, G, L, S, T, M, or V; L762 replaced with A,
G, I, S, T, M, or V; N763 replaced with Q; G764 replaced with A, I,
L, S, T, M, or V; D765 replaced with E; Y766 replaced with F, or W;
T767 replaced with A, G, I, L, S, M, or V; L768 replaced with A, G,
I, S, T, M, or V; S769 replaced with A, G, I, L, T, M, or V; T770
replaced with A, G, I, L, S, M, or V; L771 replaced with A, G, I,
S, T, M, or V; E772 replaced with D; Q773 replaced with N; D774
replaced with E; 1775 replaced with A, G, L, S, T, M, or V; M776
replaced with A, G, I, L, S, T, or V; Y777 replaced with F, or W;
K778 replaced with H, or R; G779 replaced with A, I, L, S, T, M, or
V; V780 replaced with A, G, I, L, S, T, or M; V781 replaced with A,
G, I, L, S, T, or M; L782 replaced with A, G, I, S, T, M, or V;
R783 replaced with H, or K; Y784 replaced with F, or W; S785
replaced with A, G, I, L, T, M, or V; G786 replaced with A, I, L,
S, T, M, or V; S787 replaced with A, G, I, L, T, M, or V; S788
replaced with A, G, I, L, T, M, or V; A789 replaced with G, I, L,
S, T, M, or V; A790 replaced with G, I, L, S, T, M, or V; L791
replaced with A, G, I, S, T, M, or V; E792 replaced with D; R793
replaced with H, or K; I794 replaced with A, G, L, S, T, M, or V;
R795 replaced with H, or K; S796 replaced with A, G, I, L, T, M, or
V; F797 replaced with W, or Y; S798 replaced with A, G, I, L, T, M,
or V; L800 replaced with A, G, I, S, T, M, or V; K801 replaced with
H, or R; E802 replaced with D; L804 replaced with A, G, I, S, T, M,
or V; T805 replaced with A, G, I, L, S, M, or V; I806 replaced with
A, G, L, S, T, M, or V; Q807 replaced with N; V808 replaced with A,
G, I, L, S, T, or M; L809 replaced with A, G, I, S, T, M, or V;
T810 replaced with A, G, I, L, S, M, or V; V811 replaced with A, G,
I, L, S, T, or M; G812 replaced with A, I, L, S, T, M, or V; N813
replaced with Q; A814 replaced with G, I, L, S, T, M, or V; L815
replaced with A, G, I, S, T, M, or V; R816 replaced with H, or K;
K818 replaced with H, or R; I819 replaced with A, G, L, S, T, M, or
V; K820 replaced with H, or R; Y821 replaced with F, or W; T822
replaced with A, G, I, L, S, M, or V; Y823 replaced with F, or W;
F824 replaced with W, or Y; V825 replaced with A, G, I, L, S, T, or
M; K826 replaced with H, or R; K827 replaced with H, or R; K828
replaced with H, or R; K829 replaced with H, or R; E830 replaced
with D; S831 replaced with A, G, I, L, T, M, or V; F832 replaced
with W, or Y; N833 replaced with Q; A834 replaced with G, I, L, S,
T, M, or V; I835 replaced with A, G, L, S, T, M, or V; T837
replaced with A, G, I, L, S, M, or V; F838 replaced with W, or Y;
S839 replaced with A, G, I, L, T, M, or V; A840 replaced with G, I,
L, S, T, M, or V; W841 replaced with F, or Y; V842 replaced with A,
G, I, L, S, T, or M; I843 replaced with A, G, L, S, T, M, or V;
E844 replaced with D; E845 replaced with D; W846 replaced with F,
or Y; G847 replaced with A, I, L, S, T, M, or V; E848 replaced with
D; S850 replaced with A, G, I, L, T, M, or V; K851 replaced with H,
or R; S852 replaced with A, G, I, L, T, M, or V; E854 replaced with
D; L855 replaced with A, G, I, S, T, M, or V; G856 replaced with A,
I, L, S, T, M, or V; W857 replaced with F, or Y; Q858 replaced with
N; R859 replaced with H, or K; R860 replaced with H, or K; L861
replaced with A, G, I, S, T, M, or V; V862 replaced with A, G, I,
L, S, T, or M; E863 replaced with D; R865 replaced with H, or K;
D866 replaced with E; I867 replaced with A, G, L, S, T, M, or V;
N868 replaced with Q; G869 replaced with A, I, L, S, T, M, or V;
Q870 replaced with N; A872 replaced with G, I, L, S, T, M, or V;
S873 replaced with A, G, I, L, T, M, or V; E874 replaced with D;
A876 replaced with G, I, L, S, T, M, or V; K877 replaced with H, or
R; E878 replaced with D; V879 replaced with A, G, I, L, S, T, or M;
K880 replaced with H, or R; A882 replaced with G, I, L, S, T, M, or
V; S883 replaced with A, G, I, L, T, M, or V; T884 replaced with A,
G, I, L, S, M, or V; R885 replaced with H, or K; A888 replaced with
G, I, L, S, T, M, or V; D889 replaced with E; H890 replaced with K,
or R; Q894 replaced with N; W895 replaced with F, or Y; Q896
replaced with N; L897 replaced with A, G, I, S, T, M, or V; G898
replaced with A, I, L, S, T, M, or V; E899 replaced with D; W900
replaced with F, or Y; S901 replaced with A, G, I, L, T, M, or V;
S902 replaced with A, G, I, L, T, M, or V; S904 replaced with A, G,
I, L, T, M, or V; K905 replaced with H, or R; T906 replaced with A,
G, I, L, S, M, or V; G908 replaced with A, I, L, S, T, M, or V;
K909 replaced with H, or R; G910 replaced with A, I, L, S, T, M, or
V; Y911 replaced with F, or W; K912 replaced with H, or R; K913
replaced with H, or R; R914 replaced with H, or K; S915 replaced
with A, G, I, L, T, M, or V; L916 replaced with A, G, I, S, T, M,
or V; K917 replaced with H, or R; L919 replaced with A, G, I, S, T,
M, or V; S920 replaced with A, G, I, L, T, M, or V; H921 replaced
with K, or R; D922 replaced with E; G923 replaced with A, I, L, S,
T, M, or V; G924 replaced with A, I, L, S, T, M, or V; V925
replaced with A, G, I, L, S, T, or M; L926 replaced with A, G, I,
S, T, M, or V; S927 replaced with A, G, I, L, T, M, or V; H928
replaced with K, or R; E929 replaced with D; S930 replaced with A,
G, I, L, T, M, or V; D932 replaced with E; L934 replaced with A, G,
I, S, T, M, or V; K935 replaced with H, or R; K936 replaced with H,
or R; K938 replaced with H, or R; H939 replaced with K, or R; F940
replaced with W, or Y; I941 replaced with A, G, L, S, T, M, or V;
D942 replaced with E; F943 replaced with W, or Y; T945 replaced
with A, G, I, L, S, M, or V; M946 replaced with A, G, I, L, S, T,
or V; A947 replaced with G, I, L, S, T, M, or V; E948 replaced with
D; S950 replaced with A, G, I, L, T, M, or V.
Also preferred are METH1 polypeptides with one or more of the
following non-conservative substitutions: M1 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G2 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N3 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; A4 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; E5 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; R6 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; A7 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P8 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; G9 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; S10 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R11
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
S12 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F13
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
G14 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P15
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; V16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P17
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; T18 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L19
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L20 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L21 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L22 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A23 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A24 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A25 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L26
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L27 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A28 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; V29 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S30 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; D31 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; A32 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L33 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R35
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P36 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; S37 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E38 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E39 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; D40 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E41 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; E42 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L43 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; V44 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V45 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P46 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; E47 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L48 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E49 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R50 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A51 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P52 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G53 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H54 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G55 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T56 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T57 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; R58 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L59 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; R60 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; L61 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; H62 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; A63 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; F64 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; D65 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; Q66 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; Q67 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L68 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; D69 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L70 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E71 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L72 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; R73 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P74 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; D75 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S76
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S77 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F78 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L79 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; A80 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; P81 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G82 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; F83 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; T84 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L85 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q86 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; N87 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; V88 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G89 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; R90 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; K91 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; S92 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G93 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; S94 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E95 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T96 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P97 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; L98 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P99 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; E100 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T101 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; D102 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; L103 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A104 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; H105 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; C106 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; F107 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; Y108 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; S109 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; G110 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; T111 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; V112 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; N113 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; G114 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; D115 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; P116 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; S117 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S118 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A120 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A121
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L122 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S123 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L124 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C125 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; E126 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G127 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; V128 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; R129 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G130 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A131 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; F132 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; Y133 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; L134 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; L135 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E137 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; A138 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Y139 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; F140 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; I141 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Q142 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; P143 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; L144 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P145 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; A146 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A147 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; S148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; E149 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; R150 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; L151 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A152 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T153 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A154
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A155 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P156 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G157
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E158 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K159
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P160 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; P161 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; A162 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; P163 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; L164 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Q165 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; F166 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; H167 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; L168 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L169 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; R170 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; R171 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; N172 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; R173 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q174 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G175
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D176 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V177
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G178 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G179 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T180 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C181 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; G182 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V183 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; V184 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; D185 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; D186 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E187 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; P188 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R189 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P190
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; T191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G192 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K193
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
A194 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E195
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; T196 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E197
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; D198 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E199 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; D200 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E201 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; G202 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; T203 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; E204 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; G205 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; E206 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; D207 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; E208 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G209 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P210 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; Q211 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; W212
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
S213 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P214
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; Q215 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; D216 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; P217 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; A218 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L219 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Q220 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; G221 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; V222 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G223 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q224 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; P225 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; T226 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G227 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T228 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G229
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S230 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; I231 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; R232 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K233 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K234 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R235 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F236
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
V237 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S238
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S239 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H240 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R241 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y242
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
V243 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E244
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; T245 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M246
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L247 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V248 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A249 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; D250 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; Q251 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; S252 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; M253 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A254 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E255 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; F256 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; H257 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; G258 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S259 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; G260 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L261 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; K262 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; H263 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Y264 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; L265 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L266 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T267 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L268
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F269 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S270
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V271 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A272 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A273 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R274 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L275 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; Y276 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K277 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H278 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P279
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; S280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R282
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
N283 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; S284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V285 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S286
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L287 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V288 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V289 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V290 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; K291 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; 1292 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L293 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V294 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I295 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H296
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
D297 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; E298 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; Q299 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; K300 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G301 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P302 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; E303 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V304 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T305 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S306 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N
[0123].sub.3O.sub.7 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; A308 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A309 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L310 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T311 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L312
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R313 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N314
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; F315 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; C316 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; N317 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; W318 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; Q319 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K320 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q321 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; H322
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
N323 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; P324 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; P325 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; S326 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; D327 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; R328 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; D329 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A330 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; E331 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H332 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y333 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D334
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; T335 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A336
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I337 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L338 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F339 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; T340 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R341 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; Q342 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D343 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L344
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C345 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
G346 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S347
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q348 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; T349
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C350 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
D351 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T352 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L353 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G354
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M355 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A356 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; D357 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V358 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; G359 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T360 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; V361 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; C362 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; D363 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; P364 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S365 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R366 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S367 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; C368 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S369 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; V370 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; 1371 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; E372 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; D373 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; D374 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G375 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L376 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; Q377 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; A378 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A379 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; F380 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; T381 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T382 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A383 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H384
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
E385 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; L386 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G387 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H388
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
V389 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F390
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
N391 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; M392 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P393 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; H394 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; D395 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; D396 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; A397 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K398 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; Q399 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; C400 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; A401 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S402 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L403 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; N404 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; G405 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V406 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N.sub.407 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; Q408 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; D409 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S410 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H411 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; M412 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; M413 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A414 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S415 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; M416 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L417 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S418 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N419
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; L420 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D421
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; H422 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; S423 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q424 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; P425 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; W426 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; S427 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P428 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; C429 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S430 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; A431 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; Y432 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; M433 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; I434 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T435 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S436 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F437
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L438 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D439
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; N440 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; G441 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; H442 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; G443 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E444 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; C445 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; L446 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; M447 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D448 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; K449 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; P450 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; Q451 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; N452 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P453 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; I454
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q455 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L456
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P457 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
G458 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D459
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L460 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P461
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; G462 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T463 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S464
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y465 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D466
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; A467 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N468
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; R469 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; Q470 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; C471 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; Q472 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; F473 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T474 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F475 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; G476 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E477 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D478 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S479 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K480 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H481 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C482
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; P483 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; D484 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; A485 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A486 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S487 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T488 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C489
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; S490 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T491 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L492
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W493 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C494
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; T495 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G496 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T497
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S498 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G499 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; G500 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V501 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L502 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; V503 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C504 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; Q505 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; T506 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; K507 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; H508 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; F509 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; P510 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W511 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A512 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; D513 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G514 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; T515 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S516 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C517 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; G518 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; E519 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G520 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; K521 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; W522 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; C523 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; I524 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; N525 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G526 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; K527 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C528 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V529
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N530 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; K531
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
T532 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D533
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; R534 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; K535 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; H536 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; F537 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; D538 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T539 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P540 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; F541 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H542 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G543 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S544 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; W545 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; G546 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; M547 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; W548 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; G549 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P550 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; W551 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; G552 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; D553 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; C554 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S555 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R556 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T557 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C558 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G559
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G560 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G561 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V562 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; Q563 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; Y564 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; T565 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; M566 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; R567 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; E568 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; C569 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; D570 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N571
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; P572 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; V573 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P574 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; K575 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; N576 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G577
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G578 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; K579 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y580 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C581
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; E582 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; G583 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K584 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; R585 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; V586 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; R587 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Y588 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; R589 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; S590 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C591 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; N592 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; L593 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; E594 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; D595 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C596 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P597
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; D598 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; N599 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; N600 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; G601 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; K602 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T603 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; F604 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; R605 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E606 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E607 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q608 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C609
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; E610 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; A611 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; H612 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; N613 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; E614 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; F615 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; S616 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K617 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A618 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S619 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; F620 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; G621 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; S622 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G623 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P624
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; A625 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V626 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E627
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; W628 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; I629 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P630 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; K631 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; Y632 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; A633 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; G634 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V635 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S636 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P637
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; K638 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; D639 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; R640 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C641 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; K642 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L643 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; I644 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C645 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; Q646 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A647 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; K648 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G649 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; I650 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; G651 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Y652 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; F653 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; F654 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; V655 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L656 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Q657 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; P658 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; K659 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V660 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V661 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; D662 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G663 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T664 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P665 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; C666 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S667 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P668 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; D669 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S670 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T671 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; S672 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V673 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C674 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; V675 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q676 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; G677 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q678 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; C679 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V680 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; K681 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; A682 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G683 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C684 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; D685 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R686 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I687 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; I688 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; D689 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S690 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; K691 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; K692 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K693 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F694 replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D695 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K696
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
C697 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; G698 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; V699 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C700
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; G701 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G702 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N703
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; G704 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S705
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T706 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C707 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K708
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
K709 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; I710 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S711 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G712
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S713 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V714 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T715 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S716 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A717 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; K718 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; P719 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; G720 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; Y721 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; H722 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; D723 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; I724 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; I725 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; T726 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I727 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P728 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; T729 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G730 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A731 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T732
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N733 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I734
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E735 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V736
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K737 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q738
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; R739 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; N740 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; Q741 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; R742 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G743 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S744 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; R745 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; N746 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; N747 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; G748 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S749 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; F750 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; L751 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A752 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; I753 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K754 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; A755 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A756 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D757
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; G758 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T759
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y760 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; 1761
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L762 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; N763 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G764 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D765 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y766 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; T767
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L768 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S769 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T770 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L771 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E772 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; Q773 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; D774 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I775 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; M776 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; Y777 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; K778 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; G779 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V780 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; V781 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L782 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; R783 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; Y784 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; S785 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G786 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S787 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S788
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A789 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A790 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L791 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; E792 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; R793 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I794 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; R795 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S796 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; F797 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; S798 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P799 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; L800 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; K801 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; E802 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P803 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L804
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T805 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; I806 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q807 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V808 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L809 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T810 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; V811 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G812 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N813 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; A814 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L815 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R816
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P817 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; K818 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; I819 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; K820 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Y821 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; T822 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Y823 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; F824 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; V825 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; K826 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; K827 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; K828 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; K829 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; E830 replaced with H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S831 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; F832 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; N833 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; A834 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; I835 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P836 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T837 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; F838 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S839 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A840 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; W841 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; V842 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; I843 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; E844 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E845 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; W846 replaced with D, E, H, K, R,
N, Q, A,
G, I, L, S, T, M, V, P, or C; G847 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; E848 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; C849 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S850 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; K851 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; S852 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; C853 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E854 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L855 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G856 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; W857 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q858 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R859 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R860
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L861 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V862
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E863 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C864
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; R865 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; D866 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; I867 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; N868 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; G869 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; Q870 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; P871 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; A872 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; S873 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; E874 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; C875 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or P; A876 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K877 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; E878 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V879 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; K880 replaced with D, E, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; P881 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A882 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S883 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T884 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R885 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; P886 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; C887 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; A888
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D889 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H890
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P891 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; C892 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or P; P893 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; Q894 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; W895 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q896 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L897
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G898 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E899 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W900 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S901
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S902 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C903 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; S904
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K905 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T906
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C907 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
G908 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K909
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
G910 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y911
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
K912 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; K913 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; R914 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; S915 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L916 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K917 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; C918 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; L919 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; S920 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H921 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; D922 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; G923 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G924 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
V925 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L926
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S927 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H928 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E929 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S930
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C931 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
D932 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; P933 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; L934 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; K935 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; K936 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; P937 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; K938 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H939 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F940 replaced with
D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; I941 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D942 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F943 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C944
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; T945 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
M946 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A947
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E948 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C949
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; S950 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
[0124] Also preferred are METH2 polypeptides with one or more of
the following conservative amino acid substitutions: M1 replaced
with A, G, I, L, S, T, or V; F2 replaced with W, or Y; A4 replaced
with G, I, L, S, T, M, or V; A6 replaced with G, I, L, S, T, M, or
V; A7 replaced with G, I, L, S, T, M, or V; R9 replaced with H, or
K; W10 replaced with F, or Y; L11 replaced with A, G, I, S, T, M,
or V; F13 replaced with W, or Y; L14 replaced with A, G, I, S, T,
M, or V; L15 replaced with A, G, I, S, T, M, or V; L16 replaced
with A, G, I, S, T, M, or V; L17 replaced with A, G, I, S, T, M, or
V; L18 replaced with A, G, I, S, T, M, or V; L19 replaced with A,
G, I, S, T, M, or V; L20 replaced with A, G, I, S, T, M, or V; L21
replaced with A, G, I, S, T, M, or V; L22 replaced with A, G, I, S,
T, M, or V; L24 replaced with A, G, I, S, T, M, or V; A25 replaced
with G, I, L, S, T, M, or V; R26 replaced with H, or K; G27
replaced with A, I, L, S, T, M, or V; A28 replaced with G, I, L, S,
T, M, or V; A30 replaced with G, I, L, S, T, M, or V; R31 replaced
with H, or K; A33 replaced with G, I, L, S, T, M, or V; A34
replaced with G, I, L, S, T, M, or V; G35 replaced with A, I, L, S,
T, M, or V; G36 replaced with A, I, L, S, T, M, or V; Q37 replaced
with N; A38 replaced with G, I, L, S, T, M, or V; S39 replaced with
A, G, I, L, T, M, or V; E40 replaced with D; L41 replaced with A,
G, I, S, T, M, or V; V42 replaced with A, G, I, L, S, T, or M; V43
replaced with A, G, I, L, S, T, or M; T45 replaced with A, G, I, L,
S, M, or V; R46 replaced with H, or K; L47 replaced with A, G, I,
S, T, M, or V; G49 replaced with A, I, L, S, T, M, or V; S50
replaced with A, G, I, L, T, M, or V; A51 replaced with G, I, L, S,
T, M, or V; G52 replaced with A, I, L, S, T, M, or V; E53 replaced
with D; L54 replaced with A, G, I, S, T, M, or V; A55 replaced with
G, I, L, S, T, M, or V; L56 replaced with A, G, I, S, T, M, or V;
H57 replaced with K, or R; L58 replaced with A, G, I, S, T, M, or
V; S59 replaced with A, G, I, L, T, M, or V; A60 replaced with G,
I, L, S, T, M, or V; F61 replaced with W, or Y; G62 replaced with
A, I, L, S, T, M, or V; K63 replaced with H, or R; G64 replaced
with A, I, L, S, T, M, or V; F65 replaced with W, or Y; V66
replaced with A, G, I, L, S, T, or M; L67 replaced with A, G, I, S,
T, M, or V; R68 replaced with H, or K; L69 replaced with A, G, I,
S, T, M, or V; A70 replaced with G, I, L, S, T, M, or V; D72
replaced with E; D73 replaced with E; S74 replaced with A, G, I, L,
T, M, or V; F75 replaced with W, or Y; L76 replaced with A, G, I,
S, T, M, or V; A77 replaced with G, I, L, S, T, M, or V; E79
replaced with D; F80 replaced with W, or Y; K81 replaced with H, or
R; 182 replaced with A, G, L, S, T, M, or V; E83 replaced with D;
R84 replaced with H, or K; L85 replaced with A, G, I, S, T, M, or
V; G86 replaced with A, I, L, S, T, M, or V; G87 replaced with A,
I, L, S, T, M, or V; S88 replaced with A, G, I, L, T, M, or V; G89
replaced with A, I, L, S, T, M, or V; R90 replaced with H, or K;
A91 replaced with G, I, L, S, T, M, or V; T92 replaced with A, G,
I, L, S, M, or V; G93 replaced with A, I, L, S, T, M, or V; G94
replaced with A, I, L, S, T, M, or V; E95 replaced with D; R96
replaced with H, or K; G97 replaced with A, I, L, S, T, M, or V;
L98 replaced with A, G, I, S, T, M, or V; R99 replaced with H, or
K; G100 replaced with A, I, L, S, T, M, or V; F102 replaced with W,
or Y; F103 replaced with W, or Y; S104 replaced with A, G, I, L, T,
M, or V; G105 replaced with A, I, L, S, T, M, or V; T106 replaced
with A, G, I, L, S, M, or V; V107 replaced with A, G, I, L, S, T,
or M; N108 replaced with Q; G109 replaced with A, I, L, S, T, M, or
V; E110 replaced with D; E112 replaced with D; S113 replaced with
A, G, I, L, T, M, or V; L114 replaced with A, G, I, S, T, M, or V;
A115 replaced with G, I, L, S, T, M, or V; A116 replaced with G, I,
L, S, T, M, or V; V17 replaced with A, G, I, L, S, T, or M; S118
replaced with A, G, I, L, T, M, or V; L119 replaced with A, G, I,
S, T, M, or V; R121 replaced with H, or K; G122 replaced with A, I,
L, S, T, M, or V; L123 replaced with A, G, I, S, T, M, or V; S124
replaced with A, G, I, L, T, M, or V; G125 replaced with A, I, L,
S, T, M, or V; S126 replaced with A, G, I, L, T, M, or V; F127
replaced with W, or Y; L128 replaced with A, G, I, S, T, M, or V;
L129 replaced with A, G, I, S, T, M, or V; D130 replaced with E;
G131 replaced with A, I, L, S, T, M, or V; E132 replaced with D;
E133 replaced with D; F134 replaced with W, or Y; T135 replaced
with A, G, I, L, S, M, or V; I136 replaced with A, G, L, S, T, M,
or V; Q137 replaced with N; Q139 replaced with N; G140 replaced
with A, I, L, S, T, M, or V; A141 replaced with G, I, L, S, T, M,
or V; G142 replaced with A, I, L, S, T, M, or V; G143 replaced with
A, I, L, S, T, M, or V; S144 replaced with A, G, I, L, T, M, or V;
L145 replaced with A, G, I, S, T, M, or V; A146 replaced with G, I,
L, S, T, M, or V; Q147 replaced with N; H149 replaced with K, or R;
R150 replaced with H, or K; L151 replaced with A, G, I, S, T, M, or
V; Q152 replaced with N; R153 replaced with H, or K; W154 replaced
with F, or Y; G155 replaced with A, I, L, S, T, M, or V; A157
replaced with G, I, L, S, T, M, or V; G158 replaced with A, I, L,
S, T, M, or V; A159 replaced with G, I, L, S, T, M, or V; R160
replaced with H, or K; L162 replaced with A, G, I, S, T, M, or V;
R164 replaced with H, or K; G165 replaced with A, I, L, S, T, M, or
V; E167 replaced with D; W168 replaced with F, or Y; E169 replaced
with D; V170 replaced with A, G, I, L, S, T, or M; E171 replaced
with D; T172 replaced with A, G, I, L, S, M, or V; G173 replaced
with A, I, L, S, T, M, or V; E174 replaced with D; G175 replaced
with A, I, L, S, T, M, or V; Q176 replaced with N; R177 replaced
with H, or K; Q178 replaced with N; E179 replaced with D; R180
replaced with H, or K; G181 replaced with A, I, L, S, T, M, or V;
D182 replaced with E; H183 replaced with K, or R; Q184 replaced
with N; E185 replaced with D; D186 replaced with E; S187 replaced
with A, G, I, L, T, M, or V; E188 replaced with D; E189 replaced
with D; E190 replaced with D; S191 replaced with A, G, I, L, T, M,
or V; Q192 replaced with N; E193 replaced with D; E194 replaced
with D; E195 replaced with D; A196 replaced with G, I, L, S, T, M,
or V; E197 replaced with D; G198 replaced with A, I, L, S, T, M, or
V; A199 replaced with G, I, L, S, T, M, or V; S200 replaced with A,
G, I, L, T, M, or V; E201 replaced with D; L206 replaced with A, G,
I, S, T, M, or V; G207 replaced with A, I, L, S, T, M, or V; A208
replaced with G, I, L, S, T, M, or V; T209 replaced with A, G, I,
L, S, M, or V; S210 replaced with A, G, I, L, T, M, or V; R211
replaced with H, or K; T212 replaced with A, G, I, L, S, M, or V;
K213 replaced with H, or R; R214 replaced with H, or K; F215
replaced with W, or Y; V216 replaced with A, G, I, L, S, T, or M;
S217 replaced with A, G, I, L, T, M, or V; E218 replaced with D;
A219 replaced with G, I, L, S, T, M, or V; R220 replaced with H, or
K; F221 replaced with W, or Y; V222 replaced with A, G, I, L, S, T,
or M; E223 replaced with D; T224 replaced with A, G, I, L, S, M, or
V; L225 replaced with A, G, I, S, T, M, or V; L226 replaced with A,
G, I, S, T, M, or V; V227 replaced with A, G, I, L, S, T, or M;
A228 replaced with G, I, L, S, T, M, or V; D229 replaced with E;
A230 replaced with G, I, L, S, T, M, or V; S231 replaced with A, G,
I, L, T, M, or V; M232 replaced with A, G, I, L, S, T, or V; A233
replaced with G, I, L, S, T, M, or V; A234 replaced with G, I, L,
S, T, M, or V; F235 replaced with W, or Y; Y236 replaced with F, or
W; G237 replaced with A, I, L, S, T, M, or V; A238 replaced with G,
I, L, S, T, M, or V; D239 replaced with E; L240 replaced with A, G,
I, S, T, M, or V; Q241 replaced with N; N242 replaced with Q; H243
replaced with K, or R; I244 replaced with A, G, L, S, T, M, or V;
L245 replaced with A, G, I, S, T, M, or V; T246 replaced with A, G,
I, L, S, M, or V; L247 replaced with A, G, I, S, T, M, or V; M248
replaced with A, G, I, L, S, T, or V; S249 replaced with A, G, I,
L, T, M, or V; V250 replaced with A, G, I, L, S, T, or M; A251
replaced with G, I, L, S, T, M, or V; A252 replaced with G, I, L,
S, T, M, or V; R253 replaced with H, or K; I254 replaced with A, G,
L, S, T, M, or V; Y255 replaced with F, or W; K256 replaced with H,
or R; H257 replaced with K, or R; S259 replaced with A, G, I, L, T,
M, or V; 1260 replaced with A, G, L, S, T, M, or V; K261 replaced
with H, or R; N262 replaced with Q; S263 replaced with A, G, I, L,
T, M, or V; I264 replaced with A, G, L, S, T, M, or V; N265
replaced with Q; L266 replaced with A, G, I, S, T, M, or V; M267
replaced with A, G, I, L, S, T, or V; V268 replaced with A, G, I,
L, S, T, or M; V269 replaced with A, G, I, L, S, T, or M; K270
replaced with H, or R; V271 replaced with A, G, I, L, S, T, or M;
L272 replaced with A, G, I, S, T, M, or V; I273 replaced with A, G,
L, S, T, M, or V; V274 replaced with A, G, I, L, S, T, or M; E275
replaced with D; D276 replaced with E; E277 replaced with D; K278
replaced with H, or R; W279 replaced with F, or Y; G280 replaced
with A, I, L, S, T, M, or V; E282 replaced with D; V283 replaced
with A, G, I, L, S, T, or M; S284 replaced with A, G, I, L, T, M,
or V; D285 replaced with E; N286 replaced with Q; G287 replaced
with A, I, L, S, T, M, or V; G288 replaced with A, I, L, S, T, M,
or V; L289 replaced with A, G, I, S, T, M, or V; T290 replaced with
A, G, I, L, S, M, or V; L291 replaced with A, G, I, S, T, M, or V;
R292 replaced with H, or K; N293 replaced with Q; F294 replaced
with W, or Y; N296 replaced with Q; W297 replaced with F, or Y;
Q298 replaced with N; R299 replaced with H, or K; R300 replaced
with H, or K; F301 replaced with W, or Y; N.sub.3O.sub.2 replaced
with Q; Q303 replaced with N; S305 replaced with A, G, I, L, T, M,
or V; D306 replaced with E; R307 replaced with H, or K; H308
replaced with K, or R; E310 replaced with D; H311 replaced with K,
or R; Y312 replaced with F, or W; D313 replaced with E; T314
replaced with A, G, I, L, S, M, or V; A315 replaced with G, I, L,
S, T, M, or V; I316 replaced with A, G, L, S, T, M, or V; L317
replaced with A, G, I, S, T, M, or V; L318 replaced with A, G, I,
S, T, M, or V; T319 replaced with A, G, I, L, S, M, or V; R320
replaced with H, or K; Q321 replaced with N; N322 replaced with Q;
F323 replaced with W, or Y; G325 replaced with A, I, L, S, T, M, or
V; Q326 replaced with N; E327 replaced with D; G328 replaced with
A, I, L, S, T, M, or V; L329 replaced with A, G, I, S, T, M, or V;
D331 replaced with E; T332 replaced with A, G, I, L, S, M, or V;
L333 replaced with A, G, I, S, T, M, or V; G334 replaced with A, I,
L, S, T, M, or V; V335 replaced with A, G, I, L, S, T, or M; A336
replaced with G, I, L, S, T, M, or V; D337 replaced with E; I338
replaced with A, G, L, S, T, M, or V; G339 replaced with A, I, L,
S, T, M, or V; T340 replaced with A, G, I, L, S, M, or V; I341
replaced with A, G, L, S, T, M, or V; D343 replaced with E; N345
replaced with Q; K346 replaced with H, or R; S347 replaced with A,
G, I, L, T, M, or V; S349 replaced with A, G, I, L, T, M, or V;
V350 replaced with A, G, I, L, S, T, or M; I351 replaced with A, G,
L, S, T, M, or V; E352 replaced with D; D353 replaced with E; E354
replaced with D; G355 replaced with A, I, L, S, T, M, or V; L356
replaced with A, G, I, S, T, M, or V; Q357 replaced with N; A358
replaced with G, I, L, S, T, M, or V; A359 replaced with G, I, L,
S, T, M, or V; H360 replaced with K, or R; T361 replaced with A, G,
I, L, S, M, or V; L362 replaced with A, G, I, S, T, M, or V; A363
replaced with G, I, L, S, T, M, or V; H364 replaced with K, or R;
E365 replaced with D; L366 replaced with A, G, I, S, T, M, or V;
G367 replaced with A, I, L, S, T, M, or V; H368 replaced with K, or
R; V369 replaced with A, G, I, L, S, T, or M; L370 replaced with A,
G, I, S, T, M, or V; S371 replaced with A, G, I, L, T, M, or V;
M372 replaced with A, G, I, L, S, T, or V; H374 replaced with K, or
R; D375 replaced with E; D376 replaced with E; S377 replaced with
A, G, I, L, T, M, or V; K378 replaced with H, or R; T381 replaced
with A, G, I, L, S, M, or V; R382 replaced with H, or K; L383
replaced with A, G, I, S, T, M, or V; F384 replaced with W, or Y;
G385 replaced with A, I, L, S, T, M, or V; M387 replaced with A, G,
I, L, S, T, or V; G388 replaced with A, I, L, S, T, M, or V; K389
replaced with H, or R; H390 replaced with K, or R; H391 replaced
with K, or R; V392 replaced with A, G, I, L, S, T, or M; M393
replaced with A, G, I, L, S, T, or V; A394 replaced with G, I, L,
S, T, M, or V; L396 replaced with A, G, I, S, T, M, or V; F397
replaced with W, or Y; V398 replaced with A, G, I, L, S, T, or M;
H399 replaced with K, or R; L400 replaced with A, G, I, S, T, M, or
V; N401 replaced with Q; Q402 replaced with N; T403 replaced with
A, G, I, L, S, M, or V; L404 replaced with A, G, I, S, T, M, or V;
W406 replaced with F, or Y; S407 replaced with A, G, I, L, T, M, or
V; S410 replaced with A, G, I, L, T, M, or V; A411 replaced with G,
I, L, S, T, M, or V; M412 replaced with A, G, I, L, S, T, or V;
Y413 replaced with F, or W; L414 replaced with A, G, I, S, T, M, or
V; T415 replaced with A, G, I, L, S, M, or V; E416 replaced with D;
L417 replaced with A, G, I, S, T, M, or V; L418 replaced with A, G,
I, S, T, M, or V; D419 replaced with E; G420 replaced with A, I, L,
S, T, M, or V; G421 replaced with A, I, L, S, T, M, or V; H422
replaced with K, or R; G423 replaced with A, I, L, S, T, M, or V;
D424 replaced with E; L426 replaced with A, G, I, S, T, M, or V;
L427 replaced with A, G, I, S, T, M, or V; D428 replaced with E;
A429 replaced with G, I, L, S, T, M, or V; G431 replaced with A, I,
L, S, T, M, or V; A432 replaced with G, I, L, S, T, M, or V; A433
replaced with G, I, L, S, T, M, or V; L434 replaced with A, G, I,
S, T, M, or V; L436 replaced with A, G, I, S, T, M, or V; T438
replaced with A, G, I, L, S, M, or V; G439 replaced with A, I, L,
S, T, M, or V; L440 replaced with A, G, I, S, T, M, or V; G442
replaced with A, I, L, S, T, M, or V; R443 replaced with H, or K;
M444 replaced with A, G, I, L, S, T, or V; A445 replaced with G, I,
L, S, T, M, or V; L446 replaced with A, G, I, S, T, M, or V; Y447
replaced with F, or W; Q448 replaced with N; L449 replaced with A,
G, I, S, T, M, or V; D450 replaced with E; Q451 replaced with N;
Q452 replaced with N; R454 replaced with H, or K; Q455 replaced
with N; I456 replaced with A, G, L, S, T, M, or V; F457 replaced
with W, or Y; G458 replaced with A, I, L, S, T, M, or V; D460
replaced with E; F461 replaced with W, or Y; R462 replaced with H,
or K; H463 replaced with K, or R; N466 replaced with Q; T467
replaced with A, G, I, L, S, M, or V; S468 replaced with A, G, I,
L, T, M, or V; A469 replaced with G, I, L, S, T, M, or V; Q470
replaced with N; D471 replaced with E; V472 replaced with A, G, I,
L, S, T, or M; A474 replaced with G, I, L, S, T, M, or V; Q475
replaced with N; L476 replaced with A, G, I, S, T, M, or V; W477
replaced with F, or Y; H479 replaced with K, or R; T480 replaced
with A, G, I, L, S, M, or V; D481 replaced with E; G482 replaced
with A, I, L, S, T, M, or V; A483 replaced with G, I, L, S, T, M,
or V; E484 replaced with D; L486 replaced with A, G, I, S, T, M, or
V; H488 replaced with K, or R; T489 replaced with A, G, I, L, S, M,
or V; K490 replaced with H, or R; N491 replaced with Q; G492
replaced with A, I, L, S, T, M, or V; S493 replaced with A, G, I,
L, T, M, or V; L494 replaced with A, G, I, S, T, M, or V; W496
replaced with F, or Y; A497 replaced with G, I, L, S, T, M, or V;
D498 replaced with E; G499 replaced with A, I, L, S, T, M, or V;
T500 replaced with A, G, I, L, S, M, or V; G503 replaced with A, I,
L, S, T, M, or V; G505 replaced with A, I, L, S, T, M, or V; H506
replaced with K, or R; L507 replaced with A, G, I, S, T, M, or V;
S509 replaced with A, G, I, L, T, M, or V; E510 replaced with D;
G511 replaced with A, I, L, S, T, M, or V; S512 replaced with A, G,
I, L, T, M, or V; L514 replaced with A, G, I, S, T, M, or V; E516
replaced with D; E517 replaced with D; E518 replaced with D; V519
replaced with A, G, I, L, S, T, or M; E520 replaced with D; R521
replaced with H, or K; K523 replaced with H, or R; V525 replaced
with A, G, I, L, S, T, or M; V526 replaced with A, G, I, L, S, T,
or M; D527 replaced with E; G528 replaced with A, I, L, S, T, M, or
V; G529 replaced with A, I, L, S, T, M, or V; W530 replaced with F,
or Y; A531 replaced with G, I, L, S, T, M, or V; W533 replaced with
F, or Y; G534 replaced with A, I, L, S, T, M, or V; W536 replaced
with F, or Y; G537 replaced with A, I, L, S, T, M, or V; E538
replaced with D; S540 replaced with A, G, I, L, T, M, or V; R541
replaced with H, or K; T542 replaced with A, G, I, L, S, M, or V;
G544 replaced with A, I, L, S, T, M, or V; G545 replaced with A, I,
L, S, T, M, or V; G546 replaced with A, I, L, S, T, M, or V; V547
replaced with A, G, I, L, S, T, or M; Q548 replaced with N; F549
replaced with W, or Y; S550 replaced with A, G, I, L, T, M, or V;
H551 replaced with K, or R; R552 replaced with H, or K; E553
replaced with D; K555 replaced with H, or R; D556 replaced with E;
E558 replaced with D; Q560 replaced with N; N561 replaced with Q;
G562 replaced with A, I, L, S, T, M, or V; G563 replaced with A, I,
L, S, T, M, or V; R564 replaced with H, or K; Y565 replaced with F,
or W; L567 replaced with A, G, I, S, T, M, or V; G568 replaced with
A, I, L, S, T, M, or V; R569 replaced with H, or K; R570 replaced
with H, or K; A571 replaced with G, I, L, S, T, M, or V; K572
replaced with H, or R; Y573 replaced with F, or W; Q574 replaced
with N; S575 replaced with A, G, I, L, T, M, or V; H577 replaced
with K, or R; T578 replaced with A, G, I, L, S, M, or V; E579
replaced with D; E580 replaced with D; D584 replaced with E; G585
replaced with A, I, L, S, T, M, or V; K586 replaced with H, or R;
S587 replaced with A, G, I, L, T, M, or V; F588 replaced with W, or
Y; R589 replaced with H, or K; E590 replaced with D; Q591 replaced
with
N; Q592 replaced with N; E594 replaced with D; K595 replaced with
H, or R; Y596 replaced with F, or W; N597 replaced with Q; A598
replaced with G, I, L, S, T, M, or V; Y599 replaced with F, or W;
N600 replaced with Q; Y601 replaced with F, or W; T602 replaced
with A, G, I, L, S, M, or V; D603 replaced with E; M604 replaced
with A, G, I, L, S, T, or V; D605 replaced with E; G606 replaced
with A, I, L, S, T, M, or V; N
.sub.607 replaced with Q; L608 replaced with A, G, I, S, T, M, or
V; L609 replaced with A, G, I, S, T, M, or V; Q610 replaced with N;
W611 replaced with F, or Y; V612 replaced with A, G, I, L, S, T, or
M; K614 replaced with H, or R; Y615 replaced with F, or W; A616
replaced with G, I, L, S, T, M, or V; G617 replaced with A, I, L,
S, T, M, or V; V618 replaced with A, G, I, L, S, T, or M; S619
replaced with A, G, I, L, T, M, or V; R621 replaced with H, or K;
D622 replaced with E; R623 replaced with H, or K; K625 replaced
with H, or R; L626 replaced with A, G, I, S, T, M, or V; F627
replaced with W, or Y; R629 replaced with H, or K; A630 replaced
with G, I, L, S, T, M, or V; R631 replaced with H, or K; G632
replaced with A, I, L, S, T, M, or V; R633 replaced with H, or K;
S634 replaced with A, G, I, L, T, M, or V; E635 replaced with D;
F636 replaced with W, or Y; K637 replaced with H, or R; V638
replaced with A, G, I, L, S, T, or M; F639 replaced with W, or Y;
E640 replaced with D; A641 replaced with G, I, L, S, T, M, or V;
K642 replaced with H, or R; V643 replaced with A, G, I, L, S, T, or
M; I644 replaced with A, G, L, S, T, M, or V; D645 replaced with E;
G646 replaced with A, I, L, S, T, M, or V; T647 replaced with A, G,
I, L, S, M, or V; L648 replaced with A, G, I, S, T, M, or V; G650
replaced with A, I, L, S, T, M, or V; E652 replaced with D; T653
replaced with A, G, I, L, S, M, or V; L654 replaced with A, G, I,
S, T, M, or V; A655 replaced with G, I, L, S, T, M, or V; I656
replaced with A, G, L, S, T, M, or V; V658 replaced with A, G, I,
L, S, T, or M; R659 replaced with H, or K; G660 replaced with A, I,
L, S, T, M, or V; Q661 replaced with N; V663 replaced with A, G, I,
L, S, T, or M; K664 replaced with H, or R; A665 replaced with G, I,
L, S, T, M, or V; G666 replaced with A, I, L, S, T, M, or V; D668
replaced with E; H669 replaced with K, or R; V670 replaced with A,
G, I, L, S, T, or M; V671 replaced with A, G, I, L, S, T, or M;
D672 replaced with E; S673 replaced with A, G, I, L, T, M, or V;
R675 replaced with H, or K; K676 replaced with H, or R; L677
replaced with A, G, I, S, T, M, or V; D678 replaced with E; K679
replaced with H, or R; G681 replaced with A, I, L, S, T, M, or V;
V682 replaced with A, G, I, L, S, T, or M; G684 replaced with A, I,
L, S, T, M, or V; G685 replaced with A, I, L, S, T, M, or V; K686
replaced with H, or R; G687 replaced with A, I, L, S, T, M, or V;
N688 replaced with Q; S689 replaced with A, G, I, L, T, M, or V;
R691 replaced with H, or K; K692 replaced with H, or R; V693
replaced with A, G, I, L, S, T, or M; S694 replaced with A, G, I,
L, T, M, or V; G695 replaced with A, I, L, S, T, M, or V; S696
replaced with A, G, I, L, T, M, or V; L697 replaced with A, G, I,
S, T, M, or V; T698 replaced with A, G, I, L, S, M, or V; T700
replaced with A, G, I, L, S, M, or V; N701 replaced with Q; Y702
replaced with F, or W; G703 replaced with A, I, L, S, T, M, or V;
Y704 replaced with F, or W; N.sub.7O.sub.5 replaced with Q; D706
replaced with E; I707 replaced with A, G, L, S, T, M, or V; V708
replaced with A, G, I, L, S, T, or M; T709 replaced with A, G, I,
L, S, M, or V; I710 replaced with A, G, L, S, T, M, or V; A712
replaced with G, I, L, S, T, M, or V; G713 replaced with A, I, L,
S, T, M, or V; A714 replaced with G, I, L, S, T, M, or V; T715
replaced with A, G, I, L, S, M, or V; N716 replaced with Q; I717
replaced with A, G, L, S, T, M, or V; D718 replaced with E; V719
replaced with A, G, I, L, S, T, or M; K720 replaced with H, or R;
Q721 replaced with N; R722 replaced with H, or K; S723 replaced
with A, G, I, L, T, M, or V; H724 replaced with K, or R; G726
replaced with A, I, L, S, T, M, or V; V727 replaced with A, G, I,
L, S, T, or M; Q728 replaced with N; N729 replaced with Q; D730
replaced with E; G731 replaced with A, I, L, S, T, M, or V; N732
replaced with Q; Y733 replaced with F, or W; L734 replaced with A,
G, I, S, T, M, or V; A735 replaced with G, I, L, S, T, M, or V;
L736 replaced with A, G, I, S, T, M, or V; K737 replaced with H, or
R; T738 replaced with A, G, I, L, S, M, or V; A739 replaced with G,
I, L, S, T, M, or V; D740 replaced with E; G741 replaced with A, I,
L, S, T, M, or V; Q742 replaced with N; Y743 replaced with F, or W;
L744 replaced with A, G, I, S, T, M, or V; L745 replaced with A, G,
I, S, T, M, or V; N746 replaced with Q; G747 replaced with A, I, L,
S, T, M, or V; N748 replaced with Q; L749 replaced with A, G, I, S,
T, M, or V; A750 replaced with G, I, L, S, T, M, or V; I751
replaced with A, G, L, S, T, M, or V; S752 replaced with A, G, I,
L, T, M, or V; A753 replaced with G, I, L, S, T, M, or V; I754
replaced with A, G, L, S, T, M, or V; E755 replaced with D; Q756
replaced with N; D757 replaced with E; I758 replaced with A, G, L,
S, T, M, or V; L759 replaced with A, G, I, S, T, M, or V; V760
replaced with A, G, I, L, S, T, or M; K761 replaced with H, or R;
G762 replaced with A, I, L, S, T, M, or V; T763 replaced with A, G,
I, L, S, M, or V; I764 replaced with A, G, L, S, T, M, or V; L765
replaced with A, G, I, S, T, M, or V; K766 replaced with H, or R;
Y767 replaced with F, or W; S768 replaced with A, G, I, L, T, M, or
V; G769 replaced with A, I, L, S, T, M, or V; S770 replaced with A,
G, I, L, T, M, or V; 1771 replaced with A, G, L, S, T, M, or V;
A772 replaced with G, I, L, S, T, M, or V; T773 replaced with A, G,
I, L, S, M, or V; L774 replaced with A, G, I, S, T, M, or V; E775
replaced with D; R776 replaced with H, or K; L777 replaced with A,
G, I, S, T, M, or V; Q778 replaced with N; S779 replaced with A, G,
I, L, T, M, or V; F780 replaced with W, or Y; R781 replaced with H,
or K; L783 replaced with A, G, I, S, T, M, or V; E785 replaced with
D; L787 replaced with A, G, I, S, T, M, or V; T788 replaced with A,
G, I, L, S, M, or V; V789 replaced with A, G, I, L, S, T, or M;
Q790 replaced with N; L791 replaced with A, G, I, S, T, M, or V;
L792 replaced with A, G, I, S, T, M, or V; T793 replaced with A, G,
I, L, S, M, or V; V794 replaced with A, G, I, L, S, T, or M; G796
replaced with A, I, L, S, T, M, or V; E797 replaced with D; V798
replaced with A, G, I, L, S, T, or M; F799 replaced with W, or Y;
K802 replaced with H, or R; V803 replaced with A, G, I, L, S, T, or
M; K804 replaced with H, or R; Y805 replaced with F, or W; T806
replaced with A, G, I, L, S, M, or V; F807 replaced with W, or Y;
F808 replaced with W, or Y; V809 replaced with A, G, I, L, S, T, or
M; N811 replaced with Q; D812 replaced with E; V813 replaced with
A, G, I, L, S, T, or M; D814 replaced with E; F815 replaced with W,
or Y; S816 replaced with A, G, I, L, T, M, or V; M817 replaced with
A, G, I, L, S, T, or V; Q818 replaced with N; S819 replaced with A,
G, I, L, T, M, or V; S820 replaced with A, G, I, L, T, M, or V;
K821 replaced with H, or R; E822 replaced with D; R823 replaced
with H, or K; A824 replaced with G, I, L, S, T, M, or V; T825
replaced with A, G, I, L, S, M, or V; T826 replaced with A, G, I,
L, S, M, or V; N827 replaced with Q; I828 replaced with A, G, L, S,
T, M, or V; I829 replaced with A, G, L, S, T, M, or V; Q830
replaced with N; L832 replaced with A, G, I, S, T, M, or V; L833
replaced with A, G, I, S, T, M, or V; H834 replaced with K, or R;
A835 replaced with G, I, L, S, T, M, or V; Q836 replaced with N;
W837 replaced with F, or Y; V838 replaced with A, G, I, L, S, T, or
M; L839 replaced with A, G, I, S, T, M, or V; G840 replaced with A,
I, L, S, T, M, or V; D841 replaced with E; W842 replaced with F, or
Y; S843 replaced with A, G, I, L, T, M, or V; E844 replaced with D;
S846 replaced with A, G, I, L, T, M, or V; S847 replaced with A, G,
I, L, T, M, or V; T848 replaced with A, G, I, L, S, M, or V; G850
replaced with A, I, L, S, T, M, or V; A851 replaced with G, I, L,
S, T, M, or V; G852 replaced with A, I, L, S, T, M, or V; W853
replaced with F, or Y; Q854 replaced with N; R855 replaced with H,
or K; R856 replaced with H, or K; T857 replaced with A, G, I, L, S,
M, or V; V858 replaced with A, G, I, L, S, T, or M; E859 replaced
with D; R861 replaced with H, or K; D862 replaced with E; S864
replaced with A, G, I, L, T, M, or V; G865 replaced with A, I, L,
S, T, M, or V; Q866 replaced with N; A867 replaced with G, I, L, S,
T, M, or V; S868 replaced with A, G, I, L, T, M, or V; A869
replaced with G, I, L, S, T, M, or V; T870 replaced with A, G, I,
L, S, M, or V; N872 replaced with Q; K873 replaced with H, or R;
A874 replaced with G, I, L, S, T, M, or V; L875 replaced with A, G,
I, S, T, M, or V; K876 replaced with H, or R; E878 replaced with D;
D879 replaced with E; A880 replaced with G, I, L, S, T, M, or V;
K881 replaced with H, or R; E884 replaced with D; S885 replaced
with A, G, I, L, T, M, or V; Q886 replaced with N; L887 replaced
with A, G, I, S, T, M, or V; L890 replaced with A, G, I, S, T, M,
or V.
Also preferred are METH2 polypeptides with one or more of the
following conservative amino acid substitutions: M1 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; F2 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; P3 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A4 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P5 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; A6 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A7 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P8 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; R9 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; W10 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; L11 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P12 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; F13 replaced with D,
E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L14 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L15 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L16 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L17 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L18 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L19 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L20
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L21 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L22 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; P23 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L24 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A25 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R26 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G27 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A28 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; P29 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; A30 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; R31 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; P32 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; A33 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A34 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; G35 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G36 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q37 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; A38 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S39 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E40
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L41 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V42
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V43 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; P44 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T45 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; R46 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L47 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P48 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G49 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S50 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; A51 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G52 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; E53 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; L54 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A55 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L56 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H57
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
L58 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S59
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A60 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; F61 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; G62 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; K63 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G64 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F65 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; V66 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L67 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; R68 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; L69 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A70 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; P71 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; D72 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; D73 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S74 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; F75 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; L76 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; A77 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P78 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; E79 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F80 replaced with D, E,
H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; K81 replaced with
D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; 182 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E83 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R84 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L85
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G86 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G87 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S88 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G89 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; R90 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; A91 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T92 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G93 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G94
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E95 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R96
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
G97 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L98
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R99 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G100
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C101 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
F102 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; F103 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; S104 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; G105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T106
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V107 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; N108 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G109 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E110 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P111 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
E112 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; S113 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L114 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A115
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A116 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V117 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; S118 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L119 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C120 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; R121 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G122 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L123 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S124 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; G125 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
S126 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F127
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
L128 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L129
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D130 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G131
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E132 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E133
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; F134 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; T135 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
I136 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q137
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; P138 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; Q139 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; G140 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A141 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G142 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G143 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S144
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L145 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A146 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q147 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P148 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; H149
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
R150 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; L151 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q152 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; R153 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; W154 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; G155 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; P156 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or C; A157 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; G158 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A159 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
R160 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; P161 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; L162 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P163 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; R164 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G165 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P166 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; E167 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; W168 replaced with D, E, H,
K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; E169 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V170 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E171 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T172 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G173 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; E174 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G175 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; Q176 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R177 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q178 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E179
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; R180 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; G181 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D182 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; H183 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; Q184 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; E185 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; D186 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S187 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; E188 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E189 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E190 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S191
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q192 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; E193
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; E194 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E195 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; A196 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; E197 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; G198 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A199 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S200 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E201
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; P202 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; P203 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; P204 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; P205 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L206 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G207 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A208 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; T209 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; S210 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; R211 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T212 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K213 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; R214 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; F215 replaced with D, E, H, K, R, N, Q, A, G, I,
L, S, T, M, V, P, or C; V216 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; S217 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E218 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; A219 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; R220 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; F221 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; V222 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; E223 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; T224 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L225 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L226 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V227
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A228 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D229 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A230 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S231 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; M232 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A233 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; A234 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; F235 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; Y236 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; G237 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; A238 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D239 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; L240 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Q241 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; N242 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; H243 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; I244 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L245 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; T246 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L247 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M248
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S249 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; V250 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A251 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A252 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; R253 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; I254 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; Y255 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; K256 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; H257 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; P258 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S259 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; I260 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; K261 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; N262 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; S263 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; I264 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; N265 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; L266 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; M267 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; V268 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V269 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K270 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V271 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L272 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I273
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V274 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; E275 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D276 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; E277 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; K278 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; W279 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; G280 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; P281 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; E282 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; V283 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S284 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; D285 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; N286 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; G287 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G288 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; L289 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T290 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L291 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R292
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
N293 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; F294 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; C295 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or P; N296 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; W297 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q298 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; R299 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R300
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
F301 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; N
[0125].sub.3O.sub.2 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; Q303 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; P304 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S305 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; D306 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R307 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H308 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P309
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; E310 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; H311 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; Y312 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; D313 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T314 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A315 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; I316 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L317 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L318 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T319
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R320 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q321
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; N322 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; F323 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; C324 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; G325 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q326 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; E327 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; G328 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; L329 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C330 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; D331 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T332 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; L333 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G334 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; V335 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; A336 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
D337 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; I338 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G339 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T340
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I341 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C342 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; D343
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; P344 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; N345 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; K346 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; S347 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; C348 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; S349 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; V350 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; I351 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; E352 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; D353 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; E354 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; G355 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; L356 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; Q357 replaced with D, F, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; A358 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; A359 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; H360 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; T361 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; L362 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A363
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H364 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E365
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L366 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G367
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; H368 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V369
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L370 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S371 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; M372 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P373 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; H374 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D375 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D376 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S377
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K378 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P379
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; C380 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or P; T381 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; R382 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; L383 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; F384 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; G385 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P386 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; M387 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G388 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K389 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; H390 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; H391 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; V392 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; M393 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A394 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P395
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; L396 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F397 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; V398 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H399 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; L400 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N401 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; Q402 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; T403 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L404 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P405 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; W406 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; S407 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P408 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; C409 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; S410 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A411 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; M412 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Y413 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; L414 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T415 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E416
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; L417 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L418
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D419 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G420
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G421 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; H422 replaced with D,
E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G423 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; D424 replaced with H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; C425 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
L426 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L427
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D428 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A429
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P430 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
G431 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A432
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A433 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; L434 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; P435 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L436 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P437 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; T438 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G439 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L440 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P441 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; G442 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; R443 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; M444 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; A445 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L446 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; Y447 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; Q448 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; L449 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; D450 replaced with H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; Q451 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; Q452 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; C453 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R454 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q455
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; I456 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F457
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
G458 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P459
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; D460 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; F461 replaced with D, E, H, K, R, N, Q, A, G, I, L,
S, T, M, V, P, or C; R462 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; H463 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; C464 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P465 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; N466
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; T467 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S468
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A469 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q470 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D471 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V472
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C473 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P;
A474 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q475
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; L476 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W477
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
C478 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; H479 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; T480 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; D481 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; G482 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; A483 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E484 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; P485 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; L486 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; C487 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, or P; H488 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; T489 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; K490 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; N491 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; G492 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S493 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; L494 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P495 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; W496 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; A497 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D498 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G499 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; T500 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P501 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; C502 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; G503 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; P504 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or C; G505 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; H506 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L507 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; C508 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, or P; S509 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; E510 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; G511 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; S512 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C513 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; L514 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; P515 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or C; E516 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E517 replaced with H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E518 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V519
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E520 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R521
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
P522 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; K523 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; P524 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; V525 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; V526 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; D527 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; G528 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; G529 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; W530 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; A531 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
P532 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or C; W533 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; G534 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; P535 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; W536 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; G537 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E538 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C539 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; S540 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; R541 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; T542 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C543 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; G544 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G545 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; G546 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; V547 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; Q548 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W,
Y, P, or C; F549 replaced with D, E, H, K, R, N, Q, A, G, I, L, S,
T, M, V, P, or C; S550 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; H551 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; R552 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; E553 replaced with H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; C554 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, or P; K555 replaced with D, E,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D556 replaced with
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P557
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; E558 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; P559 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, or C; Q560 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; N561 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; G562 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; G563 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; R564 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; Y565 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; C566 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; L567 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; G568 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; R569 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R570 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A571
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K572 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Y573
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
Q574 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; S575 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
C576 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, or P; H577 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; T578 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; E579 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; E580 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C581 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; P582 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P583 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
D584 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; G585 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K586 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; S587 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F588 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; R589 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; E590 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; Q591 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; Q592 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; C593 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; E594 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K595
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
Y596 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; N597 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; A598 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Y599 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; N600 replaced with D, E, H, K, R, A, G, I, L, S, T, M,
V, F, W, Y, P, or C; Y601 replaced with D, E, H, K, R, N, Q, A, G,
I, L, S, T, M, V, P, or C; T602 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D603 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; M604 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D605 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G606 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N
.sub.6O.sub.7 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; L608 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; L609 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q610 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; W611 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; V612 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; P613 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; K614 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; Y615 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; A616 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; G617 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; V618 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; S619 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P620
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; R621 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; D622 replaced with H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; R623 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; C624 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; K625 replaced with D, E, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L626 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; F627 replaced with D, E, H, K,
R, N, Q, A, G, I, L, S, T, M, V, P, or C; C628 replaced with D, E,
H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; R629 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A630
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R631 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G632
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R633 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S634
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E635 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F636
replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;
K637 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; V638 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F639 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; E640 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; A641 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K642 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; V643 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; I644 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D645
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; G646 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T647
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L648 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C649 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G650
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P651 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C;
E652 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; T653 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L654 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A655
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I656 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; C657 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; V658
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R659 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G660
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q661 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; C662
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; V663 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
K664 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; A665 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G666 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C667
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; D668 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; H669 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; V670 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; V671 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; D672 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; S673 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P674 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; R675 replaced with D, E, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, P, or C; K676 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; L677 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; D678 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; K679 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; C680 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; G681 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V682 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; C683 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or P; G684 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; G685 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; K686 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G687 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N688 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; S689 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; C690 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or P; R691 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; K692 replaced with D, E, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V693 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; S694 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; G695 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; S696 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L697 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T698 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P699
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; T700 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
N701 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; Y702 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; G703 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; Y704 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; N.sub.7O.sub.5 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; D706 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; I707 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; V708 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; T709 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; I710 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P711 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; A712 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; G713 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A714 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T715
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N716 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; I717
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D718 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V719
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K720 replaced
with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; Q721
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; R722 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; S723 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
H724 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,
or C; P725 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N,
Q, F, W, Y, or C; G726 replaced with D, E, H, K, R, N, Q, F, W, Y,
P, or C; V727 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q728 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; N729 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
F, W, Y, P, or C; D730 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; G731 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; N732 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, F, W, Y, P, or C; Y733 replaced with D, E, H, K, R, N, Q,
A, G, I, L, S, T, M, V, P, or C; L734 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; A735 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; L736 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; K737 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; T738 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; A739 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D740
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; G741 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q742
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; Y743 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; L744 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L745 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N746
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; G747 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N748
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; L749 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A750
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I751 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; S752 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; A753 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; 1754 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E755 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; Q756 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, F, W, Y, P, or C; D757 replaced with H, K, R, A,
G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; I758 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; L759 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; V760 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; K761 replaced with D, E, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, P, or C; G762 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T763 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I764 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
L765 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K766
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
Y767 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; S768 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G769 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S770
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I771 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; A772 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; T773 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; L774 replaced with D, E, H, K, R, N, Q,
F, W, Y, P, or C; E775 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; R776 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; L777 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; Q778 replaced with D, E, H, K, R, A, G, I,
L, S, T, M, V, F, W, Y, P, or C; S779 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; F780 replaced with D, E, H, K, R, N, Q, A,
G, I, L, S, T, M, V, P, or C; R781 replaced with D, E, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; P782 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L783 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; P784 replaced with D, E, H,
K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; E785 replaced
with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P786
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or C; L787 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
T788 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V789
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q790 replaced
with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L791
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L792 replaced
with D, E, H, K, R, N, Q, F, W, Y, P, or C; T793 replaced with D,
E, H, K, R, N, Q, F, W, Y, P, or C; V794 replaced with D, E, H, K,
R, N, Q, F, W, Y, P, or C; P795 replaced with D, E, H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, or C; G796 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; E797 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or C; V798 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; F799 replaced with D, E, H, K, R, N,
Q, A, G, I, L, S, T, M, V, P, or C; P800 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; P801 replaced with
D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; K802
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
V803 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K804
replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;
Y805 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; T806 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
F807 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,
or C; F808 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,
V, P, or C; V809 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; P810 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or C; N811 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; D812 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; V813 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; D814 replaced with H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; F815 replaced with D, E, H, K, R,
N, Q, A, G, I, L, S, T, M, V, P, or C; S816 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; M817 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; Q818 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, F, W, Y, P, or C; S819 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; S820 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; K821 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; E822 replaced with H, K, R, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; R823 replaced with D, E, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, P, or C; A824 replaced with D, E, H, K, R,
N, Q, F, W, Y, P, or C; T825 replaced with D, E, H, K, R, N, Q, F,
W, Y, P, or C; T826 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; N827 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,
W, Y, P, or C; I828 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; I829 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q830 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; P831 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; L832 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; L833 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; H834 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; A835 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
Q836 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,
P, or C; W837 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,
M, V, P, or C; V838 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; L839 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
G840 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D841
replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or
C; W842 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,
P, or C; S843 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E844 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; C845 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; S846 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; S847 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; T848 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C849
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
or P; G850 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
A851 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G852
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W853 replaced
with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; Q854
replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or
C; R855 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; R856 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,
W, Y, P, or C; T857 replaced with D, E, H, K, R, N, Q, F, W, Y, P,
or C; V858 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;
E859 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; C860 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or P; R861 replaced with D, E, A, G, I, L, S, T, M,
V, N, Q, F, W, Y, P, or C; D862 replaced with H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, P, or C; P863 replaced with D, E, H, K,
R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; S864 replaced with
D, E, H, K, R, N, Q, F, W, Y, P, or C; G865 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; Q866 replaced with D, E, H, K, R, A,
G, I, L, S, T, M, V, F, W, Y, P, or C; A867 replaced with D, E, H,
K, R, N, Q, F, W, Y, P, or C; S868 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; A869 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; T870 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; C871 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, or P; N872 replaced with D, E, H, K, R, A, G, I, L, S, T,
M, V, F, W, Y, P, or C; K873 replaced with D, E, A, G, I, L, S, T,
M, V, N, Q, F, W, Y, P, or C; A874 replaced with D, E, H, K, R, N,
Q, F, W, Y, P, or C; L875 replaced with D, E, H, K, R, N, Q, F, W,
Y, P, or C; K876 replaced with D, E, A, G, I, L, S, T, M, V, N, Q,
F, W, Y, P, or C; P877 replaced with D, E, H, K, R, A, G, I, L, S,
T, M, V, N, Q, F, W, Y, or C; E878 replaced with H, K, R, A, G, I,
L, S, T, M, V, N, Q, F, W, Y, P, or
C; D879 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,
Y, P, or C; A880 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or
C; K881 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y,
P, or C; P882 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,
N, Q, F, W, Y, or C; C883 replaced with D, E, H, K, R, A, G, I, L,
S, T, M, V, N, Q, F, W, Y, or P; E884 replaced with H, K, R, A, G,
I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S885 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; Q886 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, F, W, Y, P, or C; L887 replaced with D, E,
H, K, R, N, Q, F, W, Y, P, or C; C888 replaced with D, E, H, K, R,
A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P889 replaced with D,
E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; L890
replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C.
[0126] METH1 or METH2 polypeptides may contain 50, 40, 30, 10, 20,
19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative or non-conservative amino acid substitutions.
Additionally, METH1 or METH2 polypeptides may contain both
conservative or non-conservative substitutions, in any combination.
A METH1 or METH2 polypeptide may contain 50, 40, 30, 10, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative amino acids substitutions, and 50, 40, 30, 10, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
non-conservative amino acid substitutions in the same polypeptide.
For example, a particular polypeptide may contain 10 conservative
amino acid substitutions and 10 non-conservative amino acid
substitutions. Polynucleotides encoding such METH1 or METH2
polypeptides with substitutions are also encompassed within the
present invention.
[0127] The substitutions may be made in full-length METH1 or METH2,
mature METH1 or METH2, and any other METH1 or METH2 variant
disclosed herein, including METH1 or METH2 polypeptides with N-
and/or C-terminal amino acid deletions; METH1 or METH2 polypeptides
which lack one or more domains; or hybrid METH1/METH2
molecules.
[0128] Amino acids in the METH1 and METH2 proteins of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as in
vitro or in vivo inhibition of angiogenesis. Sites that are
critical for inhibition of angiogenesis can also be determined by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith et al., J. Mol. Biol.
224:899-904 (1992) and de Vos et al., Science 255:306-312
(1992)).
[0129] Particularly preferred are polypeptides with amino acid
substitutions at the boundaries of each domain (for example, at the
boundary of the metalloprotease domain). Amino acid substitutions
at these boundaries may be made to change the activity of the
protein, for example, to prevent cleavage. Amino acid substitutions
may also be made which do not affect the activity of the protein.
For example, the following amino acids may be replaced in METH1,
with the following amino acids: L-19 may be replaced with A, C, D,
E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; L-20 may be
replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W
or Y; L-21 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P,
Q, R, S, T, V, W or Y; L-22 may be replaced with A, C, D, E, F, G,
H, I, K, M, N, P, Q, R, S, T, V, W or Y; A-23 may be replaced with
may be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; A-24 may be replaced with C, D, E, F, G, H, I, K, L,
M, N, P, Q, R, S, T, V, W or Y; A-25 may be replaced with C, D, E,
F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L-26 may be
replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W
or Y; L-27 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P,
Q, R, S, T, V, W or Y; A-28 may be replaced with C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, W or Y; V-29 may be replaced with
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; S-30 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V,
W or Y; D-31 may be replaced with A, C, E, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; A-32 may be replaced with C, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L-33 may be replaced
with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;
G-34 may be replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R,
S, T, V, W or Y; R-35 may be replaced with A, C, D, E, F, G, H, I,
K, L, M, N, P, Q, S, T, V, W or Y; P-36 may be replaced with A, C,
D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; S-37 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W
or Y; E-38 may be replaced with A, C, D, F, G, H, I, K, L, M, N, P,
Q, R, S, T, V, W or Y; E-39 may be replaced with A, C, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; P-225 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y;
T-226 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, V, W or Y; G-227 may be replaced with A, C, D, E, F, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; T-228 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y; G-229 may
be replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; S-230 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, T, V, W or Y; 1-231 may be replaced with A, C, D, E, F,
G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; R-232 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y;
K-233 may be replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q,
R, S, T, V, W or Y; K-234 may be replaced with A, C, D, E, F, G, H,
I, L, M, N, P, Q, R, S, T, V, W or Y; R-235 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; F-236 may
be replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; V-237 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, W or Y; S-238 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; S-239 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;
H-240 may be replaced with A, C, D, E, F, G, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; R-241 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, S, T, V, W or Y; Y-242 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or W; V-243 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
W or Y; E-244 may be replaced with A, C, D, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; T-245 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, S, V, W or Y; K-449 may be replaced
with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y;
P-450 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W or Y; Q-451 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, R, S, T, V, W or Y; N-452 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; P-453 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V,
W or Y; 1-454 may be replaced with A, C, D, E, F, G, H, K, L, M, N,
P, Q, R, S, T, V, W or Y; Q-455 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, R, S, T, V, W or Y; L-456 may be replaced
with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;
P-457 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q,
R, S, T, V, W or Y; G-458 may be replaced with A, C, D, E, F, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; D-459 may be replaced with A,
C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L-460 may
be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,
W or Y; P-461 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, Q, R, S, T, V, W or Y; G-462 may be replaced with A, C, D, E, F,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; T-463 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y;
S-464 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, T, V, W or Y; Y-465 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, or W; D-466 may be replaced with
A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; A-467
may be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; N-468 may be replaced with A, C, D, E, F, G, H, I, K,
L, M, P, Q, R, S, T, V, W or Y; R-469 may be replaced with A, C, D,
E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; R-534 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W
or Y; K-535 may be replaced with A, C, D, E, F, G, H, I, L, M, N,
P, Q, R, S, T, V, W or Y; H-536 may be replaced with A, C, D, E, F,
G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; F-537 may be replaced
with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
D-538 may be replaced with A, C, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; T-539 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, V, W or Y; P-540 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; F-541 may
be replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; H-542 may be replaced with A, C, D, E, F, G, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; G-543 may be replaced with A, C, D, E, F,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; S-544 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;
W-545 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, T, V, or Y; G-546 may be replaced with A, C, D, E, F, H,
I, K, L, M, N, P, Q, R, S, T, V, W or Y; M-547 may be replaced with
A, C, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; W-548
may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T, V, or Y; G-549 may be replaced with A, C, D, E, F, H, I, K,
L, M, N, P, Q, R, S, T, V, W or Y; P-550 may be replaced with A, C,
D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; W-551 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
or Y; G-552 may be replaced with A, C, D, E, F, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; D-553 may be replaced with A, C, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; G-554 may be replaced
with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
S-831 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, T, V, W or Y; F-832 may be replaced with A, C, D, E, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; N-833 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; A-834 may
be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; 1-835 may be replaced with A, C, D, E, F, G, H, K, L, M, N,
P, Q, R, S, T, V, W or Y; P-836 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; T-837 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y;
F-838 may be replaced with A, C, D, E, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; S-839 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, T, V, W or Y; A-840 may be replaced with C,
D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; W-841 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V or Y; V-842 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, W or Y; 1-843 may be replaced with A, C, D, E, F,
G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; E-844 may be replaced
with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
E-845 may be replaced with A, C, D, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; W-846 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, or Y; G-847 may be replaced with
A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; E-848
may be replaced with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; C-849 may be replaced with A, D, E, F, G, H, I, K, L,
M, N, P, Q, R, S, T, V, W or Y; S-850 may be replaced with A, C, D,
E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; K-851 may be
replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W
or Y; R-885 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, S, T, V, W or Y; P-886 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; C-887 may be replaced
with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
A-888 may be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; D-889 may be replaced with A, C, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; H-890 may be replaced with A,
C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; P-891 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V,
W or Y; C-892 may be replaced with A, D, E, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; P-893 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; Q-894 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y;
W-895 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, T, V, or Y; Q-896 may be replaced with A, C, D, E, F, G,
H, I, K, L, M, N, P, R, S, T, V, W or Y; L-897 may be replaced with
A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; G-898
may be replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S,
T, V, W or Y; E-899 may be replaced with A, C, D, F, G, H, I, K, L,
M, N, P, Q, R, S, T, V, W or Y; W-900 may be replaced with A, C, D,
E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; S-901 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W
or Y; S-902 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, T, V, W or Y; C-903 may be replaced with A, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; S-904 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y;
and/or K-905 may be replaced with A, C, D, E, F, G, H, I, L, M, N,
P, Q, R, S, T, V, W or Y.
[0130] In addition, the following amino acids may be replaced in
METH2 with the following amino acids: L-14 may be replaced with A,
C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; L-15 may be
replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W
or Y; L-16 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P,
Q, R, S, T, V, W or Y; L-17 may be replaced with A, C, D, E, F, G,
H, I, K, M, N, P, Q, R, S, T, V, W or Y; L-18 may be replaced with
A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; L-19 may
be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,
W or Y; L-20 may be replaced with A, C, D, E, F, G, H, I, K, M, N,
P, Q, R, S, T, V, W or Y; L-21 may be replaced with A, C, D, E, F,
G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; L-22 may be replaced
with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;
P-23 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R,
S, T, V, W or Y; L-24 may be replaced with A, C, D, E, F, G, H, I,
K, M, N, P, Q, R, S, T, V, W or Y; A-25 may be replaced with C, D,
E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; R-26 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W
or Y; G-27 may be replaced with A, C, D, E, F, H, I, K, L, M, N, P,
Q, R, S, T, V, W or Y; A-28 may be replaced with C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, S, T, V, W or Y; P-29 may be replaced with
A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; A-30 may
be replaced with C, D, F, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; R-31 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, S, T, V, W or Y; P-32 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; A-33 may be replaced
with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
A-34 may be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R,
S, T, V, W or Y; P-204 may be replaced with A, C, D, E, F, G, H, I,
K, L, M, N, Q, R, S, T, V, W or Y; P-205 may be replaced with A, C,
D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; L-206 may be
replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W
or Y; G-207 may be replaced with A, C, D, E, F, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; A-208 may be replaced with C, D, E, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; T-209 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y;
S-210 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, T, V, W or Y; R-211 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, S, T, V, W or Y; T-212 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y; K-213 may
be replaced with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V,
W or Y; R-214 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, S, T, V, W or Y; F-215 may be replaced with A, C, D, E, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; V-216 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
S-217 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, T, V, W or Y; E-218 may be replaced with A, C, D, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; A-219 may be replaced with C,
D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; R-220 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V,
W or Y; F-221 may be replaced with A, C, D, E, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; V-222 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; E-223 may be replaced
with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
T-224 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, V, W or Y; P-430 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, Q, R, S, T, V, W or Y; G-431 may be replaced with A,
C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; A-432 may
be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; A-433 may be replaced with C, D, E, F, G, H, I, K, L, M, N,
P, Q, R, S, T, V, W or Y; L-434 may be replaced with A, C, D, E, F,
G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; P-435 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y;
L-436 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q,
R, S, T, V, W or Y; P-437 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, Q, R, S, T, V, W or Y; T-438 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y; G-439 may
be replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; L-440 may be replaced with A, C, D, E, F, G, H, I, K, M, N,
P, Q, R, S, T, V, W or Y; P-441 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; G-442 may be replaced
with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
R-443 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, S, T, V, W or Y; M-444 may be replaced with A, C, D, E, F, G, H,
I, K, L, N, P, Q, R, S, T, V, W or Y; A-445 may be replaced with C,
D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L-446 may
be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V,
W or Y; Y-447 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V or W; Q-448 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, R, S, T, V, W or Y; L-449 may be replaced
with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;
D-450 may be replaced with A, C, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; E-520 may be replaced with A, C, D, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; R-521 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; P-522 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V,
W or Y; K-523 may be replaced with A, C, D, E, F, G, H, I, L, M, N,
P, Q, R, S, T, V, W or Y; P-524 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, Q, R, S, T, V, W or Y; V-525 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
V-526 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, S, T, W or Y; D-527 may be replaced with A, C, E, F, G, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; G-528 may be replaced with A,
C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; G-529 may
be replaced with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; W-530 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V, or Y; A-531 may be replaced with C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; P-532 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W
or Y; W-533 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V, or Y; G-534 may be replaced with A, C, D, E,
F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; P-535 may be
replaced with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W
or Y; W-536 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, S, T, V or Y; G-537 may be replaced with A, C, D, E, F,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; E-538 may be replaced
with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
C-539 may be replaced with A, D, E, F, G, H, I, K, L, M, N, P, Q,
R, S, T, V, W or Y; S-540 may be replaced with A, C, D, E, F, G, H,
I, K, L, M, N, P, Q, R, T, V, W or Y; N-827 may be replaced with A,
C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; 1-828 may
be replaced with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V,
W or Y; 1-829 may be replaced with A, C, D, E, F, G, H, K, L, M, N,
P, Q, R, S, T, V, W or Y; Q-830 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, R, S, T, V, W or Y; P-831 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y;
L-832 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q,
R, S, T, V, W or Y; L-833 may be replaced with A, C, D, E, F, G, H,
I, K, M, N, P, Q, R, S, T, V, W or Y; H-834 may be replaced with A,
C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; A-835 may
be replaced with C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
W or Y; Q-836 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, R, S, T, V, W or Y; W-837 may be replaced with A, C, D, E, F,
G, H, I, K, L, M, N, P, Q, R, S, T, V, or Y; V-838 may be replaced
with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
L-839 may be replaced with A, C, D, E, F, G, H, I, K, M, N, P, Q,
R, S, T, V, W or Y; G-840 may be replaced with A, C, D, E, F, H, I,
K, L, M, N, P, Q, R, S, T, V, W or Y; D-841 may be replaced with A,
C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; W-842 may
be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V or Y; S-843 may be replaced with A, C, D, E, F, G, H, I, K, L, M,
N, P, Q, R, T, V, W or Y; E-844 may be replaced with A, C, D, F, G,
H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; C-845 may be replaced
with A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
S-846 may be replaced with A, C, D, E, F, G, H, I, K, L, M, N, P,
Q, R, T, V, W or Y; and/or S-847 may be replaced with A, C, D, E,
F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y.
[0131] METH1 or METH2 polypeptide variants, including substitution,
deletion and/or addition variants, which contain amino acid
substitutions can be tested for activity in any of the assays
described herein, for example, the chorioallantoic assay or the
cornea pocket assay. Preferred are METH1 or METH2 polypeptides with
conservative substitutions that: maintain all the activities and/or
properties of the wild type protein; or have one or more enhanced
activities and/or properties compared to the wild type protein.
Also preferred are METH1 or METH2 polypeptides with nonconservative
substitutions which: lack an activity and/or property of the wild
type protein, while maintaining all other activities and/or
properties; or lack more than one activity and/or property of the
wild type protein.
[0132] For example, activities or properties of METH1 or METH2 that
may be altered in METH1 or METH2 polypeptides with conservative or
nonconservative substitutions include, but are not limited to:
stimulation of angiogenesis; stimulation of epithelial cell
proliferation; antibody binding; ligand binding; stability;
solubility; and/or properties which affect purification.
[0133] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell or from a native source. For example, a recombinantly
produced version of the METH1 or METH2 polypeptide can be
substantially purified by the one-step method described in Smith
and Johnson, Gene 67:31-40 (1988).
[0134] The polypeptides of the present invention include the METH1
polypeptide encoded by the deposited cDNA including the leader; the
mature METH1 polypeptide encoded by the deposited the cDNA minus
the leader (i.e., the mature protein); a polypeptide comprising
amino acids about 1 to about 950 in SEQ ID NO:2; a polypeptide
comprising amino acids about 2 to about 950 in SEQ ID NO:2; a
polypeptide comprising amino acids about 29 to about 950 in SEQ ID
NO:2; a polypeptide comprising amino acids about 30 to about 950 in
SEQ ID NO:2; a polypeptide comprising the metalloprotease domain of
METH1, amino acids 235 to 459 in SEQ ID NO:2; a polypeptide
comprising the disintegrin domain of METH1, amino acids 460 to 544
in SEQ ID NO:2; a polypeptide comprising the first TSP-like domain
of METH1, amino acids 545 to 598 in SEQ ID NO:2; a polypeptide
comprising the second TSP-like domain of METH1, amino acids 841 to
894 in SEQ ID NO:2; a polypeptide comprising the third TSP-like
domain of METH1, amino acids 895 to 934 in SEQ ID NO:2; a
polypeptide comprising amino acids 536 to 613 in SEQ ID NO:2; a
polypeptide comprising amino acids 549 to 563 in SEQ ID NO:2; the
METH2 polypeptide encoded by the deposited cDNA including the
leader; the mature METH2 polypeptide encoded by the deposited the
cDNA minus the leader (i.e., the mature protein); a polypeptide
comprising amino acids about 1 to about 890 in SEQ ID NO:4; a
polypeptide comprising amino acids about 2 to about 890 in SEQ ID
NO:4; a polypeptide comprising amino acids about 24 to about 890 in
SEQ ID NO:4; a polypeptide comprising amino acids about 112 to
about 890 in SEQ ID NO:4; a polypeptide comprising the
metalloprotease domain of METH2, amino acids 214 to 439 in SEQ ID
NO:4; a polypeptide comprising the disintegrin domain of METH2,
amino acids 440 to 529 in SEQ ID NO:4; a polypeptide comprising the
first TSP-like domain of METH2, amino acids 530 to 583 in SEQ ID
NO:4; a polypeptide comprising the second TSP-like domain of METH2,
amino acids 837 to 890 in SEQ ID NO:4; a polypeptide comprising
amino acids 280 to 606 in SEQ ID NO:4; a polypeptide comprising
amino acids 529 to 548 in SEQ ID NO:4; as well as polypeptides
which are at least 95% identical, and more preferably at least 96%,
97%, 98% or 99% identical to the polypeptides described above and
also include portions of such polypeptides with at least 30 amino
acids and more preferably at least 50 amino acids.
[0135] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
METH1 or METH2 polypeptide is intended that the amino acid sequence
of the polypeptide is identical to the reference sequence except
that the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference amino acid of
the METH1 or METH2 polypeptide. In other words, to obtain a
polypeptide having an amino acid sequence at least 95% identical to
a reference amino acid sequence, up to 5% of the amino acid
residues in the reference sequence may be deleted or substituted
with another amino acid, or a number of amino acids up to 5% of the
total amino acid residues in the reference sequence may be inserted
into the reference sequence. These alterations of the reference
sequence may occur at the amino or carboxy terminal positions of
the reference amino acid sequence or anywhere between those
terminal positions, interspersed either individually among residues
in the reference sequence or in one or more contiguous groups
within the reference sequence.
[0136] As a practical matter, whether any particular polypeptide is
at least 95%, 96%, 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:4 or to the
amino acid sequence encoded by deposited cDNA clones can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). When using Bestfit or any
other sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0137] A preferred method for determining the best overall match
between a query sequence (a sequence of the present invention) and
a subject sequence, also referred to as a global sequence
alignment, can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al., Comp. App. Biosci.
6:237-245 (1990). In a sequence alignment, the query and subject
sequences are either both nucleotide sequences or both amino acid
sequences. The result of said global sequence alignment is in
percent identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty 5, Gap Size Penalty=0.05, Window Size=500 or the length of
the subject amino acid sequence, whichever is shorter.
[0138] If the subject sequence is shorter than the query sequence
due to N- or C-terminal deletions, not because of internal
deletions, a manual correction must be made to the results. This is
because the FASTDB program does not account for N- and C-terminal
truncations of the subject sequence when calculating global percent
identity. For subject sequences truncated at the N- and C-termini,
relative to the query sequence, the percent identity is corrected
by calculating the number of residues of the query sequence that
are N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total residues of the query sequence. Whether a residue is
matched/aligned is determined by the results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This final percent identity score is what is used for the purposes
of the present invention. Only residues to the N- and C-termini of
the subject sequence, which are not matched/aligned with the query
sequence, are considered for the purposes of manually adjusting the
percent identity score. That is, only query residue positions
outside the farthest N- and C-terminal residues of the subject
sequence.
[0139] For example, a 90 amino acid residue subject sequence is
aligned with a 100 residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the subject
sequence and therefore, the FASTDB alignment does not show a
match/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues
at the N- and C-termini not matched/total number of residues in the
query sequence) so 10% is subtracted from the percent identity
score calculated by the FASTDB program. If the remaining 90
residues were perfectly matched, the final percent identity would
be 90%. In another example, a 90 residue subject sequence is
compared with a 100 residue query sequence. This time, the
deletions are internal, so there are no residues at the or
C-termini of the subject sequence which are not matched/aligned
with the query. In this case, the percent identity calculated by
FASTDB is not manually corrected. Once again, only residue
positions outside the and C-terminal ends of the subject sequence,
as displayed in the FASTDB alignment, which are not matched/aligned
with the query sequence are manually corrected for. No other manual
corrections are made for the purposes of the present invention.
[0140] The polypeptides of the present invention are useful as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0141] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide described herein.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0142] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in the
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G. et al., "Antibodies that react with predetermined
sites on proteins", Science 219:660-666 (1983). Peptides capable of
eliciting protein-reactive sera are frequently represented in the
primary sequence of a protein, can be characterized by a set of
simple chemical rules, and are confined neither to immunodominant
regions of intact proteins (i.e., immunogenic epitopes) nor to the
amino or carboxyl terminals.
[0143] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. See, for instance, Wilson et al., Cell 37:767-778
(1984) at 777.
[0144] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about at least
about 15 to about 30 amino acids contained within the amino acid
sequence of a polypeptide of the invention.
[0145] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means. Houghten, R.
A., "General method for the rapid solid-phase synthesis of large
numbers of peptides: specificity of antigen-antibody interaction at
the level of individual amino acids", Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis
(SMPS)" process is further described in U.S. Pat. No. 4,631,211 to
Houghten et al. (1986).
[0146] As one of skill in the art will appreciate, METH1 or METH2
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric METH1 or METH2 protein or protein fragment alone
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).
METH1 and METH2 Polynucleotide and Polypeptide Fragments
[0147] In the present invention, a "polynucleotide fragment" refers
to a short polynucleotide having a nucleic acid sequence contained
in the deposited clones or shown in SEQ ID NO:1 or SEQ ID NO: 3.
The short nucleotide fragments are preferably at least about 15 nt,
and more preferably at least about 20 nt, still more preferably at
least about 30 nt, and even more preferably, at least about 40 nt
in length. A fragment "at least 20 nt in length," for example, is
intended to include 20 or more contiguous bases from the cDNA
sequence contained in the deposited clones or the nucleotide
sequence shown in SEQ ID NO:1 or SEQ ID NO:3. These nucleotide
fragments are useful as diagnostic probes and primers as discussed
herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000
nucleotides) are preferred.
[0148] Moreover, representative examples of METH1 or METH2
polynucleotide fragments include, for example, fragments having a
sequence from about nucleotide number 1-50, 51-100, 101-150,
151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,
501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900,
901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200,
1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500,
1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800,
1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of
SEQ ID NO:1 or SEQ ID NO:3 or the cDNA contained in the deposited
clones. In this context "about" includes the particularly recited
ranges, larger or smaller by several (5, 4, 3, 2, or 1)
nucleotides, at either terminus or at both termini. Preferably,
these fragments encode a polypeptide which has biological activity.
More preferably, these polynucleotides can be used as probes or
primers as discussed herein.
[0149] In the present invention, a "polypeptide fragment" refers to
a short amino acid sequence contained in SEQ ID NO:2 or SEQ ID NO:4
or encoded by the cDNA contained in the deposited clones. Protein
fragments may be "free-standing," or comprised within a larger
polypeptide of which the fragment forms a part or region, most
preferably as a single continuous region. Representative examples
of polypeptide fragments of the invention, include, for example,
fragments from about amino acid number 1-20, 21-40, 41-60, 61-80,
81-100, 102-120, 121-140, 141-160, 161-180, 181-200, 201-220,
221-240, 241-260, 261-280, or 281 to the end of the coding region
of SEQ ID NO:2 or SEQ ID NO:4. Moreover, polypeptide fragments can
be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,
or 150 amino acids in length. In this context "about" includes the
particularly recited ranges, larger or smaller by several (5, 4, 3,
2, or 1) amino acids, at either extreme or at both extremes.
[0150] Preferred polypeptide fragments include the secreted METH1
or METH2 protein as well as the mature form. Further preferred
polypeptide fragments include the secreted METH1 or METH2 protein
or the mature form having a continuous series of deleted residues
from the amino or the carboxy terminus, or both. For example, any
number of amino acids, ranging from 1-60, can be deleted from the
amino terminus of either the secreted METH1 or METH2 polypeptide or
the mature form. Similarly, any number of amino acids, ranging from
1-30, can be deleted from the carboxy terminus of the secreted
METH1 or METH2 protein or mature form. Furthermore, any combination
of the above amino and carboxy terminus deletions are preferred.
Similarly, polynucleotide fragments encoding these METH1 or METH2
polypeptide fragments are also preferred.
Particularly, N-terminal deletions of the METH1 polypeptide can be
described by the general formula m-950, where m is an integer from
2 to 949, where m corresponds to the position of the amino acid
residue identified in SEQ ID NO:2. Preferably, N-terminal deletions
of the METH1 polypeptide of the invention shown as SEQ ID NO:2
include polypeptides comprising the amino acid sequence of
residues: G-2 to S-950; N-3 to S-950; A-4 to S-950; E-5 to S-950;
R-6 to S-950; A-7 to S-950; P-8 to S-950; G-9 to S-950; S-10 to
S-950; R-11 to S-950; S-12 to S-950; F-13 to S-950; G-14 to S-950;
P-15 to S-950; V-16 to S-950; P-17 to S-950; T-18 to S-950; L-19 to
S-950; L-20 to S-950; L-21 to S-950; L-22 to S-950; A-23 to S-950;
A-24 to S-950; A-25 to S-950; L-26 to S-950; L-27 to S-950; A-28 to
S-950; V-29 to S-950; S-30 to S-950; D-31 to S-950; A-32 to S-950;
L-33 to S-950; G-34 to S-950; R-35 to S-950; P-36 to S-950; S-37 to
S-950; E-38 to S-950; E-39 to S-950; D-40 to S-950; E-41 to S-950;
E-42 to S-950; L-43 to S-950; V-44 to S-950; V-45 to S-950; P-46 to
S-950; E-47 to S-950; L-48 to S-950; E-49 to S-950; R-50 to S-950;
A-51 to S-950; P-52 to S-950; G-53 to S-950; H-54 to S-950; G-55 to
S-950; T-56 to S-950; T-57 to S-950; R-58 to S-950; L-59 to S-950;
R-60 to S-950; L-61 to S-950; H-62 to S-950; A-63 to S-950; F-64 to
S-950; D-65 to S-950; Q-66 to S-950; Q-67 to S-950; L-68 to S-950;
D-69 to S-950; L-70 to S-950; E-71 to S-950; L-72 to S-950; R-73 to
S-950; P-74 to S-950; D-75 to S-950; S-76 to S-950; S-77 to S-950;
F-78 to S-950; L-79 to S-950; A-80 to S-950; P-81 to S-950; G-82 to
S-950; F-83 to S-950; T-84 to S-950; L-85 to S-950; Q-86 to S-950;
N-87 to S-950; V-88 to S-950; G-89 to S-950; R-90 to S-950; K-91 to
S-950; S-92 to S-950; G-93 to S-950; S-94 to S-950; E-95 to S-950;
T-96 to S-950; P-97 to S-950; L-98 to S-950; P-99 to S-950; E-100
to S-950; T-101 to S-950; D-102 to S-950; L-103 to S-950; A-104 to
S-950; H-105 to S-950; C-106 to S-950; F-107 to S-950; Y-108 to
S-950; S-109 to S-950; G-110 to S-950; T-111 to S-950; V-112 to
S-950; N-113 to S-950; G-114 to S-950; D-115 to S-950; P-116 to
S-950; S-117 to S-950; S-118 to S-950; A-119 to S-950; A-120 to
S-950; A-121 to S-950; L-122 to S-950; S-123 to S-950; L-124 to
S-950; C-125 to S-950; E-126 to S-950; G-127 to S-950; V-128 to
S-950; R-129 to S-950; G-130 to S-950; A-131 to S-950; F-132 to
S-950; Y-133 to S-950; L-134 to S-950; L-135 to S-950; G-136 to
S-950; E-137 to S-950; A-138 to S-950; Y-139 to S-950; F-140 to
S-950; I-141 to S-950; Q-142 to S-950; P-143 to S-950; L-144 to
S-950; P-145 to S-950; A-146 to S-950; A-147 to S-950; S-148 to
S-950; E-149 to S-950; R-150 to S-950; L-151 to S-950; A-152 to
S-950; T-153 to S-950; A-154 to S-950; A-155 to S-950; P-156 to
S-950; G-157 to S-950; E-158 to S-950; K-159 to S-950; P-160 to
S-950; P-161 to S-950; A-162 to S-950; P-163 to S-950; L-164 to
S-950; Q-165 to S-950; F-166 to S-950; H-167 to S-950; L-168 to
S-950; L-169 to S-950; R-170 to S-950; R-171 to S-950; N-172 to
S-950; R-173 to S-950; Q-174 to S-950; G-175 to S-950; D-176 to
S-950; V-177 to S-950; G-178 to S-950; G-179 to S-950; T-180 to
S-950; C-181 to S-950; G-182 to S-950; V-183 to S-950; V-184 to
S-950; D-185 to S-950; D-186 to S-950; E-187 to S-950; P-188 to
S-950; R-189 to S-950; P-190 to S-950; T-191 to S-950; G-192 to
S-950; K-193 to S-950; A-194 to S-950; E-195 to S-950; T-196 to
S-950; E-197 to S-950; D-198 to S-950; E-199 to S-950; D-200 to
S-950; E-201 to S-950; G-202 to S-950; T-203 to S-950; E-204 to
S-950; G-205 to S-950; E-206 to S-950; D-207 to S-950; E-208 to
S-950; G-209 to S-950; P-210 to S-950; Q-211 to S-950; W-212 to
S-950; S-213 to S-950; P-214 to S-950; Q-215 to S-950; D-216 to
S-950; P-217 to S-950; A-218 to S-950; L-219 to S-950; Q-220 to
S-950; G-221 to S-950; V-222 to S-950; G-223 to S-950; Q-224 to
S-950; P-225 to S-950; T-226 to S-950; G-227 to S-950; T-228 to
S-950; G-229 to S-950; S-230 to S-950; I-231 to S-950; R-232 to
S-950; K-233 to S-950; K-234 to S-950; R-235 to S-950; F-236 to
S-950; V-237 to S-950; S-238 to S-950; S-239 to S-950; H-240 to
S-950; R-241 to S-950; Y-242 to S-950; V-243 to S-950; E-244 to
S-950; T-245 to S-950; M-246 to S-950; L-247 to S-950; V-248 to
S-950; A-249 to S-950; D-250 to S-950; Q-251 to S-950; S-252 to
S-950; M-253 to S-950; A-254 to S-950; E-255 to S-950; F-256 to
S-950; H-257 to S-950; G-258 to S-950; S-259 to S-950; G-260 to
S-950; L-261 to S-950; K-262 to S-950; H-263 to S-950; Y-264 to
S-950; L-265 to S-950; L-266 to S-950; T-267 to S-950; L-268 to
S-950; F-269 to S-950; S-270 to S-950; V-271 to S-950; A-272 to
S-950; A-273 to S-950; R-274 to S-950; L-275 to S-950; Y-276 to
S-950; K-277 to S-950; H-278 to S-950; P-279 to S-950; S-280 to
S-950; I-281 to S-950; R-282 to S-950; N-283 to S-950; S-284 to
S-950; V-285 to S-950; S-286 to S-950; L-287 to S-950; V-288 to
S-950; V-289 to S-950; V-290 to S-950; K-291 to S-950; I-292 to
S-950; L-293 to S-950; V-294 to S-950; I-295 to S-950; H-296 to
S-950; D-297 to S-950; E-298 to S-950; Q-299 to S-950; K-300 to
S-950; G-301 to S-950; P-302 to S-950; E-303 to S-950; V-304 to
S-950; T-305 to S-950; S-306 to S-950; N-307 to S-950; A-308 to
S-950; A-309 to S-950; L-310 to S-950; T-311 to S-950; L-312 to
S-950; R-313 to S-950; N-314 to S-950; F-315 to S-950; C-316 to
S-950; N-317 to S-950; W-318 to S-950; Q-319 to S-950; K-320 to
S-950; Q-321 to S-950; H-322 to S-950; N-323 to S-950; P-324 to
S-950; P-325 to S-950; S-326 to S-950; D-327 to S-950; R-328 to
S-950; D-329 to S-950; A-330 to S-950; E-331 to S-950; H-332 to
S-950; Y-333 to S-950; D-334 to S-950; T-335 to S-950; A-336 to
S-950; I-337 to S-950; L-338 to S-950; F-339 to S-950; T-340 to
S-950; R-341 to S-950; Q-342 to S-950; D-343 to S-950; L-344 to
S-950; C-345 to S-950; G-346 to S-950; S-347 to S-950; Q-348 to
S-950; T-349 to S-950; C-350 to S-950; D-351 to S-950; T-352 to
S-950; L-353 to S-950; G-354 to S-950; M-355 to S-950; A-356 to
S-950; D-357 to S-950; V-358 to S-950; G-359 to S-950; T-360 to
S-950; V-361 to S-950; C-362 to S-950; D-363 to S-950; P-364 to
S-950; S-365 to S-950; R-366 to S-950; S-367 to S-950; C-368 to
S-950; S-369 to S-950; V-370 to S-950; I-371 to S-950; E-372 to
S-950; D-373 to S-950; D-374 to S-950; G-375 to S-950; L-376 to
S-950; Q-377 to S-950; A-378 to S-950; A-379 to S-950; F-380 to
S-950; T-381 to S-950; T-382 to S-950; A-383 to S-950; H-384 to
S-950; E-385 to S-950; L-386 to S-950; G-387 to S-950; H-388 to
S-950; V-389 to S-950; F-390 to S-950; N-391 to S-950; M-392 to
S-950; P-393 to S-950; H-394 to S-950; D-395 to S-950; D-396 to
S-950; A-397 to S-950; K-398 to S-950; Q-399 to S-950; C-400 to
S-950; A-401 to S-950; S-402 to S-950; L-403 to S-950; N-404 to
S-950; G-405 to S-950; V-406 to S-950; N-407 to S-950; Q-408 to
S-950; D-409 to S-950; S-410 to S-950; H-411 to S-950; M-412 to
S-950; M-413 to S-950; A-414 to S-950; S-415 to S-950; M-416 to
S-950; L-417 to S-950; S-418 to S-950; N-419 to S-950; L-420 to
S-950; D-421 to S-950; H-422 to S-950; S-423 to S-950; Q-424 to
S-950; P-425 to S-950; W-426 to S-950; S-427 to S-950; P-428 to
S-950; C-429 to S-950; S-430 to S-950; A-431 to S-950; Y-432 to
S-950; M-433 to S-950; I-434 to S-950; T-435 to S-950; S-436 to
S-950; F-437 to S-950; L-438 to S-950; D-439 to S-950; N-440 to
S-950; G-441 to S-950; H-442 to S-950; G-443 to S-950; E-444 to
S-950; C-445 to S-950; L-446 to S-950; M-447 to S-950; D-448 to
S-950; K-449 to S-950; P-450 to S-950; Q-451 to S-950; N-452 to
S-950; P-453 to S-950; I-454 to S-950; Q-455 to S-950; L-456 to
S-950; P-457 to S-950; G-458 to S-950; D-459 to S-950; L-460 to
S-950; P-461 to S-950; G-462 to S-950; T-463 to S-950; S-464 to
S-950; Y-465 to S-950; D-466 to S-950; A-467 to S-950; N-468 to
S-950; R-469 to S-950; Q-470 to S-950; C-471 to S-950; Q-472 to
S-950; F-473 to S-950; T-474 to S-950; F-475 to S-950; G-476 to
S-950; E-477 to S-950; D-478 to S-950; S-479 to S-950; K-480 to
S-950; H-481 to S-950; C-482 to S-950; P-483 to S-950; D-484 to
S-950; A-485 to S-950; A-486 to S-950; S-487 to S-950; T-488 to
S-950; C-489 to S-950; S-490 to S-950; T-491 to S-950; L-492 to
S-950; W-493 to S-950; C-494 to S-950; T-495 to S-950; G-496 to
S-950; T-497 to S-950; S-498 to S-950; G-499 to S-950; G-500 to
S-950; V-501 to S-950; L-502 to S-950; V-503 to S-950; C-504 to
S-950; Q-505 to S-950; T-506 to S-950; K-507 to S-950; H-508 to
S-950; F-509 to S-950; P-510 to S-950; W-511 to S-950; A-512 to
S-950; D-513 to S-950; G-514 to S-950; T-515 to S-950; S-516 to
S-950; C-517 to S-950; G-518 to S-950; E-519 to S-950; G-520 to
S-950; K-521 to S-950; W-522 to S-950; C-523 to S-950; I-524 to
S-950; N-525 to S-950; G-526 to S-950; K-527 to S-950; C-528 to
S-950; V-529 to S-950; N-530 to S-950; K-531 to S-950; T-532 to
S-950; D-533 to S-950; R-534 to S-950; K-535 to S-950; H-536 to
S-950; F-537 to S-950; D-538 to S-950; T-539 to S-950; P-540 to
S-950; F-541 to S-950; H-542 to S-950; G-543 to S-950; S-544 to
S-950; W-545 to S-950; G-546 to S-950; M-547 to S-950; W-548 to
S-950; G-549 to S-950; P-550 to S-950; W-551 to S-950; G-552 to
S-950; D-553 to S-950; C-554 to S-950; S-555 to S-950; R-556 to
S-950; T-557 to S-950; C-558 to S-950; G-559 to S-950; G-560 to
S-950; G-561 to S-950; V-562 to S-950; Q-563 to S-950; Y-564 to
S-950; T-565 to S-950; M-566 to S-950; R-567 to S-950; E-568 to
S-950; C-569 to S-950; D-570 to S-950; N-571 to S-950; P-572 to
S-950; V-573 to S-950; P-574 to S-950; K-575 to S-950; N-576 to
S-950; G-577 to S-950; G-578 to S-950; K-579 to S-950; Y-580 to
S-950; C-581 to S-950; E-582 to S-950; G-583 to S-950; K-584 to
S-950; R-585 to S-950; V-586 to S-950; R-587 to S-950; Y-588 to
S-950; R-589 to S-950; S-590 to S-950; C-591 to S-950; N-592 to
S-950; L-593 to S-950; E-594 to S-950; D-595 to S-950; C-596 to
S-950; P-597 to S-950; D-598 to S-950; N-599 to S-950; N-600 to
S-950; G-601 to S-950; K-602 to S-950; T-603 to S-950; F-604 to
S-950; R-605 to S-950; E-606 to S-950; E-607 to S-950; Q-608 to
S-950; C-609 to S-950; E-610 to S-950; A-611 to S-950; H-612 to
S-950; N-613 to S-950; E-614 to S-950; F-615 to S-950; S-616 to
S-950; K-617 to S-950; A-618 to S-950; S-619 to S-950; F-620 to
S-950; G-621 to S-950; S-622 to S-950; G-623 to S-950; P-624 to
S-950; A-625 to S-950; V-626 to S-950; E-627 to S-950; W-628 to
S-950; I-629 to S-950; P-630 to S-950; K-631 to S-950; Y-632 to
S-950; A-633 to S-950; G-634 to S-950; V-635 to S-950; S-636 to
S-950; P-637 to S-950; K-638 to S-950; D-639 to S-950; R-640 to
S-950; C-641 to S-950; K-642 to S-950; L-643 to S-950; I-644 to
S-950; C-645 to S-950; Q-646 to S-950; A-647 to S-950; K-648 to
S-950; G-649 to S-950; I-650 to S-950; G-651 to S-950; Y-652 to
S-950; F-653 to S-950; F-654 to S-950; V-655 to S-950; L-656 to
S-950; Q-657 to S-950; P-658 to S-950; K-659 to S-950; V-660 to
S-950; V-661 to S-950; D-662 to S-950; G-663 to S-950; T-664 to
S-950; P-665 to S-950; C-666 to S-950; S-667 to S-950; P-668 to
S-950; D-669 to S-950; S-670 to S-950; T-671 to S-950; S-672 to
S-950; V-673 to S-950; C-674 to S-950; V-675 to S-950; Q-676 to
S-950; G-677 to S-950; Q-678 to S-950; C-679 to S-950; V-680 to
S-950; K-681 to S-950; A-682 to S-950; G-683 to S-950; C-684 to
S-950; D-685 to S-950; R-686 to S-950; I-687 to S-950; I-688 to
S-950; D-689 to S-950; S-690 to S-950; K-691 to S-950; K-692 to
S-950; K-693 to S-950; F-694 to S-950; D-695 to S-950; K-696 to
S-950; C-697 to S-950; G-698 to S-950; V-699 to S-950; C-700 to
S-950; G-701 to S-950; G-702 to S-950; N-703 to S-950; G-704 to
S-950; S-705 to S-950; T-706 to S-950; C-707 to S-950; K-708 to
S-950; K-709 to S-950; I-710 to S-950; S-711 to S-950; G-712 to
S-950; S-713 to S-950; V-714 to S-950; T-715 to S-950; S-716 to
S-950; A-717 to S-950; K-718 to S-950; P-719 to S-950; G-720 to
S-950; Y-721 to S-950; H-722 to S-950; D-723 to S-950; I-724 to
S-950; I-725 to S-950; T-726 to S-950; I-727 to S-950; P-728 to
S-950; T-729 to S-950; G-730 to S-950; A-731 to S-950; T-732 to
S-950; N-733 to S-950; I-734 to S-950; E-735 to S-950; V-736 to
S-950; K-737 to S-950; Q-738 to S-950; R-739 to S-950; N-740 to
S-950; Q-741 to S-950; R-742 to S-950; G-743 to S-950; S-744 to
S-950; R-745 to S-950; N-746 to S-950; N-747 to S-950; G-748 to
S-950; S-749 to S-950; F-750 to S-950; L-751 to S-950; A-752 to
S-950; I-753 to S-950; K-754 to S-950; A-755 to S-950; A-756 to
S-950; D-757 to S-950; G-758 to S-950; T-759 to S-950; Y-760 to
S-950; I-761 to S-950; L-762 to S-950; N-763 to S-950; G-764 to
S-950; D-765 to S-950; Y-766 to S-950; T-767 to S-950; L-768 to
S-950; S-769 to S-950; T-770 to S-950; L-771 to S-950; E-772 to
S-950; Q-773 to S-950; D-774 to S-950; I-775 to S-950; M-776 to
S-950; Y-777 to S-950; K-778 to S-950; G-779 to S-950; V-780 to
S-950; V-781 to S-950; L-782 to S-950; R-783 to S-950; Y-784 to
S-950; S-785 to S-950; G-786 to S-950; S-787 to S-950; S-788 to
S-950; A-789 to S-950; A-790 to S-950; L-791 to S-950; E-792 to
S-950; R-793 to S-950; I-794 to S-950; R-795 to S-950; S-796 to
S-950; F-797 to S-950; S-798 to S-950; P-799 to S-950; L-800 to
S-950; K-801 to S-950; E-802 to S-950; P-803 to S-950; L-804 to
S-950; T-805 to S-950; I-806 to S-950; Q-807 to S-950; V-808 to
S-950; L-809 to S-950; T-810 to S-950; V-811 to S-950; G-812 to
S-950; N-813 to S-950; A-814 to S-950; L-815 to S-950; R-816 to
S-950; P-817 to S-950; K-818 to S-950; I-819 to S-950; K-820 to
S-950; Y-821 to S-950; T-822 to S-950; Y-823 to S-950; F-824 to
S-950; V-825 to S-950; K-826 to S-950; K-827 to S-950; K-828 to
S-950; K-829 to S-950; E-830 to S-950; S-831 to S-950; F-832 to
S-950; N-833 to S-950; A-834 to S-950; I-835 to S-950; P-836 to
S-950; T-837 to S-950; F-838 to S-950; S-839 to S-950; A-840 to
S-950; W-841 to S-950; V-842 to S-950; I-843 to S-950; E-844 to
S-950; E-845 to S-950; W-846 to S-950; G-847 to S-950; E-848 to
S-950; C-849 to S-950; S-850 to S-950; K-851 to S-950; S-852 to
S-950; C-853 to S-950; E-854 to S-950; L-855 to S-950; G-856 to
S-950; W-857 to S-950; Q-858 to S-950; R-859 to S-950; R-860 to
S-950; L-861 to S-950; V-862 to S-950; E-863 to S-950; C-864 to
S-950; R-865 to S-950; D-866 to S-950; I-867 to S-950; N-868 to
S-950; G-869 to S-950; Q-870 to S-950; P-871 to S-950; A-872 to
S-950; S-873 to S-950; E-874 to S-950; C-875 to S-950; A-876 to
S-950; K-877 to S-950; E-878 to S-950; V-879 to S-950; K-880 to
S-950; P-881 to S-950; A-882 to S-950; S-883 to S-950; T-884 to
S-950; R-885 to S-950; P-886 to S-950; C-887 to S-950; A-888 to
S-950; D-889 to S-950; H-890 to S-950; P-891 to S-950; C-892 to
S-950; P-893 to S-950; Q-894 to S-950; W-895 to S-950; Q-896 to
S-950; L-897 to S-950; G-898 to S-950; E-899 to S-950; W-900 to
S-950; S-901 to S-950; S-902 to S-950; C-903 to S-950; S-904 to
S-950; K-905 to S-950; T-906 to S-950; C-907 to S-950; G-908 to
S-950; K-909 to S-950; G-910 to S-950; Y-911 to S-950; K-912 to
S-950; K-913 to S-950; R-914 to S-950; S-915 to S-950; L-916 to
S-950; K-917 to S-950; C-918 to S-950; L-919 to S-950; S-920 to
S-950; H-921 to S-950; D-922 to S-950; G-923 to S-950; G-924 to
S-950; V-925 to S-950; L-926 to S-950; S-927 to S-950; H-928 to
S-950; E-929 to S-950; S-930 to S-950; C-931 to S-950; D-932 to
S-950; P-933 to S-950; L-934 to S-950; K-935 to S-950; K-936 to
S-950; P-937 to S-950; K-938 to S-950; H-939 to S-950; F-940 to
S-950; I-941 to S-950; D-942 to S-950; F-943 to S-950; C-944 to
S-950; T-945 to S-950; of SEQ ID NO:2.
[0152] Moreover, C-terminal deletions of the METH1 polypeptide can
also be described by the general formula 1-n.sub.1, where n.sub.1
is an integer from 2 to 950, where n corresponds to the position of
amino acid residue identified in SEQ ID NO:2. Preferably,
C-terminal deletions of the METH1 polypeptide of the invention
shown as SEQ ID NO:2 include polypeptides comprising the amino acid
sequence of residues: M-1 to C-949; M-1 to E-948; M-1 to A-947; M-1
to M-946; M-1 to T-945; M-1 to C-944; M-1 to F-943; M-1 to D-942;
M-1 to I-941; M-1 to F-940; M-1 to H-939; M-1 to K-938; M-1 to
P-937; M-1 to K-936; M-1 to K-935; M-1 to L-934; M-1 to P-933; M-1
to D-932; M-1 to C-931; M-1 to S-930; M-1 to E-929; M-1 to H-928;
M-1 to S-927; M-1 to L-926; M-1 to V-925; M-1 to G-924; M-1 to
G-923; M-1 to D-922; M-1 to H-921; M-1 to S-920; M-1 to L-919; M-1
to C-918; M-1 to K-917; M-1 to L-916; M-1 to S-915; M-1 to R-914;
M-1 to K-913; M-1 to K-912; M-1 to Y-911; M-1 to G-910; M-1 to
K-909; M-1 to G-908; M-1 to C-907; M-1 to T-906; M-1 to K-905; M-1
to S-904; M-1 to C-903; M-1 to S-902; M-1 to S-901; M-1 to W-900;
M-1 to E-899; M-1 to G-898; M-1 to L-897; M-1 to Q-896; M-1 to
W-895; M-1 to Q-894; M-1 to P-893; M-1 to C-892; M-1 to P-891; M-1
to H-890; M-1 to D-889; M-1 to A-888; M-1 to C-887; M-1 to P-886;
M-1 to R-885; M-1 to T-884; M-1 to S-883; M-1 to A-882; M-1 to
P-881; M-1 to K-880; M-1 to V-879; M-1 to E-878; M-1 to K-877; M-1
to A-876; M-1 to C-875; M-1 to E-874; M-1 to S-873; M-1 to A-872;
M-1 to P-871; M-1 to Q-870; M-1 to G-869; M-1 to N-868; M-1 to
I-867; M-1 to D-866; M-1 to R-865; M-1 to C-864; M-1 to E-863; M-1
to V-862; M-1 to L-861; M-1 to R-860; M-1 to R-859; M-1 to Q-858;
M-1 to W-857; M-1 to G-856; M-1 to L-855; M-1 to E-854; M-1 to
C-853; M-1 to S-852; M-1 to K-851; M-1 to S-850; M-1 to C-849; M-1
to E-848; M-1 to G-847; M-1 to W-846; M-1 to E-845; M-1 to E-844;
M-1 to I-843; M-1 to V-842; M-1 to W-841; M-1 to A-840; M-1 to
S-839; M-1 to F-838; M-1 to T-837; M-1 to P-836; M-1 to I-835; M-1
to A-834; M-1 to N-833; M-1 to F-832; M-1 to S-831; M-1 to E-830;
M-1 to K-829; M-1 to K-828; M-1 to K-827; M-1 to K-826; M-1 to
V-825; M-1 to F-824; M-1 to Y-823; M-1 to T-822; M-1 to Y-821; M-1
to K-820; M-1 to I-819; M-1 to K-818; M-1 to P-817; M-1 to R-816;
M-1 to L-815; M-1 to A-814; M-1 to N-813; M-1 to G-812; M-1 to
V-811; M-1 to T-810; M-1 to L-809; M-1 to V-808; M-1 to Q-807; M-1
to I-806; M-1 to T-805; M-1 to L-804; M-1 to P-803; M-1 to E-802;
M-1 to K-801; M-1 to L-800; M-1 to P-799; M-1 to S-798; M-1 to
F-797; M-1 to S-796; M-1 to R-795; M-1 to I-794; M-1 to R-793; M-1
to E-792; M-1 to L-791; M-1 to A-790; M-1 to A-789; M-1 to S-788;
M-1 to S-787; M-1 to G-786; M-1 to S-785; M-1 to Y-784; M-1 to
R-783; M-1 to L-782; M-1 to V-781; M-1 to V-780; M-1 to G-779; M-1
to K-778; M-1 to Y-777; M-1 to M-776; M-1 to I-775; M-1 to D-774;
M-1 to Q-773; M-1 to E-772; M-1 to L-771; M-1 to T-770; M-1 to
S-769; M-1 to L-768; M-1 to T-767; M-1 to Y-766; M-1 to D-765; M-1
to G-764; M-1 to N-763; M-1 to L-762; M-1 to I-761; M-1 to Y-760;
M-1 to T-759; M-1 to G-758; M-1 to D-757; M-1 to A-756; M-1 to
A-755; M-1 to K-754; M-1 to I-753; M-1 to A-752; M-1 to L-751; M-1
to F-750; M-1 to S-749; M-1 to G-748; M-1 to N-747; M-1 to N-746;
M-1 to R-745; M-1 to S-744; M-1 to G-743; M-1 to R-742; M-1 to
Q-741; M-1 to N-740; M-1 to R-739; M-1 to Q-738; M-1 to K-737; M-1
to V-736; M-1 to E-735; M-1 to I-734; M-1 to N-733; M-1 to T-732;
M-1 to A-731; M-1 to G-730; M-1 to T-729; M-1 to P-728; M-1 to
I-727; M-1 to T-726; M-1 to I-725; M-1 to I-724; M-1 to D-723; M-1
to H-722; M-1 to Y-721; M-1 to G-720; M-1 to P-719; M-1 to K-718;
M-1 to A-717; M-1 to S-716; M-1 to T-715; M-1 to V-714; M-1 to
S-713; M-1 to G-712; M-1 to S-711; M-1 to I-710; M-1 to K-709; M-1
to K-708; M-1 to C-707; M-1 to T-706; M-1 to S-705; M-1 to G-704;
M-1 to N-703; M-1 to G-702; M-1 to G-701; M-1 to C-700; M-1 to
V-699; M-1 to G-698; M-1 to C-697; M-1 to K-696; M-1 to D-695; M-1
to F-694; M-1 to K-693; M-1 to K-692; M-1 to K-691; M-1 to S-690;
M-1 to D-689; M-1 to I-688; M-1 to I-687; M-1 to R-686; M-1 to
D-685; M-1 to C-684; M-1 to G-683; M-1 to A-682; M-1 to K-681; M-1
to V-680; M-1 to C-679; M-1 to Q-678; M-1 to G-677; M-1 to Q-676;
M-1 to V-675; M-1 to C-674; M-1 to V-673; M-1 to S-672; M-1 to
T-671; M-1 to S-670; M-1 to D-669; M-1 to P-668; M-1 to S-667; M-1
to C-666; M-1 to P-665; M-1 to T-664; M-1 to G-663; M-1 to D-662;
M-1 to V-661; M-1 to V-660; M-1 to K-659; M-1 to P-658; M-1 to
Q-657; M-1 to L-656; M-1 to V-655; M-1 to F-654; M-1 to F-653; M-1
to Y-652; M-1 to G-651; M-1 to I-650; M-1 to G-649; M-1 to K-648;
M-1 to A-647; M-1 to Q-646; M-1 to C-645; M-1 to I-644; M-1 to
L-643; M-1 to K-642; M-1 to C-641; M-1 to R-640; M-1 to D-639; M-1
to K-638; M-1 to P-637; M-1 to S-636; M-1 to V-635; M-1 to G-634;
M-1 to A-633; M-1 to Y-632; M-1 to K-631; M-1 to P-630; M-1 to
I-629; M-1 to W-628; M-1 to E-627; M-1 to V-626; M-1 to A-625; M-1
to P-624; M-1 to G-623; M-1 to S-622; M-1 to G-621; M-1 to F-620;
M-1 to S-619; M-1 to A-618; M-1 to K-617; M-1 to S-616; M-1 to
F-615; M-1 to E-614; M-1 to N-613; M-1 to H-612; M-1 to A-611; M-1
to E-610; M-1 to C-609; M-1 to Q-608; M-1 to E-607; M-1 to E-606;
M-1 to R-605; M-1 to F-604; M-1 to T-603; M-1 to K-602; M-1 to
G-601; M-1 to N-600; M-1 to N-599; M-1 to D-598; M-1 to P-597; M-1
to C-596; M-1 to D-595; M-1 to E-594; M-1 to L-593; M-1 to N-592;
M-1 to C-591; M-1 to S-590; M-1 to R-589; M-1 to Y-588; M-1 to
R-587; M-1 to V-586; M-1 to R-585; M-1 to K-584; M-1 to G-583; M-1
to E-582; M-1 to C-581; M-1 to Y-580; M-1 to K-579; M-1 to G-578;
M-1 to G-577; M-1 to N-576; M-1 to K-575; M-1 to P-574; M-1 to
V-573; M-1 to P-572; M-1 to N-571; M-1 to D-570; M-1 to C-569; M-1
to E-568; M-1 to R-567; M-1 to M-566; M-1 to T-565; M-1 to Y-564;
M-1 to Q-563; M-1 to V-562; M-1 to G-561; M-1 to G-560; M-1 to
G-559; M-1 to C-558; M-1 to T-557; M-1 to R-556; M-1 to S-555; M-1
to C-554; M-1 to D-553; M-1 to G-552; M-1 to W-551; M-1 to P-550;
M-1 to G-549; M-1 to W-548; M-1 to M-547; M-1 to G-546; M-1 to
W-545; M-1 to S-544; M-1 to G-543; M-1 to H-542; M-1 to F-541; M-1
to P-540; M-1 to T-539; M-1 to D-538; M-1 to F-537; M-1 to H-536;
M-1 to K-535; M-1 to R-534; M-1 to D-533; M-1 to T-532; M-1 to
K-531; M-1 to N-530; M-1 to V-529; M-1 to C-528; M-1 to K-527; M-1
to G-526; M-1 to N-525; M-1 to I-524; M-1 to C-523; M-1 to W-522;
M-1 to K-521; M-1 to G-520; M-1 to E-519; M-1 to G-518; M-1 to
C-517; M-1 to S-516; M-1 to T-515; M-1 to G-514; M-1 to D-513; M-1
to A-512; M-1 to W-511; M-1 to P-510; M-1 to F-509; M-1 to H-508;
M-1 to K-507; M-1 to T-506; M-1 to Q-505; M-1 to C-504; M-1 to
V-503; M-1 to L-502; M-1 to V-501; M-1 to G-500; M-1 to G-499; M-1
to S-498; M-1 to T-497; M-1 to G-496; M-1 to T-495; M-1 to C-494;
M-1 to W-493; M-1 to L-492; M-1 to T-491; M-1 to S-490; M-1 to
C-489; M-1 to T-488; M-1 to S-487; M-1 to A-486; M-1 to A-485; M-1
to D-484; M-1 to P-483; M-1 to C-482; M-1 to H-481; M-1 to K-480;
M-1 to S-479; M-1 to D-478; M-1 to E-477; M-1 to G-476; M-1 to
F-475; M-1 to T-474; M-1 to F-473; M-1 to Q-472; M-1 to C-471; M-1
to Q-470; M-1 to R-469; M-1 to N-468; M-1 to A-467; M-1 to D-466;
M-1 to Y-465; M-1 to S-464; M-1 to T-463; M-1 to G-462; M-1 to
P-461; M-1 to L-460; M-1 to D-459; M-1 to G-458; M-1 to P-457; M-1
to L-456; M-1 to Q-455; M-1 to 1-454; M-1 to P-453; M-1 to N-452;
M-1 to Q-451; M-1 to P-450; M-1 to K-449; M-1 to D-448; M-1 to
M-447; M-1 to L-446; M-1 to C-445; M-1 to E-444; M-1 to G-443; M-1
to H-442; M-1 to G-441; M-1 to N-440; M-1 to D-439; M-1 to L-438;
M-1 to F-437; M-1 to S-436; M-1 to T-435; M-1 to I-434; M-1 to
M-433; M-1 to Y-432; M-1 to A-431; M-1 to S-430; M-1 to C-429; M-1
to P-428; M-1 to S-427; M-1 to W-426; M-1 to P-425; M-1 to Q-424;
M-1 to S-423; M-1 to H-422; M-1 to D-421; M-1 to L-420; M-1 to
N-419; M-1 to S-418; M-1 to L-417; M-1 to M-416; M-1 to S-415; M-1
to A-414; M-1 to M-413; M-1 to M-412; M-1 to H-411; M-1 to S-410;
M-1 to D-409; M-1 to Q-408; M-1 to N-407; M-1 to V-406; M-1 to
G-405; M-1 to N-404; M-1 to L-403; M-1 to S-402; M-1 to A-401; M-1
to C-400; M-1 to Q-399; M-1 to K-398; M-1 to A-397; M-1 to D-396;
M-1 to D-395; M-1 to H-394; M-1 to P-393; M-1 to M-392; M-1 to
N-391; M-1 to F-390; M-1 to V-389; M-1 to H-388; M-1 to G-387; M-1
to L-386; M-1 to E-385; M-1 to H-384; M-1 to A-383; M-1 to T-382;
M-1 to T-381; M-1 to F-380; M-1 to A-379; M-1 to A-378; M-1 to
Q-377; M-1 to L-376; M-1 to G-375; M-1 to D-374; M-1 to D-373; M-1
to E-372; M-1 to I-371; M-1 to V-370; M-1 to S-369; M-1 to C-368;
M-1 to S-367; M-1 to R-366; M-1 to S-365; M-1 to P-364; M-1 to
D-363; M-1 to C-362; M-1 to V-361; M-1 to T-360; M-1 to G-359; M-1
to V-358; M-1 to D-357; M-1 to A-356; M-1 to M-355; M-1 to G-354;
M-1 to L-353; M-1 to T-352; M-1 to D-351; M-1 to C-350; M-1 to
T-349; M-1 to Q-348; M-1 to S-347; M-1 to G-346; M-1 to C-345; M-1
to L-344; M-1 to D-343; M-1 to Q-342; M-1 to R-341; M-1 to T-340;
M-1 to F-339; M-1 to L-338; M-1 to I-337; M-1 to A-336; M-1 to
T-335; M-1 to D-334; M-1 to Y-333; M-1 to H-332; M-1 to E-331; M-1
to A-330; M-1 to D-329; M-1 to R-328; M-1 to D-327; M-1 to S-326;
M-1 to P-325; M-1 to P-324; M-1 to N-323; M-1 to H-322; M-1 to
Q-321; M-1 to K-320; M-1 to Q-319; M-1 to W-318; M-1 to N-317; M-1
to C-316; M-1 to F-315; M-1 to N-314; M-1 to R-313; M-1 to L-312;
M-1 to T-311; M-1 to L-310; M-1 to A-309; M-1 to A-308; M-1 to
N-307; M-1 to S-306; M-1 to T-305; M-1 to V-304; M-1 to E-303; M-1
to P-302; M-1 to G-301; M-1 to K-300; M-1 to Q-299; M-1 to E-298;
M-1 to D-297; M-1 to H-296; M-1 to I-295; M-1 to V-294; M-1 to
L-293; M-1 to I-292; M-1 to K-291; M-1 to V-290; M-1 to V-289; M-1
to V-288; M-1 to L-287; M-1 to S-286; M-1 to V-285; M-1 to S-284;
M-1 to N-283; M-1 to R-282; M-1 to I-281; M-1 to S-280; M-1 to
P-279; M-1 to H-278; M-1 to K-277; M-1 to Y-276; M-1 to L-275; M-1
to R-274; M-1 to A-273; M-1 to A-272; M-1 to V-271; M-1 to S-270;
M-1 to F-269; M-1 to L-268; M-1 to T-267; M-1 to L-266; M-1 to
L-265; M-1 to Y-264; M-1 to H-263; M-1 to K-262; M-1 to L-261; M-1
to G-260; M-1 to S-259; M-1 to G-258; M-1 to H-257; M-1 to F-256;
M-1 to E-255; M-1 to A-254; M-1 to M-253; M-1 to S-252; M-1 to
Q-251; M-1 to D-250; M-1 to A-249; M-1 to V-248; M-1 to L-247; M-1
to M-246; M-1 to T-245; M-1 to E-244; M-1 to V-243; M-1 to Y-242;
M-1 to R-241; M-1 to H-240; M-1 to S-239; M-1 to S-238; M-1 to
V-237; M-1 to F-236; M-1 to R-235; M-1 to K-234; M-1 to K-233; M-1
to R-232; M-1 to I-231; M-1 to S-230; M-1 to G-229; M-1 to T-228;
M-1 to G-227; M-1 to T-226; M-1 to P-225; M-1 to Q-224; M-1 to
G-223; M-1 to V-222; M-1 to G-221; M-1 to Q-220; M-1 to L-219; M-1
to A-218; M-1 to P-217; M-1 to D-216; M-1 to Q-215; M-1 to P-214;
M-1 to S-213; M-1 to W-212; M-1 to Q-211; M-1 to P-210; M-1 to
G-209; M-1 to E-208; M-1 to D-207; M-1 to E-206; M-1 to G-205; M-1
to E-204; M-1 to T-203; M-1 to G-202; M-1 to E-201; M-1 to D-200;
M-1 to E-199; M-1 to D-198; M-1 to E-197; M-1 to T-196; M-1 to
E-195; M-1 to A-194; M-1 to K-193; M-1 to G-192; M-1 to T-191; M-1
to P-190; M-1 to R-189; M-1 to P-188; M-1 to E-187; M-1 to D-186;
M-1 to D-185; M-1 to V-184; M-1 to V-183; M-1 to G-182; M-1 to
C-181; M-1 to T-180; M-1 to G-179; M-1 to G-178; M-1 to V-177; M-1
to D-176; M-1 to G-175; M-1 to Q-174; M-1 to R-173; M-1 to N-172;
M-1 to R-171; M-1 to R-170; M-1 to L-169; M-1 to L-168; M-1 to
H-167; M-1 to F-166; M-1 to Q-165; M-1 to L-164; M-1 to P-163; M-1
to A-162; M-1 to P-161; M-1 to P-160; M-1 to K-159; M-1 to E-158;
M-1 to G-157; M-1 to P-156; M-1 to A-155; M-1 to A-154; M-1 to
T-153; M-1 to A-152; M-1 to L-151; M-1 to R-150; M-1 to E-149; M-1
to S-148; M-1 to A-147; M-1 to A-146; M-1 to P-145; M-1 to L-144;
M-1 to P-143; M-1 to Q-142; M-1 to I-141; M-1 to F-140; M-1 to
Y-139; M-1 to A-138; M-1 to E-137; M-1 to G-136; M-1 to L-135; M-1
to L-134; M-1 to Y-133; M-1 to F-132; M-1 to A-131; M-1 to G-130;
M-1 to R-129; M-1 to V-128; M-1 to G-127; M-1 to E-126; M-1 to
C-125; M-1 to L-124; M-1 to S-123; M-1 to L-122; M-1 to A-121; M-1
to A-120; M-1 to A-119; M-1 to S-118; M-1 to S-117; M-1 to P-116;
M-1 to D-115; M-1 to G-114; M-1 to N-113; M-1 to V-112; M-1 to
T-111; M-1 to G-110; M-1 to S-109; M-1 to Y-108; M-1 to F-107; M-1
to C-106; M-1 to H-105; M-1 to A-104; M-1 to L-103; M-1 to D-102;
M-1 to T-101; M-1 to E-100; M-1 to P-99; M-1 to L-98; M-1 to P-97;
M-1 to T-96; M-1 to E-95; M-1 to S-94; M-1 to G-93; M-1 to S-92;
M-1 to K-91; M-1 to R-90; M-1 to G-89; M-1 to V-88; M-1 to N-87;
M-1 to Q-86; M-1 to L-85; M-1 to T-84; M-1 to F-83; M-1 to G-82;
M-1 to P-81; M-1 to A-80; M-1 to L-79; M-1 to F-78; M-1 to S-77;
M-1 to S-76; M-1 to D-75; M-1 to P-74; M-1 to R-73; M-1 to L-72;
M-1 to E-71; M-1 to L-70; M-1 to D-69; M-1 to L-68; M-1 to Q-67;
M-1 to Q-66; M-1 to D-65; M-1 to F-64; M-1 to A-63; M-1 to H-62;
M-1 to L-61; M-1 to R-60; M-1 to L-59; M-1 to R-58; M-1 to T-57;
M-1 to T-56; M-1 to G-55; M-1 to H-54; M-1 to G-53; M-1 to P-52;
M-1 to A-51; M-1 to R-50; M-1 to E-49; M-1 to L-48; M-1 to E-47;
M-1 to P-46; M-1 to V-45; M-1 to V-44; M-1 to L-43; M-1 to E-42;
M-1 to E-41; M-1 to D-40; M-1 to E-39; M-1 to E-38; M-1 to S-37;
M-1 to P-36; M-1 to R-35; M-1 to G-34; M-1 to L-33; M-1 to A-32;
M-1 to D-31; M-1 to S-30; M-1 to V-29; M-1 to A-28; M-1 to L-27;
M-1 to L-26; M-1 to A-25; M-1 to A-24; M-1 to A-23; M-1 to L-22;
M-1 to L-21; M-1 to L-20; M-1 to L-19; M-1 to T-18; M-1 to P-17;
M-1 to V-16; M-1 to P-15; M-1 to G-14; M-1 to F-13; M-1 to S-12;
M-1 to R-11; M-1 to S-10; M-1 to G-9; M-1 to P-8; M-1 to A-7; of
SEQ ID NO:2. For example, any of the above listed N- or C-terminal
deletions can be combined to produce a N- and C-terminal deleted
METH1 polypeptide. Particularly preferred fragment of SEQ ID NO2
are H542-Q894 and K801-S950.
[0153] Deletion mutants of METH1 may also be made which comprise
all or part of the additional sequence described in SEQ ID NO:126.
For example, exemplary deletion mutants include: Q-2 to S-967; R-3
to S-967; A-4 to S-967; V-5 to S-967; P-6 to S-967; E-7 to S-967;
G-8 to S-967; F-9 to S-967; G-10 to S-967; R-11 to S-976; R-12 to
S-967; K-13 to S-967; L-14 to S-967; G-15 to S-967; S-16 to S-967;
D-17 to S-967; and M-18 to S-967.
[0154] Moreover, N-terminal deletions of the METH2 polypeptide can
be described by the general formula m.sub.2-890, where m.sub.2 is
an integer from 2 to 889, where m corresponds to the position of
the amino acid residue identified in SEQ ID NO:4. Preferably,
N-terminal deletions of the METH2 polypeptide of the invention
shown as SEQ ID NO:4 include polypeptides comprising the amino acid
sequence of residues: F-2 to L-890; P-3 to L-890; A-4 to L-890; P-5
to L-890; A-6 to L-890; A-7 to L-890; P-8 to L-890; R-9 to L-890;
W-10 to L-890; L-11 to L-890; P-12 to L-890; F-13 to L-890; L-14 to
L-890; L-15 to L-890; L-16 to L-890; L-17 to L-890; L-18 to L-890;
L-19 to L-890; L-20 to L-890; L-21 to L-890; L-22 to L-890; P-23 to
L-890; L-24 to L-890; A-25 to L-890; R-26 to L-890; G-27 to L-890;
A-28 to L-890; P-29 to L-890; A-30 to L-890; R-31 to L-890; P-32 to
L-890; A-33 to L-890; A-34 to L-890; G-35 to L-890; G-36 to L-890;
Q-37 to L-890; A-38 to L-890; S-39 to L-890; E-40 to L-890; L-41 to
L-890; V-42 to L-890; V-43 to L-890; P-44 to L-890; T-45 to L-890;
R-46 to L-890; L-47 to L-890; P-48 to L-890; G-49 to L-890; S-50 to
L-890; A-51 to L-890; G-52 to L-890; E-53 to L-890; L-54 to L-890;
A-55 to L-890; L-56 to L-890; H-57 to L-890; L-58 to L-890; S-59 to
L-890; A-60 to L-890; F-61 to L-890; G-62 to L-890; K-63 to L-890;
G-64 to L-890; F-65 to L-890; V-66 to L-890; L-67 to L-890; R-68 to
L-890; L-69 to L-890; A-70 to L-890; P-71 to L-890; D-72 to L-890;
D-73 to L-890; S-74 to L-890; F-75 to L-890; L-76 to L-890; A-77 to
L-890; P-78 to L-890; E-79 to L-890; F-80 to L-890; K-81 to L-890;
I-82 to L-890; E-83 to L-890; R-84 to L-890; L-85 to L-890; G-86 to
L-890; G-87 to L-890; S-88 to L-890; G-89 to L-890; R-90 to L-890;
A-91 to L-890; T-92 to L-890; G-93 to L-890; G-94 to L-890; E-95 to
L-890; R-96 to L-890; G-97 to L-890; L-98 to L-890; R-99 to L-890;
G-100 to L-890; C-101 to L-890; F-102 to L-890; F-103 to L-890;
S-104 to L-890; G-105 to L-890; T-106 to L-890; V-107 to L-890;
N-108 to L-890; G-109 to L-890; E-110 to L-890; P-111 to L-890;
E-112 to L-890; S-113 to L-890; L-114 to L-890; A-115 to L-890;
A-116 to L-890; V-117 to L-890; S-118 to L-890; L-119 to L-890;
C-120 to L-890; R-121 to L-890; G-122 to L-890; L-123 to L-890;
S-124 to L-890; G-125 to L-890; S-126 to L-890; F-127 to L-890;
L-128 to L-890; L-129 to L-890; D-130 to L-890; G-131 to L-890;
E-132 to L-890; E-133 to L-890; F-134 to L-890; T-135 to L-890;
I-136 to L-890; Q-137 to L-890; P-138 to L-890; Q-139 to L-890;
G-140 to L-890; A-141 to L-890; G-142 to L-890; G-143 to L-890;
S-144 to L-890; L-145 to L-890; A-146 to L-890; Q-147 to L-890;
P-148 to L-890; H-149 to L-890; R-150 to L-890; L-151 to L-890;
Q-152 to L-890; R-153 to L-890; W-154 to L-890; G-155 to L-890;
P-156 to L-890; A-157 to L-890; G-158 to L-890; A-159 to L-890;
R-160 to L-890; P-161 to L-890; L-162 to L-890; P-163 to L-890;
R-164 to L-890; G-165 to L-890; P-166 to L-890; E-167 to L-890;
W-168 to L-890; E-169 to L-890; V-170 to L-890; E-171 to L-890;
T-172 to L-890; G-173 to L-890; E-174 to L-890; G-175 to L-890;
Q-176 to L-890; R-177 to L-890; Q-178 to L-890; E-179 to L-890;
R-180 to L-890; G-181 to L-890; D-182 to L-890; H-183 to L-890;
Q-184 to L-890; E-185 to L-890; D-186 to L-890; S-187 to L-890;
E-188 to L-890; E-189 to L-890; E-190 to L-890; S-191 to L-890;
Q-192 to L-890; E-193 to L-890; E-194 to L-890; E-195 to L-890;
A-196 to L-890; E-197 to L-890; G-198 to L-890; A-199 to L-890;
S-200 to L-890; E-201 to L-890; P-202 to L-890; P-203 to L-890;
P-204 to L-890; P-205 to L-890; L-206 to L-890; G-207 to L-890;
A-208 to L-890; T-209 to L-890; S-210 to L-890; R-211 to L-890;
T-212 to L-890; K-213 to L-890; R-214 to L-890; F-215 to L-890;
V-216 to L-890; S-217 to L-890; E-218 to L-890; A-219 to L-890;
R-220 to L-890; F-221 to L-890; V-222 to L-890; E-223 to L-890;
T-224 to L-890; L-225 to L-890; L-226 to L-890; V-227 to L-890;
A-228 to L-890; D-229 to L-890; A-230 to L-890; S-231 to L-890;
M-232 to L-890; A-233 to L-890; A-234 to L-890; F-235 to L-890;
Y-236 to L-890; G-237 to L-890; A-238 to L-890; D-239 to L-890;
L-240 to L-890; Q-241 to L-890; N-242 to L-890; H-243 to L-890;
I-244 to L-890; L-245 to L-890; T-246 to L-890; L-247 to L-890;
M-248 to L-890; S-249 to L-890; V-250 to L-890; A-251 to L-890;
A-252 to L-890; R-253 to L-890; I-254 to L-890; Y-255 to L-890;
K-256 to L-890; H-257 to L-890; P-258 to L-890; S-259 to L-890;
I-260 to L-890; K-261 to L-890; N-262 to L-890; S-263 to L-890;
I-264 to L-890; N-265 to L-890; L-266 to L-890; M-267 to L-890;
V-268 to L-890; V-269 to L-890; K-270 to L-890; V-271 to L-890;
L-272 to L-890; I-273 to L-890; V-274 to L-890; E-275 to L-890;
D-276 to L-890; E-277 to L-890; K-278 to L-890; W-279 to L-890;
G-280 to L-890; P-281 to L-890; E-282 to L-890; V-283 to L-890;
S-284 to L-890; D-285 to L-890; N-286 to L-890; G-287 to L-890;
G-288 to L-890; L-289 to L-890; T-290 to L-890; L-291 to L-890;
R-292 to L-890; N-293 to L-890; F-294 to L-890; C-295 to L-890;
N-296 to L-890; W-297 to L-890; Q-298 to L-890; R-299 to L-890;
R-300 to L-890; F-301 to L-890; N-302 to L-890; Q-303 to L-890;
P-304 to L-890; S-305 to L-890; D-306 to L-890; R-307 to L-890;
H-308 to L-890; P-309 to L-890; E-310 to L-890; H-311 to L-890;
Y-312 to L-890; D-313 to L-890; T-314 to L-890; A-315 to L-890;
I-316 to L-890; L-317 to L-890; L-318 to L-890; T-319 to L-890;
R-320 to L-890; Q-321 to L-890; N-322 to L-890; F-323 to L-890;
C-324 to L-890; G-325 to L-890; Q-326 to L-890; E-327 to L-890;
G-328 to L-890; L-329 to L-890; C-330 to L-890; D-331 to L-890;
T-332 to L-890; L-333 to L-890; G-334 to L-890; V-335 to L-890;
A-336 to L-890; D-337 to L-890; I-338 to L-890; G-339 to L-890;
T-340 to L-890; I-341 to L-890; C-342 to L-890; D-343 to L-890;
P-344 to L-890; N-345 to L-890; K-346 to L-890; S-347 to L-890;
C-348 to L-890; S-349 to L-890; V-350 to L-890; I-351 to L-890;
E-352 to L-890; D-353 to L-890; E-354 to L-890; G-355 to L-890;
L-356 to L-890; Q-357 to L-890; A-358 to L-890; A-359 to L-890;
H-360 to L-890; T-361 to L-890; L-362 to L-890; A-363 to L-890;
H-364 to L-890; E-365 to L-890; L-366 to L-890; G-367 to L-890;
H-368 to L-890; V-369 to L-890; L-370 to L-890; S-371 to L-890;
M-372 to L-890; P-373 to L-890; H-374 to L-890; D-375 to L-890;
D-376 to L-890; S-377 to L-890; K-378 to L-890; P-379 to L-890;
C-380 to L-890; T-381 to L-890; R-382 to L-890; L-383 to L-890;
F-384 to L-890; G-385 to L-890; P-386 to L-890; M-387 to L-890;
G-388 to L-890; K-389 to L-890; H-390 to L-890; H-391 to L-890;
V-392 to L-890; M-393 to L-890; A-394 to L-890; P-395 to L-890;
L-396 to L-890; F-397 to L-890; V-398 to L-890; H-399 to L-890;
L-400 to L-890; N-401 to L-890; Q-402 to L-890; T-403 to L-890;
L-404 to L-890; P-405 to L-890; W-406 to L-890; S-407 to L-890;
P-408 to L-890; C-409 to L-890; S-410 to L-890; A-411 to L-890;
M-412 to L-890; Y-413 to L-890; L-414 to L-890; T-415 to L-890;
E-416 to L-890; L-417 to L-890; L-418 to L-890; D-419 to L-890;
G-420 to L-890; G-421 to L-890; H-422 to L-890; G-423 to L-890;
D-424 to L-890; C-425 to L-890; L-426 to L-890; L-427 to L-890;
D-428 to L-890; A-429 to L-890; P-430 to L-890; G-431 to L-890;
A-432 to L-890; A-433 to L-890; L-434 to L-890; P-435 to L-890;
L-436 to L-890; P-437 to L-890; T-438 to L-890; G-439 to L-890;
L-440 to L-890; P-441 to L-890; G-442 to L-890; R-443 to L-890;
M-444 to L-890; A-445 to L-890; L-446 to L-890; Y-447 to L-890;
Q-448 to L-890; L-449 to L-890; D-450 to L-890; Q-451 to L-890;
Q-452 to L-890; C-453 to L-890; R-454 to L-890; Q-455 to L-890;
I-456 to L-890; F-457 to L-890; G-458 to L-890; P-459 to L-890;
D-460 to L-890; F-461 to L-890; R-462 to L-890; H-463 to L-890;
C-464 to L-890; P-465 to L-890; N-466 to L-890; T-467 to L-890;
S-468 to L-890; A-469 to L-890; Q-470 to L-890; D-471 to L-890;
V-472 to L-890; C-473 to L-890; A-474 to L-890; Q-475 to L-890;
L-476 to L-890; W-477 to L-890; C-478 to L-890; H-479 to L-890;
T-480 to L-890; D-481 to L-890; G-482 to L-890; A-483 to L-890;
E-484 to L-890; P-485 to L-890; L-486 to L-890; C-487 to L-890;
H-488 to L-890; T-489 to L-890; K-490 to L-890; N-491 to L-890;
G-492 to L-890; S-493 to L-890; L-494 to L-890; P-495 to L-890;
W-496 to L-890; A-497 to L-890; D-498 to L-890; G-499 to L-890;
T-500 to L-890; P-501 to L-890; C-502 to L-890; G-503 to L-890;
P-504 to L-890; G-505 to L-890; H-506 to L-890; L-507 to L-890;
C-508 to L-890; S-509 to L-890; E-510 to L-890; G-511 to L-890;
S-512 to L-890; C-513 to L-890; L-514 to L-890; P-515 to L-890;
E-516 to L-890; E-517 to L-890; E-518 to L-890; V-519 to L-890;
E-520 to L-890; R-521 to L-890; P-522 to L-890; K-523 to L-890;
P-524 to L-890; V-525 to L-890; V-526 to L-890; D-527 to L-890;
G-528 to L-890; G-529 to L-890; W-530 to L-890; A-531 to L-890;
P-532 to L-890; W-533 to L-890; G-534 to L-890; P-535 to L-890;
W-536 to L-890; G-537 to L-890; E-538 to L-890; C-539 to L-890;
S-540 to L-890; R-541 to L-890; T-542 to L-890; C-543 to L-890;
G-544 to L-890; G-545 to L-890; G-546 to L-890; V-547 to L-890;
Q-548 to L-890; F-549 to L-890; S-550 to L-890; H-551 to L-890;
R-552 to L-890; E-553 to L-890; C-554 to L-890; K-555 to L-890;
D-556 to L-890; P-557 to L-890; E-558 to L-890; P-559 to L-890;
Q-560 to L-890; N-561 to L-890; G-562 to L-890; G-563 to L-890;
R-564 to L-890; Y-565 to L-890; C-566 to L-890; L-567 to L-890;
G-568 to L-890; R-569 to L-890; R-570 to L-890; A-571 to L-890;
K-572 to L-890; Y-573 to L-890; Q-574 to L-890; S-575 to L-890;
C-576 to L-890; H-577 to L-890; T-578 to L-890; E-579 to L-890;
E-580 to L-890; C-581 to L-890; P-582 to L-890; P-583 to L-890;
D-584 to L-890; G-585 to L-890; K-586 to L-890; S-587 to L-890;
F-588 to L-890; R-589 to L-890; E-590 to L-890; Q-591 to L-890;
Q-592 to L-890; C-593 to L-890; E-594 to L-890; K-595 to L-890;
Y-596 to L-890; N-597 to L-890; A-598 to L-890; Y-599 to L-890;
N-600 to L-890; Y-601 to L-890; T-602 to L-890; D-603 to L-890;
M-604 to L-890; D-605 to L-890; G-606 to L-890; N-607 to L-890;
L-608 to L-890; L-609 to L-890; Q-610 to L-890; W-611 to L-890;
V-612 to L-890; P-613 to L-890; K-614 to L-890; Y-615 to L-890;
A-616 to L-890; G-617 to L-890; V-618 to L-890; S-619 to L-890;
P-620 to L-890; R-621 to L-890; D-622 to L-890; R-623 to L-890;
C-624 to L-890; K-625 to L-890; L-626 to L-890; F-627 to L-890;
C-628 to L-890; R-629 to L-890; A-630 to L-890; R-631 to L-890;
G-632 to L-890; R-633 to L-890; S-634 to L-890; E-635 to L-890;
F-636 to L-890; K-637 to L-890; V-638 to L-890; F-639 to L-890;
E-640 to L-890; A-641 to L-890; K-642 to L-890; V-643 to L-890;
I-644 to L-890; D-645 to L-890; G-646 to L-890; T-647 to L-890;
L-648 to L-890; C-649 to L-890; G-650 to L-890; P-651 to L-890;
E-652 to L-890; T-653 to L-890; L-654 to L-890; A-655 to L-890;
I-656 to L-890; C-657 to L-890; V-658 to L-890; R-659 to L-890;
G-660 to L-890; Q-661 to L-890; C-662 to L-890; V-663 to L-890;
K-664 to L-890; A-665 to L-890; G-666 to L-890; C-667 to L-890;
D-668 to L-890; H-669 to L-890; V-670 to L-890; V-671 to L-890;
D-672 to L-890; S-673 to L-890; P-674 to L-890; R-675 to L-890;
K-676 to L-890; L-677 to L-890; D-678 to L-890; K-679 to L-890;
C-680 to L-890; G-681 to L-890; V-682 to L-890; C-683 to L-890;
G-684 to L-890; G-685 to L-890; K-686 to L-890; G-687 to L-890;
N-688 to L-890; S-689 to L-890; C-690 to L-890; R-691 to L-890;
K-692 to L-890; V-693 to L-890; S-694 to L-890; G-695 to L-890;
S-696 to L-890; L-697 to L-890; T-698 to L-890; P-699 to L-890;
T-700 to L-890; N-701 to L-890; Y-702 to L-890; G-703 to L-890;
Y-704 to L-890; N-705 to L-890; D-706 to L-890; I-707 to L-890;
V-708 to L-890; T-709 to L-890; I-710 to L-890; P-711 to L-890;
A-712 to L-890; G-713 to L-890; A-714 to L-890; T-715 to L-890;
N-716 to L-890; I-717 to L-890; D-718 to L-890; V-719 to L-890;
K-720 to L-890; Q-721 to L-890; R-722 to L-890; S-723 to L-890;
H-724 to L-890; P-725 to L-890; G-726 to L-890; V-727 to L-890;
Q-728 to L-890; N-729 to L-890; D-730 to L-890; G-731 to L-890;
N-732 to L-890; Y-733 to L-890; L-734 to L-890; A-735 to L-890;
L-736 to L-890; K-737 to L-890; T-738 to L-890; A-739 to L-890;
D-740 to L-890; G-741 to L-890; Q-742 to L-890; Y-743 to L-890;
L-744 to L-890; L-745 to L-890; N-746 to L-890; G-747 to L-890;
N-748 to L-890; L-749 to L-890; A-750 to L-890; I-751 to L-890;
S-752 to L-890; A-753 to L-890; I-754 to L-890; E-755 to L-890;
Q-756 to L-890; D-757 to L-890; I-758 to L-890; L-759 to L-890;
V-760 to L-890; K-761 to L-890; G-762 to L-890; T-763 to L-890;
I-764 to L-890; L-765 to L-890; K-766 to L-890; Y-767 to L-890;
S-768 to L-890; G-769 to L-890; S-770 to L-890; I-771 to L-890;
A-772 to L-890; T-773 to L-890; L-774 to L-890; E-775 to L-890;
R-776 to L-890; L-777 to L-890; Q-778 to L-890; S-779 to L-890;
F-780 to L-890; R-781 to L-890; P-782 to L-890; L-783 to L-890;
P-784 to L-890; E-785 to L-890; P-786 to L-890; L-787 to L-890;
T-788 to L-890; V-789 to L-890; Q-790 to L-890; L-791 to L-890;
L-792 to L-890; T-793 to L-890; V-794 to L-890; P-795 to L-890;
G-796 to L-890; E-797 to L-890; V-798 to L-890; F-799 to L-890;
P-800 to L-890; P-801 to L-890; K-802 to L-890; V-803 to L-890;
K-804 to L-890; Y-805 to L-890; T-806 to L-890; F-807 to L-890;
F-808 to L-890; V-809 to L-890; P-810 to L-890; N-811 to L-890;
D-812 to L-890; V-813 to L-890; D-814 to L-890; F-815 to L-890;
S-816 to L-890; M-817 to L-890; Q-818 to L-890; S-819 to L-890;
S-820 to L-890; K-821 to L-890; E-822 to L-890; R-823 to L-890;
A-824 to L-890; T-825 to L-890; T-826 to L-890; N-827 to L-890;
I-828 to L-890; 1-829 to L-890; Q-830 to L-890; P-831 to L-890;
L-832 to L-890; L-833 to L-890; H-834 to L-890; A-835 to L-890;
Q-836 to L-890; W-837 to L-890; V-838 to L-890; L-839 to L-890;
G-840 to L-890; D-841 to L-890; W-842 to L-890; S-843 to L-890;
E-844 to L-890; C-845 to L-890; S-846 to L-890; S-847 to L-890;
T-848 to L-890; C-849 to L-890; G-850 to L-890; A-851 to L-890;
G-852 to L-890; W-853 to L-890; Q-854 to L-890; R-855 to L-890;
R-856 to L-890; T-857 to L-890; V-858 to L-890; E-859 to L-890;
C-860 to L-890; R-861 to L-890; D-862 to L-890; P-863 to L-890;
S-864 to L-890; G-865 to L-890; Q-866 to L-890; A-867 to L-890;
S-868 to L-890; A-869 to L-890; T-870 to L-890; C-871 to L-890;
N-872 to L-890; K-873 to L-890; A-874 to L-890; L-875 to L-890;
K-876 to L-890; P-877 to L-890; E-878 to L-890; D-879 to L-890;
A-880 to L-890; K-881 to L-890; P-882 to L-890; C-883 to L-890;
E-884 to L-890; S-885 to L-890; of SEQ ID NO:4.
[0155] Moreover, C-terminal deletions of the METH2 polypeptide can
also be described by the general formula 1-n.sub.2, where n.sub.2
is an integer from 2 to 890 where n corresponds to the position of
amino acid residue identified in SEQ ID NO:4. Preferably,
C-terminal deletions of the METH2 polypeptide of the invention
shown as SEQ ID NO:4 include polypeptides comprising the amino acid
sequence of residues: M-1 to P-889; M-1 to C-888; M-1 to L-887; M-1
to Q-886; M-1 to S-885; M-1 to E-884; M-1 to C-883; M-1 to P-882;
M-1 to K-881; M-1 to A-880; M-1 to D-879; M-1 to E-878; M-1 to
P-877; M-1 to K-876; M-1 to L-875; M-1 to A-874; M-1 to K-873; M-1
to N-872; M-1 to C-871; M-1 to T-870; M-1 to A-869; M-1 to S-868;
M-1 to A-867; M-1 to Q-866; M-1 to G-865; M-1 to S-864; M-1 to
P-863; M-1 to D-862; M-1 to R-861; M-1 to C-860; M-1 to E-859; M-1
to V-858; M-1 to T-857; M-1 to R-856; M-1 to R-855; M-1 to Q-854;
M-1 to W-853; M-1 to G-852; M-1 to A-851; M-1 to G-850; M-1 to
C-849; M-1 to T-848; M-1 to S-847; M-1 to S-846; M-1 to C-845; M-1
to E-844; M-1 to S-843; M-1 to W-842; M-1 to D-841; M-1 to G-840;
M-1 to L-839; M-1 to V-838; M-1 to W-837; M-1 to Q-836; M-1 to
A-835; M-1 to H-834; M-1 to L-833; M-1 to L-832; M-1 to P-831; M-1
to Q-830; M-1 to I-829; M-1 to I-828; M-1 to N-827; M-1 to T-826;
M-1 to T-825; M-1 to A-824; M-1 to R-823; M-1 to E-822; M-1 to
K-821; M-1 to S-820; M-1 to S-819; M-1 to Q-818; M-1 to M-817; M-1
to S-816; M-1 to F-815; M-1 to D-814; M-1 to V-813; M-1 to D-812;
M-1 to N-811; M-1 to P-810; M-1 to V-809; M-1 to F-808; M-1 to
F-807; M-1 to T-806; M-1 to Y-805; M-1 to K-804; M-1 to V-803; M-1
to K-802; M-1 to P-801; M-1 to P-800; M-1 to F-799; M-1 to V-798;
M-1 to E-797; M-1 to G-796; M-1 to P-795; M-1 to V-794; M-1 to
T-793; M-1 to L-792; M-1 to L-791; M-1 to Q-790; M-1 to V-789; M-1
to T-788; M-1 to L-787; M-1 to P-786; M-1 to E-785; M-1 to P-784;
M-1 to L-783; M-1 to P-782; M-1 to R-781; M-1 to F-780; M-1 to
S-779; M-1 to Q-778; M-1 to L-777; M-1 to R-776; M-1 to E-775; M-1
to L-774; M-1 to T-773; M-1 to A-772; M-1 to I-771; M-1 to S-770;
M-1 to G-769; M-1 to S-768; M-1 to Y-767; M-1 to K-766; M-1 to
L-765; M-1 to I-764; M-1 to T-763; M-1 to G-762; M-1 to K-761; M-1
to V-760; M-1 to L-759; M-1 to I-758; M-1 to D-757; M-1 to Q-756;
M-1 to E-755; M-1 to I-754; M-1 to A-753; M-1 to S-752; M-1 to
I-751; M-1 to A-750; M-1 to L-749; M-1 to N-748; M-1 to G-747; M-1
to N-746; M-1 to L-745; M-1 to L-744; M-1 to Y-743; M-1 to Q-742;
M-1 to G-741; M-1 to D-740; M-1 to A-739; M-1 to T-738; M-1 to
K-737; M-1 to L-736; M-1 to A-735; M-1 to L-734; M-1 to Y-733; M-1
to N-732; M-1 to G-731; M-1 to D-730; M-1 to N-729; M-1 to Q-728;
M-1 to V-727; M-1 to G-726; M-1 to P-725; M-1 to H-724; M-1 to
S-723; M-1 to R-722; M-1 to Q-721; M-1 to K-720; M-1 to V-719; M-1
to D-718; M-1 to I-717; M-1 to N-716; M-1 to T-715; M-1 to A-714;
M-1 to G-713; M-1 to A-712; M-1 to P-711; M-1 to I-710; M-1 to
T-709; M-1 to V-708; M-1 to I-707; M-1 to D-706; M-1 to N-705; M-1
to Y-704; M-1 to G-703; M-1 to Y-702; M-1 to N-701; M-1 to T-700;
M-1 to P-699; M-1 to T-698; M-1 to L-697; M-1 to S-696; M-1 to
G-695; M-1 to S-694; M-1 to V-693; M-1 to K-692; M-1 to R-691; M-1
to C-690; M-1 to S-689; M-1 to N-688; M-1 to G-687; M-1 to K-686;
M-1 to G-685; M-1 to G-684; M-1 to C-683; M-1 to V-682; M-1 to
G-681; M-1 to C-680; M-1 to K-679; M-1 to D-678; M-1 to L-677; M-1
to K-676; M-1 to R-675; M-1 to P-674; M-1 to S-673; M-1 to D-672;
M-1 to V-671; M-1 to V-670; M-1 to H-669; M-1 to D-668; M-1 to
C-667; M-1 to G-666; M-1 to A-665; M-1 to K-664; M-1 to V-663; M-1
to C-662; M-1 to Q-661; M-1 to G-660; M-1 to R-659; M-1 to V-658;
M-1 to C-657; M-1 to I-656; M-1 to A-655; M-1 to L-654; M-1 to
T-653; M-1 to E-652; M-1 to P-651; M-1 to G-650; M-1 to C-649; M-1
to L-648; M-1 to T-647; M-1 to G-646; M-1 to D-645; M-1 to I-644;
M-1 to V-643; M-1 to K-642; M-1 to A-641; M-1 to E-640; M-1 to
F-639; M-1 to V-638; M-1 to K-637; M-1 to F-636; M-1 to E-635; M-1
to S-634; M-1 to R-633; M-1 to G-632; M-1 to R-631; M-1 to A-630;
M-1 to R-629; M-1 to C-628; M-1 to F-627; M-1 to L-626; M-1 to
K-625; M-1 to C-624; M-1 to R-623; M-1 to D-622; M-1 to R-621; M-1
to P-620; M-1 to S-619; M-1 to V-618; M-1 to G-617; M-1 to A-616;
M-1 to Y-615; M-1 to K-614; M-1 to P-613; M-1 to V-612; M-1 to
W-611; M-1 to Q-610; M-1 to L-609; M-1 to L-608; M-1 to N-607; M-1
to G-606; M-1 to D-605; M-1 to M-604; M-1 to D-603; M-1 to T-602;
M-1 to Y-601; M-1 to N-600; M-1 to Y-599; M-1 to A-598; M-1 to
N-597; M-1 to Y-596; M-1 to K-595; M-1 to E-594; M-1 to C-593; M-1
to Q-592; M-1 to Q-591; M-1 to E-590; M-1 to R-589; M-1 to F-588;
M-1 to S-587; M-1 to K-586; M-1 to G-585; M-1 to D-584; M-1 to
P-583; M-1 to P-582; M-1 to C-581; M-1 to E-580; M-1 to E-579; M-1
to T-578; M-1 to H-577; M-1 to C-576; M-1 to S-575; M-1 to Q-574;
M-1 to Y-573; M-1 to K-572; M-1 to A-571; M-1 to R-570; M-1 to
R-569; M-1 to G-568; M-1 to L-567; M-1 to C-566; M-1 to Y-565; M-1
to R-564; M-1 to G-563; M-1 to G-562; M-1 to N-561; M-1 to Q-560;
M-1 to P-559; M-1 to E-558; M-1 to P-557; M-1 to D-556; M-1 to
K-555; M-1 to C-554; M-1 to E-553; M-1 to R-552; M-1 to H-551; M-1
to S-550; M-1 to F-549; M-1 to Q-548; M-1 to V-547; M-1 to G-546;
M-1 to G-545; M-1 to G-544; M-1 to C-543; M-1 to T-542; M-1 to
R-541; M-1 to S-540; M-1 to C-539; M-1 to E-538; M-1 to G-537; M-1
to W-536; M-1 to P-535; M-1 to G-534; M-1 to W-533; M-1 to P-532;
M-1 to A-531; M-1 to W-530; M-1 to G-529; M-1 to G-528; M-1 to
D-527; M-1 to V-526; M-1 to V-525; M-1 to P-524; M-1 to K-523; M-1
to P-522; M-1 to R-521; M-1 to E-520; M-1 to V-519; M-1 to E-518;
M-1 to E-517; M-1 to E-516; M-1 to P-515; M-1 to L-514; M-1 to
C-513; M-1 to S-512; M-1 to G-511; M-1 to E-510; M-1 to S-509; M-1
to C-508; M-1 to L-507; M-1 to H-506; M-1 to G-505; M-1 to P-504;
M-1 to G-503; M-1 to C-502; M-1 to P-501; M-1 to T-500; M-1 to
G-499; M-1 to D-498; M-1 to A-497; M-1 to W-496; M-1 to P-495; M-1
to L-494; M-1 to S-493; M-1 to G-492; M-1 to N-491; M-1 to K-490;
M-1 to T-489; M-1 to H-488; M-1 to C-487; M-1 to L-486; M-1 to
P-485; M-1 to E-484; M-1 to A-483; M-1 to G-482; M-1 to D-481; M-1
to T-480; M-1 to H-479; M-1 to C-478; M-1 to W-477; M-1 to L-476;
M-1 to Q-475; M-1 to A-474; M-1 to C-473; M-1 to V-472; M-1 to
D-471; M-1 to Q-470; M-1 to A-469; M-1 to S-468; M-1 to T-467; M-1
to N-466; M-1 to P-465; M-1 to C-464; M-1 to H-463; M-1 to R-462;
M-1 to F-461; M-1 to D-460; M-1 to P-459; M-1 to G-458; M-1 to
F-457; M-1 to I-456; M-1 to Q-455; M-1 to R-454; M-1 to C-453; M-1
to Q-452; M-1 to Q-451; M-1 to D-450; M-1 to L-449; M-1 to Q-448;
M-1 to Y-447; M-1 to L-446; M-1 to A-445; M-1 to M-444; M-1 to
R-443; M-1 to G-442; M-1 to P-441; M-1 to L-440; M-1 to G-439; M-1
to T-438; M-1 to P-437; M-1 to L-436; M-1 to P-435; M-1 to L-434;
M-1 to A-433; M-1 to A-432; M-1 to G-431; M-1 to P-430; M-1 to
A-429; M-1 to D-428; M-1 to L-427; M-1 to L-426; M-1 to C-425; M-1
to D-424; M-1 to G-423; M-1 to H-422; M-1 to G-421; M-1 to G-420;
M-1 to D-419; M-1 to L-418; M-1 to L-417; M-1 to E-416; M-1 to
T-415; M-1 to L-414; M-1 to Y-413; M-1 to M-412; M-1 to A-411; M-1
to S-410; M-1 to C-409; M-1 to P-408; M-1 to S-407; M-1 to W-406;
M-1 to P-405; M-1 to L-404; M-1 to T-403; M-1 to Q-402; M-1 to
N-401; M-1 to L-400; M-1 to H-399; M-1 to V-398; M-1 to F-397; M-1
to L-396; M-1 to P-395; M-1 to A-394; M-1 to M-393; M-1 to V-392;
M-1 to H-391; M-1 to H-390; M-1 to K-389; M-1 to G-388; M-1 to
M-387; M-1 to P-386; M-1 to G-385; M-1 to F-384; M-1 to L-383; M-1
to R-382; M-1 to T-381; M-1 to C-380; M-1 to P-379; M-1 to K-378;
M-1 to S-377; M-1 to D-376; M-1 to D-375; M-1 to H-374; M-1 to
P-373; M-1 to M-372; M-1 to S-371; M-1 to L-370; M-1 to V-369; M-1
to H-368; M-1 to G-367; M-1 to L-366; M-1 to E-365; M-1 to H-364;
M-1 to A-363; M-1 to L-362; M-1 to T-361; M-1 to H-360; M-1 to
A-359; M-1 to A-358; M-1 to Q-357; M-1 to L-356; M-1 to G-355; M-1
to E-354; M-1 to D-353; M-1 to E-352; M-1 to I-351; M-1 to V-350;
M-1 to S-349; M-1 to C-348; M-1 to S-347; M-1 to K-346; M-1 to
N-345; M-1 to P-344; M-1 to D-343; M-1 to C-342; M-1 to I-341; M-1
to T-340; M-1 to G-339; M-1 to I-338; M-1 to D-337; M-1 to A-336;
M-1 to V-335; M-1 to G-334; M-1 to L-333; M-1 to T-332; M-1 to
D-331; M-1 to C-330; M-1 to L-329; M-1 to G-328; M-1 to E-327; M-1
to Q-326; M-1 to G-325; M-1 to C-324; M-1 to F-323; M-1 to N-322;
M-1 to Q-321; M-1 to R-320; M-1 to T-319; M-1 to L-318; M-1 to
L-317; M-1 to I-316; M-1 to A-315; M-1 to T-314; M-1 to D-313; M-1
to Y-312; M-1 to H-311; M-1 to E-310; M-1 to P-309; M-1 to H-308;
M-1 to R-307; M-1 to D-306; M-1 to S-305; M-1 to P-304; M-1 to
Q-303; M-1 to N-302; M-1 to F-301; M-1 to R-300; M-1 to R-299; M-1
to Q-298; M-1 to W-297; M-1 to N-296; M-1 to C-295; M-1 to F-294;
M-1 to N-293; M-1 to R-292; M-1 to L-291; M-1 to T-290; M-1 to
L-289; M-1 to G-288; M-1 to G-287; M-1 to N-286; M-1 to D-285; M-1
to S-284; M-1 to V-283; M-1 to E-282; M-1 to P-281; M-1 to G-280;
M-1 to W-279; M-1 to K-278; M-1 to E-277; M-1 to D-276; M-1 to
E-275; M-1 to V-274; M-1 to I-273; M-1 to L-272; M-1 to V-271; M-1
to K-270; M-1 to V-269; M-1 to V-268; M-1 to M-267; M-1 to L-266;
M-1 to N-265; M-1 to I-264; M-1 to S-263; M-1 to N-262; M-1 to
K-261; M-1 to I-260; M-1 to S-259; M-1 to P-258; M-1 to H-257; M-1
to K-256; M-1 to Y-255; M-1 to I-254; M-1 to R-253; M-1 to A-252;
M-1 to A-251; M-1 to V-250; M-1 to S-249; M-1 to M-248; M-1 to
L-247; M-1 to T-246; M-1 to L-245; M-1 to I-244; M-1 to H-243; M-1
to N-242; M-1 to Q-241; M-1 to L-240; M-1 to D-239; M-1 to A-238;
M-1 to G-237; M-1 to Y-236; M-1 to F-235; M-1 to A-234; M-1 to
A-233; M-1 to M-232; M-1 to S-231; M-1 to A-230; M-1 to D-229; M-1
to A-228; M-1 to V-227; M-1 to L-226; M-1 to L-225; M-1 to T-224;
M-1 to E-223; M-1 to V-222; M-1 to F-221; M-1 to R-220; M-1 to
A-219; M-1 to E-218; M-1 to S-217; M-1 to V-216; M-1 to F-215; M-1
to R-214; M-1 to K-213; M-1 to T-212; M-1 to R-211; M-1 to S-210;
M-1 to T-209; M-1 to A-208; M-1 to G-207; M-1 to L-206; M-1 to
P-205; M-1 to P-204; M-1 to P-203; M-1 to P-202; M-1 to E-201; M-1
to S-200; M-1 to A-199; M-1 to G-198; M-1 to E-197; M-1 to A-196;
M-1 to E-195; M-1 to E-194; M-1 to E-193; M-1 to Q-192; M-1 to
S-191; M-1 to E-190; M-1 to E-189; M-1 to E-188; M-1 to S-187; M-1
to D-186; M-1 to E-185; M-1 to Q-184; M-1 to H-183; M-1 to D-182;
M-1 to G-181; M-1 to R-180; M-1 to E-179; M-1 to Q-178; M-1 to
R-177; M-1 to Q-176; M-1 to G-175; M-1 to E-174; M-1 to G-173; M-1
to T-172; M-1 to E-171; M-1 to V-170; M-1 to E-169; M-1 to W-168;
M-1 to E-167; M-1 to P-166; M-1 to G-165; M-1 to R-164; M-1 to
P-163; M-1 to L-162; M-1 to P-161; M-1 to R-160; M-1 to A-159; M-1
to G-158; M-1 to A-157; M-1 to P-156; M-1 to G-155; M-1 to W-154;
M-1 to R-153; M-1 to Q-152; M-1 to L-151; M-1 to R-150; M-1 to
H-149; M-1 to P-148; M-1 to Q-147; M-1 to A-146; M-1 to L-145; M-1
to S-144; M-1 to G-143; M-1 to G-142; M-1 to A-141; M-1 to G-140;
M-1 to Q-139; M-1 to P-138; M-1 to Q-137; M-1 to I-136; M-1 to
T-135; M-1 to F-134; M-1 to E-133; M-1 to E-132; M-1 to G-131; M-1
to D-130; M-1 to L-129; M-1 to L-128; M-1 to F-127; M-1 to S-126;
M-1 to G-125; M-1 to S-124; M-1 to L-123; M-1 to G-122; M-1 to
R-121; M-1 to C-120; M-1 to L-119; M-1 to S-118; M-1 to V-117; M-1
to A-116; M-1 to A-115; M-1 to L-114; M-1 to S-113; M-1 to E-112;
M-1 to P-111; M-1 to E-110; M-1 to G-109; M-1 to N-108; M-1 to
V-107; M-1 to T-106; M-1 to G-105; M-1 to S-104; M-1 to F-103; M-1
to F-102; M-1 to C-101; M-1 to G-100; M-1 to R-99; M-1 to L-98; M-1
to G-97; M-1 to R-96; M-1 to E-95; M-1 to G-94; M-1 to G-93; M-1 to
T-92; M-1 to A-91; M-1 to R-90; M-1 to G-89; M-1 to S-88; M-1 to
G-87; M-1 to G-86; M-1 to L-85; M-1 to R-84; M-1 to E-83; M-1 to
I-82; M-1 to K-81; M-1 to F-80; M-1 to E-79; M-1 to P-78; M-1 to
A-77; M-1 to L-76; M-1 to F-75; M-1 to S-74; M-1 to D-73; M-1 to
D-72; M-1 to P-71; M-1 to A-70; M-1 to L-69; M-1 to R-68; M-1 to
L-67; M-1 to V-66; M-1 to F-65; M-1 to G-64; M-1 to K-63; M-1 to
G-62; M-1 to F-61; M-1 to A-60; M-1 to S-59; M-1 to L-58; M-1 to
H-57; M-1 to L-56; M-1 to A-55; M-1 to L-54; M-1 to E-53; M-1 to
G-52; M-1 to A-51; M-1 to S-50; M-1 to G-49; M-1 to P-48; M-1 to
L-47; M-1 to R-46; M-1 to T-45; M-1 to P-44; M-1 to V-43; M-1 to
V-42; M-1 to L-41; M-1 to E-40; M-1 to S-39; M-1 to A-38; M-1 to
Q-37; M-1 to G-36; M-1 to G-35; M-1 to A-34; M-1 to A-33; M-1 to
P-32; M-1 to R-31; M-1 to A-30; M-1 to P-29; M-1 to A-28; M-1 to
G-27; M-1 to R-26; M-1 to A-25; M-1 to L-24; M-1 to P-23; M-1 to
L-22; M-1 to L-21; M-1 to L-20; M-1 to L-19; M-1 to L-18; M-1 to
L-17; M-1 to L-16; M-1 to L-15; M-1 to L-14; M-1 to F-13; M-1 to
P-12; M-1 to L-11; M-1 to W-10; M-1 to R-9; M-1 to P-8; M-1 to A-7;
of SEQ ID NO:4. Preferably, any of the above listed N- or
C-terminal deletions can be combined to produce a N- and C-terminal
deleted METH2 polypeptide.
[0156] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini,
which may be described generally as having residues m.sub.1-n.sub.1
of SEQ ID NO:2 or m.sub.2-n.sub.2 SEQ ID NO:4, where n and m are
integers as described above.
[0157] The invention also provides mutants of the metalloprotease
domain of METH1, which are described by the general formula
m.sub.3-n.sub.3, where m.sub.3 is an integer from 205 to 265, and
n.sub.3 is an integer from 285 to 950, where m.sub.3 and n.sub.3
correspond to the position of the amino acid residue identified in
SEQ ID NO:2. The invention further provides mutants of the
metalloprotease domain of METH1, which are described by the general
formula m.sub.4-n.sub.4, where m.sub.4 is an integer from 1 to 409,
and n.sub.4 is an integer from 429 to 489, where m.sub.4 and
n.sub.4 correspond to the position of the amino acid residue
identified in SEQ ID NO:2.
[0158] The invention also provides mutants of the disintegrin
domain of METH1, which are described by the general formula
m.sub.5-n.sub.5, where m.sub.5 is an integer from 430 to 490, and
n.sub.5 is an integer from 510 to 950, where m.sub.5 and n.sub.5
correspond to the position of the amino acid residue identified in
SEQ ID NO:2.
[0159] The invention further provides mutants of the disintegrin
domain of METH1, which are described by the general formula
m.sub.6-n.sub.6, where m.sub.6 is an integer from 1 to 494, and
n.sub.6 is an integer from 514 to 574, where m.sub.6 and n.sub.6
correspond to the position of the amino acid residue identified in
SEQ ID NO:2.
[0160] The invention further provides mutants of the TSP1 domain of
METH1, which are described by the general formula m.sub.7-n.sub.7,
where m.sub.7 is an integer from 515 to 575, and n.sub.7 is an
integer from 595 to 950, where m.sub.7 an n.sub.7 correspond to the
position of the amino acid residue identified in SEQ ID NO:2.
[0161] The invention also provides mutants of the TSP1 domain of
METH1, which are described by the general formula m.sub.8-n.sub.8,
where m.sub.8 is an integer from 1 to 548, and n.sub.8 is an
integer from 568 to 628, where m.sub.8 and n.sub.8 correspond to
the position of the amino acid residue identified in SEQ ID
NO:2.
[0162] The invention further provides mutants of the TSP2 domain of
METH1, which are described by the general formula m.sub.9-n.sub.9,
where m.sub.9 is an integer from 801 to 871, and n.sub.9 is an
integer from 891 to 950, where m.sub.9 and n.sub.9 correspond to
the position of the amino acid residue identified in SEQ ID
NO:2.
[0163] The invention also provides mutants of the TSP2 domain of
METH1, which are described by the general formula
m.sub.10-n.sub.10, where m.sub.10 is an integer from 1 to 834, and
n.sub.10 is an integer from 864 to 924, where m.sub.10 and n.sub.10
correspond to the position of the amino acid residue identified in
SEQ ID NO:2.
[0164] The invention further provides mutants of the TSP3 domain of
METH1, which are described by the general formula
m.sub.11-n.sub.11, where m.sub.11 is an integer from 865 to 925,
and n.sub.11 is an integer from 945 to 950, where m.sub.11 and
n.sub.11 correspond to the position of the amino acid residue
identified in SEQ ID NO:2. The invention also provides mutants of
the TSP3 domain of METH1, which are described by the general
formula m.sub.12-n.sub.12, where m.sub.12 is an integer from 1 to
884, and n.sub.12 is an integer from 904 to 950, where m.sub.12 and
n.sub.12 correspond to the position of the amino acid residue
identified in SEQ ID NO:2.
[0165] The invention further provides mutants of the
metalloprotease domain of METH2, which are described by the general
formula m.sub.13-n.sub.13, where m.sub.13 is an integer from 184 to
244, and n.sub.13 is an integer from 264 to 890, where m.sub.13 and
n.sub.13 correspond to the position of the amino acid residue
identified in SEQ ID NO:4. The invention also provides mutants of
the metalloprotease domain of METH2, which are described by the
general formula m.sub.14-n.sub.14, where m.sub.14 is an integer
from 1 to 389, and n.sub.14 is an integer from 409 to 469, where
m.sub.14 and n.sub.14 correspond to the position of the amino acid
residue identified in SEQ ID NO:4.
[0166] The invention further provides mutants of the disintegrin
domain of METH2, which are described by the general formula
m.sub.15-n.sub.15, where m.sub.15 is an integer from 400 to 470,
and n.sub.15 is an integer from 490 to 890, where m.sub.15 and
n.sub.15 correspond to the position of the amino acid residue
identified in SEQ ID NO:4. The invention also provides mutants of
the disintegrin domain of METH2, which are described by the general
formula m.sub.16-n.sub.16, where m.sub.16 is an integer from 1 to
479, and n.sub.16 is an integer from 499 to 559, where m.sub.16 and
n.sub.16 correspond to the position of the amino acid residue
identified in SEQ ID NO:4.
[0167] The invention further provides mutants of the TSP1 domain of
METH2, which are described by the general formula
m.sub.17-n.sub.17, where m.sub.17 is an integer from 500 to 560,
and n.sub.17 is an integer from 580 to 890, where m.sub.17 and
n.sub.17 correspond to the position of the amino acid residue
identified in SEQ ID NO:4. The invention also provides mutants of
the TSP1 domain of METH2, which are described by the general
formula m.sub.18-n.sub.18, where m.sub.18 is an integer from 1 to
533, and n.sub.18 is an integer from 553 to 613, where m.sub.18 and
n.sub.18 correspond to the position of the amino acid residue
identified in SEQ ID NO:4.
[0168] The invention further provides mutants of the TSP2 domain of
METH2, which are described by the general formula m.sub.9-n.sub.19,
where m.sub.19 is an integer from 807 to 867, and n.sub.19 is an
integer from 887 to 890, where m.sub.19 and n.sub.19 correspond to
the position of the amino acid residue identified in SEQ ID NO:4.
The invention also provides mutants of the TSP2 domain of METH2,
which are described by the general formula m.sub.20-n.sub.20, where
m.sub.20 is an integer from 1 to 840, and n.sub.20 is an integer
from 860 to 890, where m.sub.20 and n.sub.20 correspond to the
position of the amino acid residue identified in SEQ ID NO:4.
[0169] Also preferred are METH1 or METH2 polypeptide and
polynucleotide fragments characterized by structural or functional
domains. Preferred embodiments of the invention include fragments
that comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet-forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions,
substrate binding region, and high antigenic index regions. As set
out in the Figures, such preferred regions include Garnier-Robson
alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman alpha-regions, beta-regions, and turn-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha and beta amphipathic regions, Karplus-Schulz
flexible regions, Emini surface-forming regions, and Jameson-Wolf
high antigenic index regions. Polypeptide fragments of SEQ ID NO:2
falling within conserved domains are specifically contemplated by
the present invention. (See FIGS. 10 & 11 and Tables 1 &
2.) Moreover, polynucleotide fragments encoding these domains are
also contemplated.
[0170] Other preferred fragments are biologically active METH1 or
METH2 fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
the METH1 or METH2 polypeptide. The biological activity of the
fragments may include an improved desired activity, or a decreased
undesirable activity. However, many polynucleotide sequences, such
as EST sequences, are publicly available and accessible through
sequence databases. Some of these sequences are related to SEQ ID
NO:1 or SEQ ID NO:3 and may have been publicly available prior to
conception of the present invention. Preferably, such related
polynucleotides are specifically excluded from the scope of the
present invention. To list every related sequence would be
cumbersome. Accordingly, preferably excluded from the present
invention are one or more polynucleotides comprising a nucleotide
sequence described by the general formula of a-b, where a is any
integer between 1 to 936 of SEQ ID NO:1, b is an integer of 15 to
950, where both a and b correspond to the positions of nucleotide
residues shown in SEQ ID NO:1, and where the b is greater than or
equal to a+14. Moreover, preferably excluded from the present
invention are one or more polynucleotides comprising a nucleotide
sequence described by the general formula of a-b, where a is any
integer between 1 to 876 of SEQ ID NO:3, b is an integer of 15 to
890, where both a and b correspond to the positions of nucleotide
residues shown in SEQ ID NO:3, and where the b is greater than or
equal to a+14.
[0171] The above-described fragments may be used to make fusion
proteins, for example Fc or Flag fusion proteins, as described
below.
Epitopes & Antibodies
[0172] In another aspect, the invention provides peptides and
polypeptides comprising epitope-bearing portions of the
polypeptides of the present invention. These epitopes are
immunogenic or antigenic epitopes of the polypeptides of the
present invention. An "immunogenic epitope" is defined as a part of
a protein that elicits an antibody response in vivo when the whole
polypeptide of the present invention, or fragment thereof, is the
immunogen. On the other hand, a region of a polypeptide to which an
antibody can bind is defined as an "antigenic determinant" or
"antigenic epitope." The number of in vivo immunogenic epitopes of
a protein generally is less than the number of antigenic epitopes.
See, e.g., Geysen, et al. (1983) Proc. Natl. Acad. Sci. USA
81:3998-4002. However, antibodies can be made to any antigenic
epitope, regardless of whether it is an immunogenic epitope, by
using methods such as phage display. See e.g., Petersen G. et al.
(1995) Mol. Gen. Genet. 249:425-431. Therefore, included in the
present invention are both immunogenic epitopes and antigenic
epitopes.
[0173] A list of exemplified amino acid sequences comprising
immunogenic epitopes are shown in Tables 1 and 2. It is pointed out
that Tables 1 and 2 only list amino acid residues comprising
epitopes predicted to have the highest degree of antigenicity using
the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci.
4:181-186 (said references incorporated by reference in their
entireties). The Jameson-Wolf antigenic analysis was performed
using the computer program PROTEAN, using default parameters
(Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South
Park Street Madison, Wis.). Tables 1 and 2 and portions of
polypeptides not listed in Tables 1 and 2 are not considered
non-immunogenic. The immunogenic epitopes of Tables 1 and 2 are
exemplified lists, not exhaustive lists, because other immunogenic
epitopes are merely not recognized as such by the particular
algorithm used. Amino acid residues comprising other immunogenic
epitopes may be routinely determined using algorithms similar to
the Jameson-Wolf analysis or by in vivo testing for an antigenic
response using methods known in the art. See, e.g., Geysen et al.,
supra; U.S. Pat. Nos. 4,708,781; 5,194,392; 4,433,092; and
5,480,971 (said references incorporated by reference in their
entireties).
[0174] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention.
[0175] Using DNAstar analysis, SEQ ID NO:2 was found antigenic at
amino acids: 2-14, 32-44, 47-60, 66-78, 87-103, 109-118, 146-162,
168-180, 183-219, 223-243, 275-284, 296-306, 314-334, 341-354,
357-376, 392-399, 401-410, 418-429, 438-454, 456-471, 474-488,
510-522, 524-538, 550-561, 565-626, 630-643, 659-671, 679-721,
734-749, 784-804, 813-820, 825-832, 845-854, 860-894, 899-917,
919-924 and 928-939.
[0176] Using DNAstar analysis, SEQ ID NO:4 was found antigenic at
amino acids: 26-38, 45-52, 69-76, 80-99, 105-113, 129-136, 138-217,
254-263, 273-289, 294-313, 321-331, 339-356, 371-383, 417-427,
438-443, 459-471, 479-505, 507-526, 535-546, 550-607, 615-640,
648-653, 660-667, 669-681, 683-704, 717-732, 737-743, 775-787,
797-804, 811-825, 840-867 and 870-884.
[0177] Thus, these regions of METH1 and/or METH2 are non-limiting
examples of antigenic polypeptides or peptides that can be used to
raise METH1 and/or METH2-specific antibodies include.
[0178] It is particularly pointed out that the amino acid sequences
of Tables 1 and 2 comprise immunogenic epitopes. Tables 1 and 2
list only the critical residues of immunogenic epitopes determined
by the Jameson-Wolf analysis. Thus, additional flanking residues on
either the N-terminal, C-terminal, or both N- and C-terminal ends
may be added to the sequences of Tables 1 and 2 to generate an
epitope-bearing polypeptide of the present invention. Therefore,
the immunogenic epitopes of Tables 1 and 2 may include additional
N-terminal or C-terminal amino acid residues. The additional
flanking amino acid residues may be contiguous flanking N-terminal
and/or C-terminal sequences from the polypeptides of the present
invention, heterologous polypeptide sequences, or may include both
contiguous flanking sequences from the polypeptides of the present
invention and heterologous polypeptide sequences.
[0179] Polypeptides of the present invention comprising immunogenic
or antigenic epitopes are at least 7 amino acids residues in
length. "At least" means that a polypeptide of the present
invention comprising an immunogenic or antigenic epitope may be 7
amino acid residues in length or any integer between 7 amino acids
and the number of amino acid residues of the full length
polypeptides of the invention. Preferred polypeptides comprising
immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino
acid residues in length. However, it is pointed out that each and
every integer between 7 and the number of amino acid residues of
the full length polypeptide are included in the present
invention.
[0180] The immuno and antigenic epitope-bearing fragments may be
specified by either the number of contiguous amino acid residues,
as described above, or further specified by N-terminal and
C-terminal positions of these fragments on the amino acid sequence
of SEQ ID NO:2 or 4. Every combination of a N-terminal and
C-terminal position that a fragment of, for example, at least 7 or
at least 15 contiguous amino acid residues in length could occupy
on the amino acid sequence of SEQ ID NO:2 or 4 is included in the
invention. Again, "at least 7 contiguous amino acid residues in
length" means 7 amino acid residues in length or any integer
between 7 amino acids and the number of amino acid residues of the
full length polypeptide of the present invention. Specifically,
each and every integer between 7 and the number of amino acid
residues of the full length polypeptide are included in the present
invention.
[0181] Immunogenic and antigenic epitope-bearing polypeptides of
the invention are useful, for example, to make antibodies which
specifically bind the polypeptides of the invention, and in
immunoassays to detect the polypeptides of the present invention.
The antibodies are useful, for example, in affinity purification of
the polypeptides of the present invention. The antibodies may also
routinely be used in a variety of qualitative or quantitative
immunoassays, specifically for the polypeptides of the present
invention using methods known in the art. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press; 2nd Ed. 1988).
[0182] The epitope-bearing polypeptides of the present invention
may be produced by any conventional means for making polypeptides
including synthetic and recombinant methods known in the art. For
instance, epitope-bearing peptides may be synthesized using known
methods of chemical synthesis. For instance, Houghten has described
a simple method for the synthesis of large numbers of peptides,
such as 10-20 mgs of 248 individual and distinct 13 residue
peptides representing single amino acid variants of a segment of
the HA1 polypeptide, all of which were prepared and characterized
(by ELISA-type binding studies) in less than four weeks (Houghten,
R. A. Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985)). This
"Simultaneous Multiple Peptide Synthesis (SMPS)" process is further
described in U.S. Pat. No. 4,631,211 to Houghten and coworkers
(1986). In this procedure the individual resins for the solid-phase
synthesis of various peptides are contained in separate
solvent-permeable packets, enabling the optimal use of the many
identical repetitive steps involved in solid-phase methods. A
completely manual procedure allows 500-1000 or more syntheses to be
conducted simultaneously (Houghten et al. (1985) Proc. Natl. Acad.
Sci. 82:5131-5135 at 5134).
[0183] Epitope-bearing polypeptides of the present invention are
used to induce antibodies according to methods well known in the
art including, but not limited to, in vivo immunization, in vitro
immunization, and phage display methods. See, e.g., Sutcliffe, et
al., supra; Wilson, et al., supra, and Bitle, et al. (1985) J. Gen.
Virol. 66:2347-2354. If in vivo immunization is used, animals may
be immunized with free peptide; however, anti-peptide antibody
titer may be boosted by coupling of the peptide to a macromolecular
carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
For instance, peptides containing cysteine residues may be coupled
to a carrier using a linker such as
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carriers using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.gs of peptide or carrier protein
and Freund's adjuvant. Several booster injections may be needed,
for instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0184] As one of skill in the art will appreciate, and discussed
above, the polypeptides of the present invention comprising an
immunogenic or antigenic epitope can be fused to heterologous
polypeptide sequences. For example, the polypeptides of the present
invention may be fused with the constant domain of immunoglobulins
(IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, any
combination thereof including both entire domains and portions
thereof) resulting in chimeric polypeptides. These fusion proteins
facilitate purification, and show an increased half-life in vivo.
This has been shown, e.g., for chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins. See, e.g., EPA 0,394,827; Traunecker et al. (1988)
Nature 331:84-86. Fusion proteins that have a disulfide-linked
dimeric structure due to the IgG portion can also be more efficient
in binding and neutralizing other molecules than monomeric
polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al. (1995) J. Biochem. 270:3958-3964. Nucleic acids encoding the
above epitopes can also be recombined with a gene of interest as an
epitope tag to aid in detection and purification of the expressed
polypeptide.
[0185] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. Most preferably the antibodies
are human antigen binding antibody fragments of the present
invention including, but not limited to, Fab, Fab' and F(ab')2, Fd,
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked
Fvs (sdFv) and fragments comprising either a V.sub.L or V.sub.H
domain. The antibodies may be from any animal origin including
birds and mammals. Preferably, the antibodies are human, murine,
rabbit, goat, guinea pig, camel, horse, or chicken.
[0186] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH1, CH2, and CH3 domains. Also included in the invention
are any combinations of variable region(s) and hinge region, CH1,
CH2, and CH3 domains. The present invention further includes
monoclonal, polyclonal, chimeric, humanized, and human monoclonal
and polyclonal antibodies which specifically bind the polypeptides
of the present invention. The present invention further includes
antibodies which are anti-idiotypic to the antibodies of the
present invention.
[0187] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt, A. et al. (1991) J. Immunol. 147:60-69; U.S. Pat.
Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648;
Kostelny, S. A. et al. (1992) J. Immunol. 148:1547-1553.
[0188] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which are recognized or specifically bound
by the antibody. The epitope(s) or polypeptide portion(s) may be
specified as described herein, e.g., by N-terminal and C-terminal
positions, by size in contiguous amino acid residues, or listed in
the Tables and Figures. Antibodies which specifically bind any
epitope or polypeptide of the present invention may also be
excluded. Therefore, the present invention includes antibodies that
specifically bind polypeptides of the present invention, and allows
for the exclusion of the same.
[0189] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of the polypeptides
of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Further included in the present invention are antibodies which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the
present invention may also be described or specified in terms of
their binding affinity. Preferred binding affinities include those
with a dissociation constant or Kd less than 5.times.10.sup.-6M,
10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M, 5.times.10.sup.-3M,
10.sup.-3M, 5.times.10.sup.-9M, 10.sup.-9M, 5.times.10.sup.-10M,
10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M, 5.times.10.sup.-12M,
10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M, 5.times.10.sup.-14M,
10.sup.-14M, 5.times.10.sup.-15M, and 10.sup.-15M.
[0190] Antibodies of the present invention have uses that include,
but are not limited to, methods known in the art to purify, detect,
and target the polypeptides of the present invention including both
in vitro and in vivo diagnostic and therapeutic methods. For
example, the antibodies have use in immunoassays for qualitatively
and quantitatively measuring levels of the polypeptides of the
present invention in biological samples. See, e.g., Harlow et al.,
ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference in the
entirety).
[0191] The antibodies of the present invention may be used either
alone or in combination with other compositions. The antibodies may
further be recombinantly fused to a heterologous polypeptide at the
N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions.
For example, antibodies of the present invention may be
recombinantly fused or conjugated to molecules useful as labels in
detection assays and effector molecules such as heterologous
polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO
91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396
387.
[0192] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. The term "monoclonal antibody" is
not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including eukaryotic, prokaryotic, or
phage clones, and not by the method which it is produced.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant and phage display technology.
[0193] Hybridoma techniques include those known in the art and
taught in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold
Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al.,
in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier,
N.Y., 1981) (said references incorporated by reference in their
entireties). Fab and F(ab')2 fragments may be produced by
proteolytic cleavage, using enzymes such as papain (to produce Fab
fragments) or pepsin (to produce F(ab')2 fragments).
[0194] Alternatively, antibodies of the present invention can be
produced through the application of recombinant DNA and phage
display technology or through synthetic chemistry using methods
known in the art. For example, the antibodies of the present
invention can be prepared using various phage display methods known
in the art. In phage display methods, functional antibody domains
are displayed on the surface of a phage particle which carries
polynucleotide sequences encoding them. Phage with a desired
binding property are selected from a repertoire or combinatorial
antibody library (e.g. human or murine) by selecting directly with
antigen, typically antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 with Fab, Fv or disulfide stabilized Fv
antibody domains recombinantly fused to either the phage gene III
or gene VIII protein. Examples of phage display methods that can be
used to make the antibodies of the present invention include those
disclosed in Brinkman U. et al. (1995) J. Immunol. Methods
182:41-50; Ames, R. S. et al. (1995) J. Immunol. Methods
184:177-186; Kettleborough, C. A. et al. (1994) Eur. J. Immunol.
24:952-958; Persic, L. et al. (1997) Gene 187:9-18; Burton, D. R.
et al. (1994) Advances in Immunology 57:191-280; PCT/GB91/01134; WO
90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO
95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409,
5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,
5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (said
references incorporated by reference in their entireties).
[0195] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in WO 92/22324;
Mullinax, R. L. et al. (1992) BioTechniques 12(6):864-869; and
Sawai, H. et al. (1995) AJRI 34:26-34; and Better, M. et al. (1988)
Science 240:1041-1043 (said references incorporated by reference in
their entireties).
[0196] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (1991) Methods in
Enzymology 203:46-88; Shu, L. et al. (1993) PNAS 90:7995-7999; and
Skerra, A. et al. (1988) Science 240:1038-1040. For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. Methods for producing chimeric antibodies are
known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies, S. D. et al. (1989) J.
Immunol. Methods 125:191-202; and U.S. Pat. No. 5,807,715.
Antibodies can be humanized using a variety of techniques including
CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101;
and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519
596; Padlan E. A., (1991) Molecular Immunology 28(4/5):489-498;
Studnicka G. M. et al. (1994) Protein Engineering 7(6):805-814;
Roguska M. A. et al. (1994) PNAS 91:969-973), and chain shuffling
(U.S. Pat. No. 5,565,332). Human antibodies can be made by a
variety of methods known in the art including phage display methods
described above. See also, U.S. Pat. Nos. 4,444,887, 4,716,111,
5,545,806, and 5,814,318; and WO 98/46645 (said references
incorporated by reference in their entireties).
[0197] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al. supra and WO 93/21232; EP 0 439 095; Naramura, M. et
al. (1994) Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981;
Gillies, S. O. et al. (1992) PNAS 89:1428-1432; Fell, H. P. et al.
(1991) J. Immunol. 146:2446-2452 (said references incorporated by
reference in their entireties).
[0198] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the hinge region, CH1 domain, CH2 domain, and CH3
domain or any combination of whole domains or portions thereof. The
polypeptides of the present invention may be fused or conjugated to
the above antibody portions to increase the in vivo half life of
the polypeptides or for use in immunoassays using methods known in
the art. The polypeptides may also be fused or conjugated to the
above antibody portions to form multimers. For example, Fc portions
fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher
multimeric forms can be made by fusing the polypeptides to portions
of IgA and IgM. Methods for fusing or conjugating the polypeptides
of the present invention to antibody portions are known in the art.
See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,
5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO
96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS
88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol.
154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (said
references incorporated by reference in their entireties).
[0199] The invention further relates to antibodies that act as
agonists or antagonists of the polypeptides of the present
invention. Antibodies which act as agonists or antagonists of the
polypeptides of the present invention include, for example,
antibodies which disrupt receptor/ligand interactions with the
polypeptides of the invention either partially or fully. For
example, the present invention includes antibodies that disrupt the
ability of the proteins of the invention to multimerize. In another
example, the present invention includes antibodies which allow the
proteins of the invention to multimerize, but disrupt the ability
of the proteins of the invention to bind one or more METH1 and/or
METH2 receptor(s)/ligand(s). In yet another example, the present
invention includes antibodies which allow the proteins of the
invention to multimerize, and bind METH1 and/or METH2
receptor(s)/ligand(s), but blocks biological activity associated
with the METH1 and/or METH2/receptor/ligand complex.
[0200] Antibodies which act as agonists or antagonists of the
polypeptides of the present invention also include, both
receptor-specific antibodies and ligand-specific antibodies.
Included are receptor-specific antibodies that do not prevent
ligand binding but prevent receptor activation. Receptor activation
(i.e., signaling) may be determined by techniques described herein
or otherwise known in the art. Also included are receptor-specific
antibodies which both prevent ligand binding and receptor
activation. Likewise, included are neutralizing antibodies which
bind the ligand and prevent binding of the ligand to the receptor,
as well as antibodies which bind the ligand, thereby preventing
receptor activation, but do not prevent the ligand from binding the
receptor. Further included are antibodies that activate the
receptor. These antibodies may act as agonists for either all or
less than all of the biological activities affected by
ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. See e.g., WO
96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood
92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.
58(16):3668-3678 (1998); Harrop, J. A. et al., J. Immunol.
161(4):1786-1794 (1998); Zhu, Z. et al., Cancer Res.
58(15):3209-3214 (1998); Yoon, D. Y. et al., J. Immunol.
160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci.
111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods
205(2):177-190 (1997); Liautard, J. et al., Cytokine 9(4):233-241
(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301
(1997); Taryman, R. E. et al., Neuron 14(4):755-762 (1995); Muller,
Y. A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al.,
Cytokine 8(1):14-20 (1996) (said references incorporated by
reference in their entireties).
[0201] As discussed above, antibodies to the METH1 and/or METH2
proteins of the invention can, in turn, be utilized to generate
anti-idiotype antibodies that "mimic" METH1 and/or METH2 using
techniques well known to those skilled in the art. (See, e.g.,
Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff,
J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which
bind to METH1 and/or METH2 and competitively inhibit METH1 and/or
METH2 multimerization and/or binding to ligand can be used to
generate anti-idiotypes that "mimic" the METH1 and/or METH2
multimerization and/or binding domain and, as a consequence, bind
to and neutralize METH1 and/or METH2 and/or its ligand. Such
neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes
can be used in therapeutic regimens to neutralize METH1 and/or
METH2 ligand. For example, such anti-idiotypic antibodies can be
used to bind METH1 and/or METH2, or to bind METH1 and/or METH2
ligands/receptors, and thereby block METH1 and/or METH2 biological
activity.
Fusion Proteins
[0202] Any METH1 or METH2 polypeptide can be used to generate
fusion proteins. For example, the METH1 or METH2 polypeptide, when
fused to a second protein, can be used as an antigenic tag.
Antibodies raised against the METH1 or METH2 polypeptide can be
used to indirectly detect the second protein by binding to the
METH1 or METH2. Moreover, because secreted proteins target cellular
locations based on trafficking signals, the METH1 or METH2
polypeptides can be used as a targeting molecule once fused to
other proteins.
[0203] Examples of domains that can be fused to METH1 or METH2
polypeptides include not only heterologous signal sequences, but
also other heterologous functional regions. The fusion does not
necessarily need to be direct, but may occur through linker
sequences.
[0204] In certain preferred embodiments, METH1 or METH2 proteins of
the invention comprise fusion proteins wherein the METH1 or METH2
polypeptides are those described above as m.sub.1-n.sub.1 or
m.sub.2-n.sub.2, respectively. In preferred embodiments, the
application is directed to nucleic acid molecules at least 90%,
95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences
encoding polypeptides having the amino acid sequence of the
specific N- and C-terminal deletions recited herein.
Polynucleotides encoding these polypeptides are also encompassed by
the invention.
[0205] Moreover, fusion proteins may also be engineered to improve
characteristics of the METH1 or METH2 polypeptide. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the METH1 or METH2 polypeptide to
improve stability and persistence during purification from the host
cell or subsequent handling and storage. Also, peptide moieties may
be added to the METH1 or METH2 polypeptide to facilitate
purification. Such regions may be removed prior to final
preparation of the METH1 or METH2 polypeptide. The addition of
peptide moieties to facilitate handling of polypeptides are
familiar and routine techniques in the art.
[0206] Moreover, METH1 or METH2 polypeptides, including fragments,
and specifically epitopes, can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. One reported example describes
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins. (EP A 394,827;
Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having
disulfide-linked dimeric structures (due to the IgG) can also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
(Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
[0207] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. (See, D. Bennett et al.,
J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J.
Biol. Chem. 270:9459-9471 (1995).)
[0208] Moreover, the METH1 or METH2 polypeptides can be fused to
marker sequences, such as a peptide which facilitates purification
of METH1 or METH2. In preferred embodiments, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311), among others, many of which are commercially available. As
described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824
(1989), for instance, hexa-histidine provides for convenient
purification of the fusion protein. Another peptide tag useful for
purification, the "HA" tag, corresponds to an epitope derived from
the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
[0209] Thus, any of these above fusions can be engineered using the
METH1 or METH2 polynucleotides or the polypeptides.
[0210] Biological Activities of METH1 and/or METH2
[0211] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, can be used in
assays to test for one or more biological activities. If METH1
and/or METH2 polynucleotides or polypeptides, or agonists or
antagonists of METH1 and/or METH2, do exhibit activity in a
particular assay, it is likely that METH1 and/or METH2 may be
involved in the diseases associated with the biological activity.
Therefore, METH1 and/or METH2 could be used to treat the associated
disease.
[0212] Immune Activity
[0213] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may be useful in
treating deficiencies or disorders of the immune system, by
activating or inhibiting the proliferation, differentiation, or
mobilization (chemotaxis) of immune cells. Immune cells develop
through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and
lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
The etiology of these immune deficiencies or disorders may be
genetic, somatic, such as cancer or some autoimmune disorders,
acquired (e.g., by chemotherapy or toxins), or infectious.
Moreover, METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, can be used as a
marker or detector of a particular immune system disease or
disorder.
[0214] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may be useful in
treating or detecting deficiencies or disorders of hematopoietic
cells. METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, could be used to
increase differentiation and proliferation of hematopoietic cells,
including the pluripotent stem cells, in an effort to treat those
disorders associated with a decrease in certain (or many) types
hematopoietic cells. Examples of immunologic deficiency syndromes
include, but are not limited to: blood protein disorders (e.g.
agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia,
common variable immunodeficiency, Digeorge Syndrome, HIV infection,
HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia,
thrombocytopenia, or hemoglobinuria.
[0215] Moreover, METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2, can
also be used to modulate hemostatic (the stopping of bleeding) or
thrombolytic activity (clot formation). For example, by increasing
hemostatic or thrombolytic activity, METH1 and/or METH2
polynucleotides or polypeptides, or agonists or antagonists of
METH1 and/or METH2, could be used to treat blood coagulation
disorders (e.g., afibrinogenemia, factor deficiencies), blood
platelet disorders (e.g. thrombocytopenia), or wounds resulting
from trauma, surgery, or other causes. Alternatively, METH1 and/or
METH2 polynucleotides or polypeptides, or agonists or antagonists
of METH1 and/or METH2, that can decrease hemostatic or thrombolytic
activity could be used to inhibit or dissolve clotting, important
in the treatment of heart attacks (infarction), strokes, or
scarring.
[0216] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may also be useful
in treating or detecting autoimmune disorders. Many autoimmune
disorders result from inappropriate recognition of self as foreign
material by immune cells. This inappropriate recognition results in
an immune response leading to the destruction of the host tissue.
Therefore, the administration of METH1 and/or METH2 polynucleotides
or polypeptides, or agonists or antagonists of METH1 and/or METH2,
that can inhibit an immune response, particularly the
proliferation, differentiation, or chemotaxis of T-cells, may be an
effective therapy in preventing autoimmune disorders.
[0217] Examples of autoimmune disorders that can be treated or
detected include, but are not limited to: Addison's Disease,
hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis,
dermatitis, allergic encephalomyelitis, glomerulonephritis,
Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis,
Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease,
Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus
Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Barre
Syndrome, insulin dependent diabetes mellitis, and autoimmune
inflammatory eye disease.
[0218] Similarly, allergic reactions and conditions, such as asthma
(particularly allergic asthma) or other respiratory problems, may
also be treated by METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2.
Moreover, these molecules can be used to treat anaphylaxis,
hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[0219] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may also be used to
treat and/or prevent organ rejection or graft-versus-host disease
(GVHD). Organ rejection occurs by host immune cell destruction of
the transplanted tissue through an immune response. Similarly, an
immune response is also involved in GVHD, but, in this case, the
foreign transplanted immune cells destroy the host tissues. The
administration of METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2,
that inhibits an immune response, particularly the proliferation,
differentiation, or chemotaxis of T-cells, may be an effective
therapy in preventing organ rejection or GVHD.
[0220] Similarly, METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2, may
also be used to modulate inflammation. For example, METH1 and/or
METH2 polynucleotides or polypeptides, or agonists or antagonists
of METH1 and/or METH2, may inhibit the proliferation and
differentiation of cells involved in an inflammatory response.
These molecules can be used to treat inflammatory conditions, both
chronic and acute conditions, including inflammation associated
with infection (e.g., septic shock, sepsis, or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion
injury, endotoxin lethality, arthritis, complement-mediated
hyperacute rejection, nephritis, cytokine or chemokine induced lung
injury, inflammatory bowel disease, Crohn's disease, or resulting
from over production of cytokines (e.g., TNF or IL-1.)
[0221] Hyperproliferative Disorders
[0222] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, can be used to treat
or detect hyperproliferative disorders, including neoplasms. METH1
and/or METH2 polynucleotides or polypeptides, or agonists or
antagonists of METH1 and/or METH2, may inhibit the proliferation of
the disorder through direct or indirect interactions.
Alternatively, METH1 and/or METH2 polynucleotides or polypeptides,
or agonists or antagonists of METH1 and/or METH2, may proliferate
other cells which can inhibit the hyperproliferative disorder.
[0223] For example, by increasing an immune response, particularly
increasing antigenic qualities of the hyperproliferative disorder
or by proliferating, differentiating, or mobilizing T-cells,
hyperproliferative disorders can be treated. This immune response
may be increased by either enhancing an existing immune response,
or by initiating a new immune response. Alternatively, decreasing
an immune response may also be a method of treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[0224] Examples of hyperproliferative disorders that can be treated
or detected by METH1 and/or METH2 polynucleotides or polypeptides,
or agonists or antagonists of METH1 and/or METH2, include, but are
not limited to neoplasms located in the: abdomen, bone, breast,
digestive system, liver, pancreas, peritoneum, endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and
urogenital.
[0225] Similarly, other hyperproliferative disorders can also be
treated or detected by METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2.
Examples of such hyperproliferative disorders include, but are not
limited to: hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,
Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis,
and any other hyperproliferative disease, besides neoplasia,
located in an organ system listed above.
[0226] Cardiovascular Disorders
[0227] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, encoding METH1
and/or METH2 may be used to treat cardiovascular disorders,
including peripheral artery disease, such as limb ischemia.
[0228] Cardiovascular disorders include cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous
fistula, cerebral arteriovenous malformations, congenital heart
defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects include aortic coarctation, cor triatriatum, coronary
vessel anomalies, crisscross heart, dextrocardia, patent ductus
arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic
left heart syndrome, levocardia, tetralogy of fallot, transposition
of great vessels, double outlet right ventricle, tricuspid atresia,
persistent truncus arteriosus, and heart septal defects, such as
aortopulmonary septal defect, endocardial cushion defects,
Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal
defects.
[0229] Cardiovascular disorders also include heart disease, such as
arrhythmias, carcinoid heart disease, high cardiac output, low
cardiac output, cardiac tamponade, endocarditis (including
bacterial), heart aneurysm, cardiac arrest, congestive heart
failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left
ventricular hypertrophy, right ventricular hypertrophy,
post-infarction heart rupture, ventricular septal rupture, heart
valve diseases, myocardial diseases, myocardial ischemia,
pericardial effusion, pericarditis (including constrictive and
tuberculous), pneumopericardium, postpericardiotomy syndrome,
pulmonary heart disease, rheumatic heart disease, ventricular
dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar Syndrome, cardiovascular syphilis, and cardiovascular
tuberculosis.
[0230] Arrhythmias include sinus arrhythmia, atrial fibrillation,
atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome,
bundle-branch block, sinoatrial block, long QT syndrome,
parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type
pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include paroxysmal tachycardia, supraventricular tachycardia,
accelerated idioventricular rhythm, atrioventricular nodal reentry
tachycardia, ectopic atrial tachycardia, ectopic junctional
tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia, Torsades de Pointes, and ventricular tachycardia.
[0231] Heart valve diseases include aortic valve insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral
valve prolapse, tricuspid valve prolapse, mitral valve
insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
[0232] Myocardial diseases include alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic
subvalvular stenosis, pulmonary subvalvular stenosis, restrictive
cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis,
endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion
injury, and myocarditis.
[0233] Myocardial ischemias include coronary disease, such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and
myocardial stunning.
[0234] Cardiovascular diseases also include vascular diseases such
as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,
Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome,
Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,
Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases, arteritis, enarteritis, polyarteritis nodosa,
cerebrovascular disorders, diabetic angiopathies, diabetic
retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,
hepatic veno-occlusive disease, hypertension, hypotension,
ischemia, peripheral vascular diseases, phlebitis, pulmonary
veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal
vein occlusion, Scimitar syndrome, superior vena cava syndrome,
telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer,
vasculitis, and venous insufficiency.
[0235] Aneurysms include dissecting aneurysms, false aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac
aneurysms.
[0236] Arterial occlusive diseases include arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular
dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal
artery obstruction, retinal artery occlusion, and thromboangiitis
obliterans.
[0237] Cerebrovascular disorders include carotid artery diseases,
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral arteriosclerosis, cerebral arteriovenous malformation,
cerebral artery diseases, cerebral embolism and thrombosis, carotid
artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma,
subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar insufficiency.
[0238] Embolisms include air embolisms, amniotic fluid embolisms,
cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include coronary
thrombosis, hepatic vein thrombosis, retinal vein occlusion,
carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome,
and thrombophlebitis.
[0239] Ischemia includes cerebral ischemia, ischemic colitis,
compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia.
Vasculitis includes aortitis, arteritis, Behcet's Syndrome,
Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis,
Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's granulomatosis.
[0240] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, are especially
effective for the treatment of critical limb ischemia and coronary
disease.
[0241] METH1 and/or METH2 polypeptides may be administered using
any method known in the art, including, but not limited to, direct
needle injection at the delivery site, intravenous injection,
topical administration, catheter infusion, biolistic injectors,
particle accelerators, gelfoam sponge depots, other commercially
available depot materials, osmotic pumps, oral or suppositorial
solid pharmaceutical formulations, decanting or topical
applications during surgery, aerosol delivery. Such methods are
known in the art. METH1 and/or METH2 polypeptides may be
administered as part of a pharmaceutical composition, described in
more detail below. Methods of delivering METH1 and/or METH2
polynucleotides are described in more detail herein.
[0242] Diseases at the Cellular Level
[0243] Diseases associated with increased cell survival or the
inhibition of apoptosis that could be treated or detected by METH1
and/or METH2 polynucleotides or polypeptides, as well as
antagonists or agonists of METH1 and/or METH2, include cancers
(such as follicular lymphomas, carcinomas with p53 mutations, and
hormone-dependent tumors, including, but not limited to colon
cancer, cardiac tumors, pancreatic cancer, melanoma,
retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's
sarcoma and ovarian cancer); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection. In
preferred embodiments, METH1 and/or METH2 polynucleotides,
polypeptides, and/or antagonists of the invention are used to
inhibit growth, progression, and/or metasis of cancers, in
particular those listed above.
[0244] Additional diseases or conditions associated with increased
cell survival that could be treated or detected by METH1 and/or
METH2 polynucleotides or polypeptides, or agonists or antagonists
of METH1 and/or METH2, include, but are not limited to,
progression, and/or metastases of malignancies and related
disorders such as leukemia (including acute leukemias (e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, and solid tumors including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0245] Diseases associated with increased apoptosis that could be
treated or detected by METH1 and/or METH2 polynucleotides or
polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, include AIDS; neurodegenerative disorders (such as
Alzheimer's disease, Parkinson's disease, Amyotrophic lateral
sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain
tumor or prior associated disease); autoimmune disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis,
systemic lupus erythematosus and immune-related glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as
aplastic anemia), graft v. host disease, ischemic injury (such as
that caused by myocardial infarction, stroke and reperfusion
injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and
liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock, cachexia and anorexia.
[0246] Wound Healing and Epithelial Cell Proliferation
[0247] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing METH1 and/or
METH2 polynucleotides or polypeptides, as well as agonists or
antagonists of METH1 and/or METH2, for therapeutic purposes, for
example, to stimulate epithelial cell proliferation and basal
keratinocytes for the purpose of wound healing, and to stimulate
hair follicle production and healing of dermal wounds. METH1 and/or
METH2 polynucleotides or polypeptides, as well as agonists or
antagonists of METH1 and/or METH2, may be clinically useful in
stimulating wound healing including surgical wounds, excisional
wounds, deep wounds involving damage of the dermis and epidermis,
eye tissue wounds, dental tissue wounds, oral cavity wounds,
diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,
venous stasis ulcers, burns resulting from heat exposure or
chemicals, and other abnormal wound healing conditions such as
uremia, malnutrition, vitamin deficiencies and complications
associated with systemic treatment with steroids, radiation therapy
and antineoplastic drugs and antimetabolites. METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could be used to promote dermal
reestablishment subsequent to dermal loss
[0248] METH1 and/or METH2 polynucleotides or polypeptides, as well
as agonists or antagonists of METH1 and/or METH2, could be used to
increase the adherence of skin grafts to a wound bed and to
stimulate re-epithelialization from the wound bed. The following
are types of grafts that METH1 and/or METH2 polynucleotides or
polypeptides, agonists or antagonists of METH1 and/or METH2, could
be used to increase adherence to a wound bed: autografts,
artificial skin, allografts, autodermic graft, autoepdermic grafts,
avacular grafts, Blair-Brown grafts, bone graft, brephoplastic
grafts, cutis graft, delayed graft, dermic graft, epidermic graft,
fascia graft, full thickness graft, heterologous graft, xenograft,
homologous graft, hyperplastic graft, lamellar graft, mesh graft,
mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,
pedicle graft, penetrating graft, split skin graft, thick split
graft. METH1 and/or METH2 polynucleotides or polypeptides, as well
as agonists or antagonists of METH1 and/or METH2, can be used to
promote skin strength and to improve the appearance of aged
skin.
[0249] It is believed that METH1 and/or METH2 polynucleotides or
polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, will also produce changes in hepatocyte proliferation, and
epithelial cell proliferation in the lung, breast, pancreas,
stomach, small intesting, and large intestine. METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could promote proliferation of epithelial
cells such as sebocytes, hair follicles, hepatocytes, type II
pneumocytes, mucin-producing goblet cells, and other epithelial
cells and their progenitors contained within the skin, lung, liver,
and gastrointestinal tract. METH1 and/or METH2 polynucleotides or
polypeptides, agonists or antagonists of METH1 and/or METH2, may
promote proliferation of endothelial cells, keratinocytes, and
basal keratinocytes.
[0250] METH1 and/or METH2 polynucleotides or polypeptides, as well
as agonists or antagonists of METH1 and/or METH2, could also be
used to reduce the side effects of gut toxicity that result from
radiation, chemotherapy treatments or viral infections. METH1
and/or METH2 polynucleotides or polypeptides, as well as agonists
or antagonists of METH1 and/or METH2, may have a cytoprotective
effect on the small intestine mucosa. METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, may also stimulate healing of mucositis
(mouth ulcers) that result from chemotherapy and viral
infections.
[0251] METH1 and/or METH2 polynucleotides or polypeptides, as well
as agonists or antagonists of METH1 and/or METH2, could further be
used in full regeneration of skin in full and partial thickness
skin defects, including burns, (i.e., repopulation of hair
follicles, sweat glands, and sebaceous glands), treatment of other
skin defects such as psoriasis. METH1 and/or METH2 polynucleotides
or polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, could be used to treat epidermolysis bullosa, a defect in
adherence of the epidermis to the underlying dermis which results
in frequent, open and painful blisters by accelerating
reepithelialization of these lesions. METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could also be used to treat gastric and
duodenal ulcers and help heal by scar formation of the mucosal
lining and regeneration of glandular mucosa and duodenal mucosal
lining more rapidly. Inflammatory bowel diseases, such as Crohn's
disease and ulcerative colitis, are diseases which result in
destruction of the mucosal surface of the small or large intestine,
respectively. Thus, METH1 and/or METH2 polynucleotides or
polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, could be used to promote the resurfacing of the mucosal
surface to aid more rapid healing and to prevent progression of
inflammatory bowel disease. Treatment with METH1 and/or METH2
polynucleotides or polypeptides, agonists or antagonists of METH1
and/or METH2, is expected to have a significant effect on the
production of mucus throughout the gastrointestinal tract and could
be used to protect the intestinal mucosa from injurious substances
that are ingested or following surgery. METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could be used to treat diseases associate
with the under expression of METH1 and/or METH2.
[0252] Moreover, METH1 and/or METH2 polynucleotides or
polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, could be used to prevent and heal damage to the lungs due to
various pathological states. A growth factor such as METH1 and/or
METH2 polynucleotides or polypeptides, as well as agonists or
antagonists of METH1 and/or METH2, could stimulate proliferation
and differentiation and promote the repair of alveoli and
bronchiolar epithelium to prevent or treat acute or chronic lung
damage. For example, emphysema, which results in the progressive
loss of alveoli, and inhalation injuries, i.e., resulting from
smoke inhalation and burns, that cause necrosis of the bronchiolar
epithelium and alveoli could be effectively treated using METH1
and/or METH2 polynucleotides or polypeptides, agonists or
antagonists of METH1 and/or METH2. Also, METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could be used to stimulate the proliferation
and differentiation of type II pneumocytes, which may help treat or
prevent diseases such as hyaline membrane diseases, such as infant
respiratory distress syndrome and bronchopulmonary displasia, in
premature infants.
[0253] METH1 and/or METH2 polynucleotides or polypeptides, as well
as agonists or antagonists of METH1 and/or METH2, could stimulate
the proliferation and differentiation of hepatocytes and, thus,
could be used to alleviate or treat liver diseases and pathologies
such as fulminant liver failure caused by cirrhosis, liver damage
caused by viral hepatitis and toxic substances (i.e.,
acetaminophen, carbon tetrachloride and other hepatotoxins known in
the art).
[0254] In addition, METH1 and/or METH2 polynucleotides or
polypeptides, as well as agonists or antagonists of METH1 and/or
METH2, could be used treat or prevent the onset of diabetes
mellitus. In patients with newly diagnosed Types I and II diabetes,
where some islet cell function remains, METH1 and/or METH2
polynucleotides or polypeptides, as well as agonists or antagonists
of METH1 and/or METH2, could be used to maintain the islet function
so as to alleviate, delay or prevent permanent manifestation of the
disease. Also, METH1 and/or METH2 polynucleotides or polypeptides,
as well as agonists or antagonists of METH1 and/or METH2, could be
used as an auxiliary in islet cell transplantation to improve or
promote islet cell function.
[0255] Infectious Disease
[0256] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, can be used to treat
or detect infectious agents. For example, by increasing the immune
response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be
treated. The immune response may be increased by either enhancing
an existing immune response, or by initiating a new immune
response. Alternatively, METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2, may
also directly inhibit the infectious agent, without necessarily
eliciting an immune response.
[0257] Viruses are one example of an infectious agent that can
cause disease or symptoms that can be treated or detected by METH1
and/or METH2 polynucleotides or polypeptides, or agonists or
antagonists of METH1 and/or METH2. Examples of viruses, include,
but are not limited to the following DNA and RNA viral families:
Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae,
Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus
(e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae,
Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia),
Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling
within these families can cause a variety of diseases or symptoms,
including, but not limited to: arthritis, bronchiollitis,
encephalitis, eye infections (e.g., conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active,
Delta), meningitis, opportunistic infections (e.g., AIDS),
pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever,
Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases
(e.g., Kaposi's, warts), and viremia. METH1 and/or METH2
polynucleotides or polypeptides, or agonists or antagonists of
METH1 and/or METH2, can be used to treat or detect any of these
symptoms or diseases.
[0258] Similarly, bacterial or fungal agents that can cause disease
or symptoms and that can be treated or detected by METH1 and/or
METH2 polynucleotides or polypeptides, or agonists or antagonists
of METH1 and/or METH2, include, but not limited to, the following
Gram-Negative and Gram-positive bacterial families and fungi:
Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),
Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis,
Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella,
Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis,
Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g.,
Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections
(e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas,
Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. These
bacterial or fungal families can cause the following diseases or
symptoms, including, but not limited to: bacteremia, endocarditis,
eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis,
opportunistic infections (e.g., AIDS related infections),
paronychia, prosthesis-related infections, Reiter's Disease,
respiratory tract infections, such as Whooping Cough or Empyema,
sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid
Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis,
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,
Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo,
Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound infections. METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2, can
be used to treat or detect any of these symptoms or diseases.
[0259] Moreover, parasitic agents causing disease or symptoms that
can be treated or detected by METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2,
include, but are not limited to, the following families: Amebiasis,
Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,
Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
These parasites can cause a variety of diseases or symptoms,
including, but not limited to: Scabies, Trombiculiasis, eye
infections, intestinal disease (e.g., dysentery, giardiasis), liver
disease, lung disease, opportunistic infections (e.g., AIDS
related), Malaria, pregnancy complications, and toxoplasmosis.
METH1 and/or METH2 polynucleotides or polypeptides, or agonists or
antagonists of METH1 and/or METH2, can be used to treat or detect
any of these symptoms or diseases.
[0260] Preferably, treatment using METH1 and/or METH2
polynucleotides or polypeptides, or agonists or antagonists of
METH1 and/or METH2, could either be by administering an effective
amount of METH1 and/or METH2 polypeptide to the patient, or by
removing cells from the patient, supplying the cells with METH1
and/or METH2 polynucleotide, and returning the engineered cells to
the patient (ex vivo therapy). Moreover, the METH1 and/or METH2
polypeptide or polynucleotide can be used as an antigen in a
vaccine to raise an immune response against infectious disease.
[0261] Regeneration
[0262] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, can be used to
differentiate, proliferate, and attract cells, leading to the
regeneration of tissues. (See, Science 276:59-87 (1997).) The
regeneration of tissues could be used to repair, replace, or
protect tissue damaged by congenital defects, trauma (wounds,
burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery,
including cosmetic plastic surgery, fibrosis, reperfusion injury,
or systemic cytokine damage.
[0263] Tissues that could be regenerated using the present
invention include organs (e.g., pancreas, liver, intestine, kidney,
skin, endothelium), muscle (smooth, skeletal or cardiac),
vasculature (including vascular and lymphatics), nervous,
hematopoietic, and skeletal (bone, cartilage, tendon, and ligament)
tissue. Preferably, regeneration occurs without or decreased
scarring. Regeneration also may include angiogenesis.
[0264] Moreover, METH1 and/or METH2 polynucleotides or
polypeptides, or agonists or antagonists of METH1 and/or METH2, may
increase regeneration of tissues difficult to heal. For example,
increased tendon/ligament regeneration would quicken recovery time
after damage. METH1 and/or METH2 polynucleotides or polypeptides,
or agonists or antagonists of METH1 and/or METH2, of the present
invention could also be used prophylactically in an effort to avoid
damage. Specific diseases that could be treated include of
tendinitis, carpal tunnel syndrome, and other tendon or ligament
defects. A further example of tissue regeneration of non-healing
wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
[0265] Similarly, nerve and brain tissue could also be regenerated
by using METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, to proliferate and
differentiate nerve cells. Diseases that could be treated using
this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal
cord disorders, head trauma, cerebrovascular disease, and stoke).
Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropathy (e.g., resulting from chemotherapy or other
medical therapies), localized neuropathies, and central nervous
system diseases (e.g., Alzheimer's disease, Parkinson's disease,
Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome), could all be treated using the METH1 and/or METH2
polynucleotides or polypeptides, or agonists or antagonists of
METH1 and/or METH2.
[0266] Chemotaxis
[0267] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may have chemotaxis
activity. A chemotaxic molecule attracts or mobilizes cells (e.g.,
monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils, epithelial and/or endothelial cells) to a particular
site in the body, such as a site of inflammation, infection, or
hyperproliferation. The mobilized cells can then fight off and/or
heal the particular trauma or abnormality.
[0268] METH1 and/or METH2 polynucleotides or polypeptides, or
agonists or antagonists of METH1 and/or METH2, may increase
chemotaxic activity of particular cells. These chemotactic
molecules can then be used to treat inflammation, infection,
hyperproliferative disorders, or any immune system disorder by
increasing the number of cells targeted to a particular location in
the body. For example, chemotaxic molecules can be used to treat
wounds and other trauma to tissues by attracting immune cells to
the injured location. As a chemotactic molecule, METH1 and/or METH2
could also attract fibroblasts, which can be used to treat
wounds.
[0269] It is also contemplated that METH1 and/or METH2
polynucleotides or polypeptides, or agonists or antagonists of
METH1 and/or METH2, may inhibit chemotactic activity. These
molecules could also be used to treat disorders. Thus, METH1 and/or
METH2 polynucleotides or polypeptides, or agonists or antagonists
of METH1 and/or METH2, could be used as an inhibitor of
chemotaxis.
[0270] Binding Activity
[0271] METH1 and/or METH2 polypeptides may be used to screen for
molecules that bind to METH1 and/or METH2 or for molecules to which
METH1 and/or METH2 binds. The binding of METH1 and/or METH2 and the
molecule may activate (agonist), increase, inhibit (antagonist), or
decrease activity of the METH1 and/or METH2 or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides,
proteins (e.g., receptors), or small molecules.
[0272] Preferably, the molecule is closely related to the natural
ligand of METH1 and/or METH2, e.g., a fragment of the ligand, or a
natural substrate, a ligand, a structural or functional mimetic.
(See, Coligan et al., Current Protocols in Immunology 1(2): Chapter
5 (1991).) Similarly, the molecule can be closely related to the
natural receptor to which METH1 and/or METH2 binds, or at least, a
fragment of the receptor capable of being bound by METH1 and/or
METH2 (e.g., active site). In either case, the molecule can be
rationally designed using known techniques.
[0273] Preferably, the screening for these molecules involves
producing appropriate cells which express METH1 and/or METH2,
either as a secreted protein or on the cell membrane. Preferred
cells include cells from mammals, yeast, Drosophila, or E. coli.
Cells expressing METH1 and/or METH2 (or cell membrane containing
the expressed polypeptide) are then preferably contacted with a
test compound potentially containing the molecule to observe
binding, stimulation, or inhibition of activity of either METH1
and/or METH2 or the molecule.
[0274] The assay may simply test binding of a candidate compound to
METH1 and/or METH2, wherein binding is detected by a label, or in
an assay involving competition with a labeled competitor. Further,
the assay may test whether the candidate compound results in a
signal generated by binding to METH1 and/or METH2.
[0275] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing METH1 and/or METH2, measuring METH1 and/or
METH2/molecule activity or binding, and comparing the METH1 and/or
METH2/molecule activity or binding to a standard.
[0276] Preferably, an ELISA assay can measure METH1 and/or METH2
level or activity in a sample (e.g., biological sample) using a
monoclonal or polyclonal antibody. The antibody can measure METH1
and/or METH2 level or activity by either binding, directly or
indirectly, to METH1 and/or METH2 or by competing with METH1 and/or
METH2 for a substrate.
[0277] Additionally, the receptor to which METH1 and/or METH2 binds
can be identified by numerous methods known to those of skill in
the art, for example, ligand panning and FACS sorting (Coligan, et
al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For
example, expression cloning is employed wherein polyadenylated RNA
is prepared from a cell responsive to the polypeptides, for
example, NIH3T3 cells which are known to contain multiple receptors
for the FGF family proteins, and SC-3 cells, and a cDNA library
created from this RNA is divided into pools and used to transfect
COS cells or other cells that are not responsive to the
polypeptides. Transfected cells which are grown on glass slides are
exposed to the polypeptide of the present invention, after they
have been labelled. The polypeptides can be labeled by a variety of
means including iodination or inclusion of a recognition site for a
site-specific protein kinase.
[0278] Following fixation and incubation, the slides are subjected
to auto-radiographic analysis. Positive pools are identified and
sub-pools are prepared and re-transfected using an iterative
sub-pooling and re-screening process, eventually yielding a single
clones that encodes the putative receptor.
[0279] As an alternative approach for receptor identification, the
labeled polypeptides can be photoaffinity linked with cell membrane
or extract preparations that express the receptor molecule.
Cross-linked material is resolved by PAGE analysis and exposed to
X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and
subjected to protein microsequencing. The amino acid sequence
obtained from microsequencing would be used to design a set of
degenerate oligonucleotide probes to screen a cDNA library to
identify the genes encoding the putative receptors.
[0280] Moreover, the techniques of gene-shuffling, motif-shuffling,
exon-shuffling, and/or codon-shuffling (collectively referred to as
"DNA shuffling") may be employed to modulate the activities of
METH1 or METH2 thereby effectively generating agonists and
antagonists of METH1 or METH2. See generally, U.S. Pat. Nos.
5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; and
Patten, P. A. et al., Curr. Opinion Biotechnol. 8:724-733 (1997);
Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O.
et al., J. Mol. Biol. 287:265-276 (1999); and Lorenzo, M. M. and
Blasco, R. Biotechniques 24(2):308-313 (1998) (each of these
patents and publications are hereby incorporated by reference). In
one embodiment, alteration of METH1 or METH2 polynucleotides and
corresponding polypeptides may be achieved by DNA shuffling. DNA
shuffling involves the assembly of two or more DNA segments into a
desired METH1 or METH2 molecule by homologous, or site-specific,
recombination.
[0281] In another embodiment, METH1 or METH2 polynucleotides and
corresponding polypeptides may be altered by being subjected to
random mutagenesis by error-prone PCR, random nucleotide insertion,
or other methods prior to recombination. In another embodiment, one
or more components, motifs, sections, parts, domains, fragments,
etc., or METH1 or METH2 may be recombined with one or more
components, motifs, sections, parts, domains, fragments, etc. of
one or more heterologous molecules. In preferred embodiments, the
heterologous molecule is a growth factor such as, for example,
platelet-derived growth factor (PDGF), insulin-like growth factor
(IGI-I), transforming growth factor (TGF)-alpha, epidermal growth
factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone
morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-6, BMP-7,
activins A and B, decapentaplegic (dpp), 60A, OP-2, dorsalin,
growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived
neutrophic factor (GDNF).
[0282] Other preferred fragments are biologically active METH1 or
METH2 fragments. Biologically active fragments are those exhibiting
activity similar, but not necessarily identical, to an activity of
the METH1 or METH2 polypeptide. The biological activity of the
fragments may include an improved desired activity, or a decreased
undesirable activity.
[0283] Additionally, this invention provides a method of screening
compounds to identify those which modulate the action of the
polypeptide of the present invention. An example of such an assay
comprises combining a mammalian fibroblast cell, the polypeptide of
the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell
would normally proliferate. A control assay may be performed in the
absence of the compound to be screened and compared to the amount
of fibroblast proliferation in the presence of the compound to
determine if the compound stimulates proliferation by determining
the uptake of 3[H] thymidine in each case. The amount of fibroblast
cell proliferation is measured by liquid scintillation
chromatography which measures the incorporation of 3[H] thymidine.
Both agonist and antagonist compounds may be identified by this
procedure.
[0284] In another method, a mammalian cell or membrane preparation
expressing a receptor for a polypeptide of the present invention is
incubated with a labeled polypeptide of the present invention in
the presence of the compound. The ability of the compound to
enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system
following interaction of a compound to be screened and the METH1
and/or METH2 receptor is measured and the ability of the compound
to bind to the receptor and elicit a second messenger response is
measured to determine if the compound is a potential agonist or
antagonist. Such second messenger systems include but are not
limited to, cAMP guanylate cyclase, ion channels or
phosphoinositide hydrolysis.
[0285] All of these above assays can be used as diagnostic or
prognostic markers. The molecules discovered using these assays can
be used to treat disease or to bring about a particular result in a
patient (e.g., blood vessel growth) by activating or inhibiting the
METH1 and/or METH2/molecule. Moreover, the assays can discover
agents which may inhibit or enhance the production of METH1 and/or
METH2 from suitably manipulated cells or tissues.
[0286] Therefore, the invention includes a method of identifying
compounds which bind to METH1 and/or METH2 comprising the steps of:
(a) incubating a candidate binding compound with METH1 and/or
METH2; and (b) determining if binding has occurred. Moreover, the
invention includes a method of identifying agonists/antagonists
comprising the steps of: (a) incubating a candidate compound with
METH1 and/or METH2, (b) assaying a biological activity, and (b)
determining if a biological activity of METH1 and/or METH2 has been
altered.
[0287] Also, one could identify molecules which bind METH1 and/or
METH2 experimentally by using the beta-pleated sheet regions
disclosed in FIGS. 10 and 11 and Tables 1 and 2. Accordingly,
specific embodiments of the invention are directed to
polynucleotides encoding polypeptides which comprise, or
alternatively consist of, the amino acid sequence of each beta
pleated sheet regions disclosed in FIG. 10/Table 1 and FIG.
11/Table 2. Additional embodiments of the invention are directed to
polynucleotides encoding METH1 and/or METH2 polypeptides which
comprise, or alternatively consist of, any combination or all of
the beta pleated sheet regions disclosed in FIG. 10/Table 1 and
FIG. 11/Table 2. Additional preferred embodiments of the invention
are directed to polypeptides which comprise, or alternatively
consist of, the METH1 and/or METH2 amino acid sequence of each of
the beta pleated sheet regions disclosed in FIG. 10/Table 1 and
FIG. 11/Table 2. Additional embodiments of the invention are
directed to METH1 and/or METH2 polypeptides which comprise, or
alternatively consist of, any combination or all of the beta
pleated sheet regions disclosed in FIG. 10/Table 1 and FIG.
11/Table 2.
[0288] Antisense And Ribozyme (Antagonists)
[0289] In specific embodiments, antagonists according to the
present invention are nucleic acids corresponding to the sequences
contained in SEQ ID NO:1 or 3, or the complementary strand thereof,
and/or to nucleotide sequences contained in the deposited clones.
In one embodiment, antisense sequence is generated internally by
the organism, in another embodiment, the antisense sequence is
separately administered (see, for example, O'Connor, J., Neurochem.
56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense
technology can be used to control gene expression through antisense
DNA or RNA, or through triple-helix formation. Antisense techniques
are discussed for example, in Okano, J., Neurochem. 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988). Triple helix formation is
discussed in, for instance, Lee et al, Nucleic Acids Research
6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et
al., Science 251:1300 (1991). The methods are based on binding of a
polynucleotide to a complementary DNA or RNA.
[0290] For example, the 5' coding portion of a polynucleotide that
encodes the mature polypeptide of the present invention may be used
to design an antisense RNA oligonucleotide of from about 10 to 40
base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
thereby preventing transcription and the production of the
receptor. The antisense RNA oligonucleotide hybridizes to the mRNA
in vivo and blocks translation of the mRNA molecule into receptor
polypeptide.
[0291] In one embodiment, the METH1 and/or METH2 antisense nucleic
acid of the invention is produced intracellularly by transcription
from an exogenous sequence. For example, a vector or a portion
thereof, is transcribed, producing an antisense nucleic acid (RNA)
of the invention. Such a vector would contain a sequence encoding
the METH1 and/or METH2 antisense nucleic acid. Such a vector can
remain episomal or become chromosomally integrated, as long as it
can be transcribed to produce the desired antisense RNA. Such
vectors can be constructed by recombinant DNA technology methods
standard in the art. Vectors can be plasmid, viral, or others know
in the art, used for replication and expression in vertebrate
cells. Expression of the sequence encoding METH1 and/or METH2, or
fragments thereof, can be by any promoter known in the art to act
in vertebrate, preferably human cells. Such promoters can be
inducible or constitutive. Such promoters include, but are not
limited to, the SV40 early promoter region (Bernoist and Chambon,
Nature 29:304-310 (1981), the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell
22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,
Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory
sequences of the metallothionein gene (Brinster, et al., Nature
296:39-42 (1982)), etc.
[0292] The antisense nucleic acids of the invention comprise a
sequence complementary to at least a portion of an RNA transcript
of a METH1 and/or METH2 gene. However, absolute complementarity,
although preferred, is not required. A sequence "complementary to
at least a portion of an RNA," referred to herein, means a sequence
having sufficient complementarity to be able to hybridize with the
RNA, forming a stable duplex; in the case of double stranded METH1
and/or METH2 antisense nucleic acids, a single strand of the duplex
DNA may thus be tested, or triplex formation may be assayed. The
ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid
Generally, the larger the hybridizing nucleic acid, the more base
mismatches with a METH1 and/or METH2 RNA it may contain and still
form a stable duplex (or triplex as the case may be). One skilled
in the art can ascertain a tolerable degree of mismatch by use of
standard procedures to determine the melting point of the
hybridized complex.
[0293] Oligonucleotides that are complementary to the 5' end of the
message, e.g., the 5' untranslated sequence up to and including the
AUG initiation codon, should work most efficiently at inhibiting
translation. However, sequences complementary to the 3'
untranslated sequences of mRNAs have been shown to be effective at
inhibiting translation of mRNAs as well. See generally, Wagner, R.,
1994, Nature 372:333-335. Thus, oligonucleotides complementary to
either the 5'- or 3'-non-translated, non-coding regions of METH1
and/or METH2 shown in FIG. 1 could be used in an antisense approach
to inhibit translation of endogenous METH1 and/or METH2 mRNA.
Oligonucleotides complementary to the 5' untranslated region of the
mRNA should include the complement of the AUG start codon.
Antisense oligonucleotides complementary to mRNA coding regions are
less efficient inhibitors of translation but could be used in
accordance with the invention. Whether designed to hybridize to the
5'-, 3'- or coding region of METH1 and/or METH2 mRNA, antisense
nucleic acids should be at least six nucleotides in length, and are
preferably oligonucleotides ranging from 6 to about 50 nucleotides
in length. In specific aspects the oligonucleotide is at least 10
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at
least 50 nucleotides.
[0294] The polynucleotides of the invention can be DNA or RNA or
chimeric mixtures or derivatives or modified versions thereof,
single-stranded or double-stranded. The oligonucleotide can be
modified at the base moiety, sugar moiety, or phosphate backbone,
for example, to improve stability of the molecule, hybridization,
etc. The oligonucleotide may include other appended groups such as
peptides (e.g., for targeting host cell receptors in vivo), or
agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;
Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication No. WO88/09810, published Dec. 15, 1988) or the
blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,
published Apr. 25, 1988), hybridization-triggered cleavage agents.
(See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or
intercalating agents. (See, e.g., Zon, 1988, Pharm. Res.
5:539-549). To this end, the oligonucleotide may be conjugated to
another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0295] The antisense oligonucleotide may comprise at least one
modified base moiety which is selected from the group including,
but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)w, and
2,6-diaminopurine.
[0296] The antisense oligonucleotide may also comprise at least one
modified sugar moiety selected from the group including, but not
limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0297] In yet another embodiment, the antisense oligonucleotide
comprises at least one modified phosphate backbone selected from
the group including, but not limited to, a phosphorothioate, a
phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester,
and a formacetal or analog thereof.
[0298] In yet another embodiment, the antisense oligonucleotide is
an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual b-units, the strands run parallel to each
other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The
oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al., 1987,
Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue
(Inoue et al., 1987, FEBS Lett. 215:327-330).
[0299] Polynucleotides of the invention may be synthesized by
standard methods known in the art, e.g. by use of an automated DNA
synthesizer (such as are commercially available from Biosearch,
Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides may be synthesized by the method of Stein et al.
(1988, Nucl. Acids Res. 16:3209), methylphosphonate
oligonucleotides can be prepared by use of controlled pore glass
polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A.
85:7448-7451), etc.
[0300] While antisense nucleotides complementary to the METH1
and/or METH2 coding region sequence could be used, those
complementary to the transcribed untranslated region are most
preferred.
[0301] Potential antagonists according to the invention also
include catalytic RNA, or a ribozyme (See, e.g., PCT International
Publication WO 90/11364, published Oct. 4, 1990; Sarver et al.,
Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at
site specific recognition sequences can be used to destroy METH1
and/or METH2 mRNAs, the use of hammerhead ribozymes is preferred.
Hammerhead ribozymes cleave mRNAs at locations dictated by flanking
regions that form complementary base pairs with the target mRNA.
The sole requirement is that the target mRNA have the following
sequence of two bases: 5'-UG-3'. The construction and production of
hammerhead ribozymes is well known in the art and is described more
fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are
numerous potential hammerhead ribozyme cleavage sites within the
nucleotide sequence of METH1 and/or METH2 (FIGS. 1 and 2).
Preferably, the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the METH1 and/or
METH2 mRNA; i.e., to increase efficiency and minimize the
intracellular accumulation of non-functional mRNA transcripts.
[0302] As in the antisense approach, the ribozymes of the invention
can be composed of modified oligonucleotides (e.g. for improved
stability, targeting, etc.) and should be delivered to cells which
express METH1 and/or METH2 in vivo. DNA constructs encoding the
ribozyme may be introduced into the cell in the same manner as
described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct
"encoding" the ribozyme under the control of a strong constitutive
promoter, such as, for example, pol III or pol II promoter, so that
transfected cells will produce sufficient quantities of the
ribozyme to destroy endogenous METH1 and/or METH2 messages and
inhibit translation. Since ribozymes, unlike antisense molecules,
are catalytic, a lower intracellular concentration is required for
efficiency.
[0303] Antagonist/agonist compounds may be employed to inhibit the
cell growth and proliferation effects of the polypeptides of the
present invention on neoplastic cells and tissues, i.e. stimulation
of angiogenesis of tumors, and, therefore, retard or prevent
abnormal cellular growth and proliferation, for example, in tumor
formation or growth.
[0304] The antagonist/agonist may also be employed to prevent
hyper-vascular diseases, and prevent the proliferation of
epithelial lens cells after extracapsular cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the
present invention may also be desirous in cases such as restenosis
after balloon angioplasty.
[0305] The antagonist/agonist may also be employed to prevent the
growth of scar tissue during wound healing.
[0306] The antagonist/agonist may also be employed to treat the
diseases described herein.
[0307] Other Activities
[0308] As stated below, METH1 and METH2 share structural and
sequence homology with members of the ADAM family. ADAM proteins
have been shown to proteolytically process membrane-anchored
proteins, including TNF (Black et al., Nature 385:729 (1997); Moss
et al., Nature 385:733 (1997)). Thus, METH1 and/or METH2 may be
useful in proteolytic processing of membrane-anchored proteins.
Membrane-anchored proteins which may be proteolytically processed
by METH1 and/or METH2 include cytokines, growth factors, cytokine
receptors and growth factor receptors.
[0309] METH1 or METH2 polypeptides or polynucleotides, or agonists
or antagonists of METH1 and/or METH2, may also increase or decrease
the differentiation or proliferation of embryonic stem cells,
besides, as discussed above, hematopoietic lineage.
[0310] METH1 or METH2 polypeptides or polynucleotides, or agonists
or antagonists of METH1 and/or METH2, may also be used to modulate
mammalian characteristics, such as body height, weight, hair color,
eye color, skin, percentage of adipose tissue, pigmentation, size,
and shape (e.g., cosmetic surgery). Similarly, METH1 or METH2
polypeptides or polynucleotides, or agonists or antagonists of
METH1 and/or METH2, may be used to modulate mammalian metabolism
affecting catabolism, anabolism, processing, utilization, and
storage of energy.
[0311] As angiogenesis is a key factor in supporting adipose
tissue, METH1 or METH2 polypeptides or polynucleotides, or agonists
or antagonists of METH1 and/or METH2 may be used to control weight,
reduce weight, treat obesity, and/or control adipose tissue in an
individual.
[0312] METH1 or METH2 polypeptides or polynucleotides, or agonists
or antagonists of METH1 and/or METH2, may be used to change a
mammal's mental state or physical state by influencing biorhythms,
circadian rhythms, depression (including depressive disorders),
tendency for violence, tolerance for pain, reproductive
capabilities (preferably by Activin or Inhibin-like activity),
hormonal or endocrine levels, appetite, libido, memory, stress, or
other cognitive qualities.
[0313] METH1 or METH2 polypeptides or polynucleotides, or agonists
or antagonists of METH1 and/or METH2, may also be used as a food
additive or preservative, such as to increase or decrease storage
capabilities, fat content, lipid, protein, carbohydrate, vitamins,
minerals, cofactors or other nutritional components.
[0314] The above-recited applications have uses in a wide variety
of hosts. Such hosts include, but are not limited to, human,
murine, rabbit, goat, guinea pig, camel, horse, mouse, rat,
hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat,
non-human primate, and human. In specific embodiments, the host is
a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig,
sheep, dog or cat. In preferred embodiments, the host is a mammal.
In most preferred embodiments, the host is a human.
Anti-Angiogenesis
[0315] As shown in Examples 4 and 5, METH1 and METH2 inhibit
angiogenesis. Thus, the present invention provides a method of
treating an angiogenesis-related disease and/or disorder,
comprising administering to an individual in need thereof a
therapeutically effective amount of a METH1 and/or METH2
polynucleotide, polypeptide, and/or agonist.
[0316] For example, METH1 and/or METH2 polynucleotides,
polypeptides, and/or agonists may be utilized in a variety of
additional methods in order to therapeutically treat a cancer or
tumor. Cancers which may be treated with METH1 and/or METH2
polynucleotides, polypeptides and/or agonists include, but are not
limited to solid tumors, including prostate, lung, breast, ovarian,
stomach, pancreas, larynx, esophagus, testes, liver, parotid,
biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,
bladder, thyroid cancer; primary tumors and metastases; melanomas;
glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung
cancer; colorectal cancer; advanced malignancies; and blood born
tumors such as leukemias. For example, METH1 and/or METH2
polynucleotides, polypeptides, and/or agonists may be delivered
topically, in order to treat cancers such as skin cancer, head and
neck tumors, breast tumors, and Kaposi's sarcoma. Within yet other
aspects, METH1 and/or METH2 polynucleotides, polypeptides, and/or
agonists may be utilized to treat superficial forms of bladder
cancer by, for example, intravesical administration. METH1 and/or
METH2 polynucleotides, polypeptides and/or agonists may be
delivered directly into the tumor, or near the tumor site, via
injection or a catheter. Of course, as the artisan of ordinary
skill will appreciate, the appropriate mode of administration will
vary according to the cancer to be treated. Other modes of delivery
are discussed herein.
[0317] METH1 and/or METH2 polynucleotides, polypeptides and/or
agonists may be useful in treating other disorders, besides
cancers, which involve angiogenesis. These disorders include, but
are not limited to: benign tumors, for example hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas; artheroscleric plaques; ocular angiogenic diseases, for
example, diabetic retinopathy, retinopathy of prematurity, macular
degeneration, corneal graft rejection, neovascular glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia (abnormal blood vessel growth) of the eye; rheumatoid
arthritis; psoriasis; delayed wound healing; endometriosis;
vasculogenesis; granulations; hypertrophic scars (keloids);
nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial angiogenesis; coronary collaterals; cerebral
collaterals; arteriovenous malformations; ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular
dysplasia; wound granulation; Crohn's disease; and
atherosclerosis.
[0318] For example, within one aspect of the present invention
methods are provided for treating hypertrophic scars and keloids,
comprising the step of administering a METH1 and/or METH2
polynucleotide, polypeptide, and/or agonist to a hypertrophic scar
or keloid. Within one embodiment of the present invention METH1
and/or METH2 polynucleotides, polypeptides, and/or agonists are
directly injected into a hypertrophic scar or keloid, in order to
prevent the progression of these lesions. This therapy is of
particular value in the prophylactic treatment of conditions which
are known to result in the development of hypertrophic scars and
keloids (e.g., burns), and is preferably initiated after the
proliferative phase has had time to progress (approximately 14 days
after the initial injury), but before hypertrophic scar or keloid
development.
[0319] As noted above, the present invention also provides methods
for treating neovascular diseases of the eye, including for
example, corneal neovascularization, neovascular glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and
macular degeneration, as well as other eye inflammatory diseases,
ocular tumors and diseases associated with choroidal or iris
neovascularization. See, e.g., reviews by Waltman et al., Am. J.
Ophthal 85:704-710 (1978) and Gartner et al., Surv. Ophthal.
22:291-312 (1978).
[0320] Thus, within one aspect of the present invention methods are
provided for treating neovascular diseases of the eye such as
corneal neovascularization (including corneal graft
neovascularization), comprising the step of administering to a
patient a therapeutically effective amount of a METH1 and/or METH2
compound (as described above) to the cornea, such that the
formation of blood vessels is inhibited. Briefly, the cornea is a
tissue which normally lacks blood vessels. In certain pathological
conditions however, capillaries may extend into the cornea from the
pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized, it also becomes clouded, resulting in a decline in
the patient's visual acuity. Visual loss may become complete if the
cornea completely opacitates.
[0321] A wide variety of disorders can result in corneal
neovascularization, including for example, corneal infections
(e.g., trachoma, herpes simplex keratitis, leishmaniasis and
onchocerciasis), immunological processes (e.g., graft rejection and
Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of
any cause), toxic and nutritional deficiency states, and as a
complication of wearing contact lenses.
[0322] Within particularly preferred embodiments of the invention,
METH1 and/or METH2 may be prepared for topical administration in
saline (combined with any of the preservatives and antimicrobial
agents commonly used in ocular preparations), and administered in
eyedrop form. The solution or suspension may be prepared in its
pure form and administered several times daily. Alternatively,
anti-angiogenic compositions, prepared as described above, may also
be administered directly to the cornea. Within preferred
embodiments, the anti-angiogenic composition is prepared with a
muco-adhesive polymer which binds to cornea. Within further
embodiments, the anti-angiogenic factors or anti-angiogenic
compositions may be utilized as an adjunct to conventional steroid
therapy. Topical therapy may also be useful prophylactically in
corneal lesions which are known to have a high probability of
inducing an angiogenic response (such as chemical burns). In these
instances the treatment, likely in combination with steroids, may
be instituted immediately to help prevent subsequent
complications.
[0323] Within other embodiments, the compounds described above may
be injected directly into the corneal stroma by an ophthalmologist
under microscopic guidance. The preferred site of injection may
vary with the morphology of the individual lesion, but the goal of
the administration would be to place the composition at the
advancing front of the vasculature (i.e., interspersed between the
blood vessels and the normal cornea). In most cases this would
involve perilimbic corneal injection to "protect" the cornea from
the advancing blood vessels. This method may also be utilized
shortly after a corneal insult in order to prophylactically prevent
corneal neovascularization. In this situation the material could be
injected in the perilimbic cornea interspersed between the corneal
lesion and its undesired potential limbic blood supply. Such
methods may also be utilized in a similar fashion to prevent
capillary invasion of transplanted corneas. In a sustained-release
form injections might only be required 2-3 times per year. A
steroid could also be added to the injection solution to reduce
inflammation resulting from the injection itself
[0324] Within another aspect of the present invention, methods are
provided for treating neovascular glaucoma, comprising the step of
administering to a patient a therapeutically effective amount of a
METH1 and/or METH2 polypeptide, polynucleotide, and/or agonist to
the eye, such that the formation of blood vessels is inhibited. In
one embodiment, the compound may be administered topically to the
eye in order to treat early forms of neovascular glaucoma. Within
other embodiments, the compound may be implanted by injection into
the region of the anterior chamber angle. Within other embodiments,
the compound may also be placed in any location such that the
compound is continuously released into the aqueous humor.
[0325] Within another aspect of the present invention, methods are
provided for treating proliferative diabetic retinopathy,
comprising the step of administering to a patient a therapeutically
effective amount of a METH1 and/or METH2 polynucleotide,
polypeptide, and/or agonist to the eyes, such that the formation of
blood vessels is inhibited. Within particularly preferred
embodiments of the invention, proliferative diabetic retinopathy
may be treated by injection into the aqueous humor or the vitreous,
in order to increase the local concentration of the METH1 and/or
METH2 polynucleotide, polypeptide, and/or agonist in the retina.
Preferably, this treatment should be initiated prior to the
acquisition of severe disease requiring photocoagulation.
[0326] Within another aspect of the present invention, methods are
provided for treating retrolental fibroplasia, comprising the step
of administering to a patient a therapeutically effective amount of
a METH1 and/or METH2 polynucleotide, polypeptide, and/or agonist to
the eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous injection
and/or via intraocular implants.
[0327] METH1 and/or METH2 polynucleotides, polypeptides and/or
agonists may be used to treat diseases that have angiogenesis as a
pathologic consequence such as cat scratch disease (Rochele minalia
quintosa), ulcers (Helicobacter pylori), Bartonellosis and
bacillary angiomatosis.
[0328] METH1 and/or METH2 polynucleotides, polypeptides and/or
agonists may be used as a birth control agent by preventing
vascularization required for embryo implantation. In one aspect of
the birth control method, an amount of the compound sufficient to
block embryo implantation is administered before or after
intercourse and fertilization have occurred, thus providing an
effective method of birth control, possibly a "morning after"
method.
[0329] METH1 and/or METH2 polynucleotides, polypeptides and/or
agonists may also be used in controlling menstruation or
administered as either a peritoneal lavage fluid or for peritoneal
implantation in the treatment of endometriosis.
[0330] METH1 and/or METH2 polynucleotides, polypeptides, and/or
agonists of the present invention may be incorporated into surgical
sutures in order to prevent stitch granulomas.
[0331] METH1 and/or and METH2 polynucleotides, polypeptides, and/or
agonists may be utilized in a wide variety of surgical procedures.
For example, within one aspect of the present invention a METH1
and/or METH2 compositions (in the form of, for example, a spray or
film) may be utilized to coat or spray an area prior to removal of
a tumor, in order to isolate normal surrounding tissues from
malignant tissue, and/or to prevent the spread of disease to
surrounding tissues. Within other aspects of the present invention,
METH1 and/or METH2 compositions (e.g., in the form of a spray) may
be delivered via endoscopic procedures in order to coat tumors, or
inhibit angiogenesis in a desired locale. Within yet other aspects
of the present invention, surgical meshes which have been coated
with anti-angiogenic compositions of the present invention may be
utilized in any procedure wherein a surgical mesh might be
utilized. For example, within one embodiment of the invention a
surgical mesh laden with an anti-angiogenic composition may be
utilized during abdominal cancer resection surgery (e.g.,
subsequent to colon resection) in order to provide support to the
structure, and to release an amount of the anti-angiogenic
factor.
[0332] Within further aspects of the present invention, methods are
provided for treating tumor excision sites, comprising
administering a METH1 and/or METH2 polynucleotide, polypeptide,
and/or agonist to the resection margins of a tumor subsequent to
excision, such that the local recurrence of cancer and the
formation of new blood vessels at the site is inhibited. Within one
embodiment of the invention, the anti-angiogenic compound is
administered directly to the tumor excision site (e.g., applied by
swabbing, brushing or otherwise coating the resection margins of
the tumor with the anti-angiogenic compound). Alternatively, the
anti-angiogenic compounds may be incorporated into known surgical
pastes prior to administration. Within particularly preferred
embodiments of the invention, the anti-angiogenic compounds are
applied after hepatic resections for malignancy, and after
neurosurgical operations.
[0333] Within one aspect of the present invention, METH1 and/or
METH2 polynucleotides, polypeptides, and/or agonists may be
administered to the resection margin of a wide variety of tumors,
including for example, breast, colon, brain and hepatic tumors. For
example, within one embodiment of the invention, anti-angiogenic
compounds may be administered to the site of a neurological tumor
subsequent to excision, such that the formation of new blood
vessels at the site are inhibited.
[0334] The METH1 and/or METH2 polynucleotides, polypeptides, and/or
agonists of the present invention may also be administered along
with other anti-angiogenic factors. Representative examples of
other anti-angiogenic factors include: Anti-Invasive Factor,
retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of Metalloproteinase-1, Tissue Inhibitor of
Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen
Activator Inhibitor-2, and various forms of the lighter "d group"
transition metals.
[0335] Lighter "d group" transition metals include, for example,
vanadium, molybdenum, tungsten, titanium, niobium, and tantalum
species. Such transition metal species may form transition metal
complexes. Suitable complexes of the above-mentioned transition
metal species include oxo transition metal complexes.
Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate
complexes include metavanadate and orthovanadate complexes such as,
for example, ammonium metavanadate, sodium metavanadate, and sodium
orthovanadate. Suitable vanadyl complexes include, for example,
vanadyl acetylacetonate and vanadyl sulfate including vanadyl
sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
Representative examples of tungsten and molybdenum complexes also
include oxo complexes. Suitable oxo tungsten complexes include
tungstate and tungsten oxide complexes. Suitable tungstate
complexes include ammonium tungstate, calcium tungstate, sodium
tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and
molybdenyl complexes. Suitable molybdate complexes include ammonium
molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium molybdate and its hydrates. Suitable molybdenum oxides
include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic
acid. Suitable molybdenyl complexes include, for example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes include hydroxo derivatives derived from, for example,
glycerol, tartaric acid, and sugars.
[0336] A wide variety of other anti-angiogenic factors may also be
utilized within the context of the present invention.
Representative examples include platelet factor 4; protamine
sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated
Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this
compound may be enhanced by the presence of steroids such as
estrogen, and tamoxifen citrate); Staurosporine; modulators of
matrix metabolism, including for example, proline analogs,
cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,
.alpha.,.alpha.-dipyridyl, .beta.-aminopropionitrile fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate;
Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3
(Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin
(Tomkinson et al., Biochem J. 286:475-480, 1992);
.beta.-Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin;
Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium
Thiomalate ("GST"; Matsubara and Ziff, J. Clin. Invest.
79:1440-1446, 1987); .beta.-anticollagenase-serum;
.alpha.2-antiplasmin (Holmes et al., J. Biol. Chem.
262(4):1659-1664, 1987); Bisantrene (National Cancer Institute);
Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid
disodium or "CCA"; Takeuchi et al., Agents Actions 36:312-316,
1992); Thalidomide; Angostatic steroid; AGM-1470;
carboxynaminolmidazole; and metalloproteinase inhibitors such as
BB94.
Diagnostic Methods
[0337] The invention also relates to the use of METH1 or METH2
polynucleotides for use as diagnostic reagents. Detection of a
mutated form of the METH1 or METH2 gene associated with a
dysfunction will provide a diagnostic tool that can add to or
define a diagnosis of a disease or susceptibility to a disease
which results from under-expression, overexpression or altered
expression of METH1 or METH2. Individuals carrying mutations in the
METH1 or METH2 gene may be detected at the DNA level by a variety
of techniques.
[0338] Nucleic acids for diagnosis may be obtained from a subject's
cells, such as from blood, urine, saliva, tissue biopsy or autopsy
material. The genomic DNA may be used directly for detection or may
be amplified enzymatically by using PCR or other amplification
techniques prior to analysis. RNA or cDNA may also be used in
similar fashion. Deletions and insertions can be detected by a
change in size of the amplified product in comparison to the normal
genotype. Point mutations can be identified by hybridizing
amplified DNA to labeled METH1 or METH2 nucleotide sequences.
Perfectly matched sequences can be distinguished from mismatched
duplexes by RNase digestion or by differences in melting
temperatures. DNA sequence differences may also be detected by
alterations in electrophoretic mobility of DNA fragments in gels,
with or without denaturing agents, or by direct DNA sequencing.
See, e.g., Myers et al., Science 230:1242 (1985). Sequence changes
at specific locations may also be revealed by nuclease protection
assays, such as RNase and S1 protection or the chemical cleavage
method. See Cotton et al., Proc Natl Acad Sci USA 85:4397-4401
(1985). In another embodiment, an array of oligonucleotides probes
comprising METH1 or METH2 nucleotide sequence or fragments thereof
can be constructed to conduct efficient screening of e.g., genetic
mutations. Array technology methods are well known and have general
applicability and can be used to address a variety of questions in
molecular genetics including gene expression, genetic linkage, and
genetic variability. (See, for example, M. Chee et al., Science
274:610-613 (1996).
[0339] The diagnostic assays offer a process for diagnosing or
determining a susceptibility to angiogenic diseases (cancer, cancer
metastasis, chronic inflammatory disorders, rheumatoid arthritis,
atherosclerosis, macular degeneration, diabetic retinopathy),
restenosis, Alzheimer's disease and tissue remodeling through
detection of mutation in the METH1 or METH2 gene by the methods
described.
[0340] In addition, angiogenic diseases (cancer, cancer metastasis,
chronic inflammatory disorders, rheumatoid arthritis,
atherosclerosis, macular degeneration, diabetic retinopathy),
restenosis, Alzheimer's disease and tissue remodeling can be
diagnosed by methods comprising determining from a sample derived
from a subject an abnormally decreased or increased level of the
METH1 or METH2 polypeptide or METH1 or METH2 mRNA. Decreased or
increased expression can be measured at the RNA level using any of
the methods well known in the art for the quantitation of
polynucleotides, such as, for example, PCR, RT-PCR, RNase
protection, Northern blotting and other hybridization methods.
Assay techniques that can be used to determine levels of a protein,
such as an METH1 or METH2 polypeptide, in a sample derived from a
host are well-known to those of skill in the art. Such assay
methods include radioimmunoassays, competitive-binding assays,
Western Blot analysis and ELISA assays.
Cancer Diagnosis and Prognosis
[0341] It is believed that certain tissues in mammals with cancer
express significantly diminished levels of the METH1 or METH2
protein and mRNA encoding the METH1 or METH2 protein when compared
to a corresponding "standard" mammal, i.e., a mammal of the same
species not having the cancer. Further, it is believed that
diminished levels of the METH1 or METH2 protein can be detected in
certain body fluids (e.g., sera, plasma, urine, and spinal fluid)
from mammals with cancer when compared to sera from mammals of the
same species not having the cancer. Thus, the invention provides a
diagnostic method useful during tumor diagnosis, which involves
assaying the expression level of the gene encoding the METH1
protein in mammalian cells or body fluid and comparing the gene
expression level with a standard METH1 gene expression level,
whereby a decrease in the gene expression level under the standard
is indicative of certain tumors. The invention also provides a
diagnostic method useful during tumor diagnosis, which involves
assaying the expression level of the gene encoding the METH2
protein in mammalian cells or body fluid and comparing the gene
expression level with a standard METH2 gene expression level,
whereby a decrease in the gene expression level under the standard
is indicative of certain tumors.
[0342] Where a tumor diagnosis has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting diminished METH1
or METH2 gene expression will experience a worse clinical outcome
relative to patients expressing the gene at a lower level.
[0343] By "assaying the expression level of the gene encoding the
METH1 or METH2 protein" is intended qualitatively or quantitatively
measuring or estimating the level of the METH1 or METH2 protein or
the level of the mRNA encoding the METH1 or METH2 protein in a
first biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the METH1 or METH2 protein level or mRNA
level in a second biological sample).
[0344] Preferably, the METH1 or METH2 protein level or mRNA level
in the first biological sample is measured or estimated and
compared to a standard METH1 or METH2 protein level or mRNA level,
the standard being taken from a second biological sample obtained
from an individual not having the cancer. As will be appreciated in
the art, once a standard METH1 or METH2 protein level or mRNA level
is known, it can be used repeatedly as a standard for
comparison.
[0345] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source which contains METH1 or METH2 protein or mRNA. Biological
samples include mammalian body fluids (such as sera, plasma, urine,
synovial fluid and spinal fluid) which contain secreted mature
METH1 or METH2 protein, and adrenal, thyroid, stomach, brain,
heart, placenta, lung, liver, muscle, kidney, pancreas, testis and
ovarian tissue (for METH1); and prostate, small intestine, colon,
brain and lung tissue (for METH2).
[0346] The present invention is useful for detecting cancer in
mammals. In particular the invention is useful during diagnosis of
the of following types of cancers in mammals: breast, ovarian,
prostate, liver, lung, pancreatic, colon, and testicular. Preferred
mammals include monkeys, apes, cats, dogs, cows, pigs, horses,
rabbits and humans. Particularly preferred are humans.
[0347] Total cellular RNA can be isolated from a biological sample
using the single-step guanidinium-thiocyanate-phenol-chloroform
method described in Chomczynski and Sacchi, Anal. Biochem.
162:156-159 (1987). Levels of mRNA encoding the METH1 or METH2
protein are then assayed using any appropriate method. These
include Northern blot analysis (Harada et al., Cell 63:303-312
(1990)), S1 nuclease mapping (Fujita et al., Cell 49:357-367
(1987)), the polymerase chain reaction (PCR), reverse transcription
in combination with the polymerase chain reaction (RT-PCR) (Makino
et al., Technique 2:295-301 (1990)), and reverse transcription in
combination with the ligase chain reaction (RT-LCR).
[0348] Assaying METH1 or METH2 protein levels in a biological
sample can occur using antibody-based techniques. For example,
METH1 or METH2 protein expression in tissues can be studied with
classical immunohistological methods (Jalkanen, M., et al., J.
Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell.
Biol. 105:3087-3096 (1987)).
[0349] Other antibody-based methods useful for detecting METH1 or
METH2 protein gene expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay
(RIA).
[0350] Suitable labels are known in the art and include enzyme
labels, such as, glucose oxidase, and radioisotopes, such as iodine
(.sup.125I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.112In), and technetium
(.sup.99mTc), and fluorescent labels, such as fluorescein and
rhodamine, and biotin.
[0351] Vaccines
[0352] Another aspect of the invention relates to a method for
inducing an immunological response in a mammal which comprises
inoculating the mammal with METH1 or METH2 polypeptide, or a
fragment thereof, adequate to produce antibody and/or T cell immune
response to protect said animal from angiogenic diseases (cancer,
cancer metastasis, chronic inflammatory disorders, rheumatoid
arthritis, altherosclerosis, macular degeneration, diabetic
retinopathy), restenosis, Alzheimer's disease and tissue
remodeling, among others. Yet another aspect of the invention
relates to a method of inducing immunological response in a mammal
which comprises delivering METH1 or METH2 polypeptide via a vector
directing expression of METH1 or METH2 polynucleotide in vivo in
order to induce such an immunological response to produce antibody
to protect such animal from diseases.
[0353] Further aspect of the invention relates to an
immunological/vaccine formulation (composition) which, when
introduced into a mammalian host, induces an immunological response
in that mammal to a METH1 or METH2 polypeptide wherein the
composition comprises a METH1 or METH2 polypeptide or METH1 or
METH2 gene. The vaccine formulation may further comprise a suitable
carrier. Since METH1 or METH2 polypeptide may be broken down in the
stomach, it is preferably administered parenterally (including
subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the recipient;
and aqueous and non-aqueous sterile suspensions which may include
suspending agents or thickening agents. The formulations may be
presented in unit-dose or multi-dose containers, for example,
sealed ampules and vials and may be stored in a freeze-dried
condition requiring only the addition of the sterile liquid carrier
immediately prior to use. The vaccine formulation may also include
adjuvant systems for enhancing the immunogenicity of the
formulation, such as oil-in water systems and other systems known
in the art. The dosage will depend on the specific activity of the
vaccine and can be readily determined by routine
experimentation.
Modes of Administration
[0354] It is recognized than an increase in the vascular supply
plays a central role in tumor progression and metastasis;
therefore, inhibitors of angiogenesis can prove effective as
adjuvant therapy for cancer patients. Some of the currently
recognized angiogenic suppressors are poor candidates for systemic
treatment due to severe collateral effect. The present inventors
have found that METH1 and METH2 are potent inhibitors of
angiogenesis both in vitro and in vivo. The advantage of METH1 and
METH1 is that these inhibitors are normally associated with
suppression of physiological angiogenesis; therefore, they offer
lack of toxicity and endothelial specificity over other angiogenic
inhibitors. Furthermore, METH1 and METH2 present a restricted
pattern of expression providing a possible advantage on organ
specificity.
[0355] Accordingly, the polypeptides of the present invention may
be employed to treat cancer. The METH1 and METH2 polypeptides of
the present invention can also be used to treat individuals with
other disorders that are related to angiogenesis, including
abnormal wound healing, inflammation, rheumatoid arthritis,
psoriasis, endometrial bleeding disorders, diabetic retinopathy,
some forms of macular degeneration, hemangiomas, and
arterial-venous malformations.
[0356] Thus, the invention provides a method of inhibiting
angiogenesis in an individual comprising administering to such an
individual a pharmaceutical composition comprising an effective
amount of an isolated METH1 polypeptide of the invention, effective
to increase the METH1 activity level in such an individual. The
invention also provides a method of inhibiting angiogenesis in an
individual comprising administering to such an individual a
pharmaceutical composition comprising an effective amount of an
isolated METH2 polypeptide of the invention, effective to increase
the METH2 activity level in such an individual.
[0357] METH1 polypeptides which may be used to inhibit angiogenesis
in this manner include: METH1 polypeptide encoded by the deposited
cDNA including the leader; the mature METH1 polypeptide encoded by
the deposited the cDNA minus the leader (i.e., the mature protein);
a polypeptide comprising amino acids about 1 to about 950 in SEQ ID
NO:2; a polypeptide comprising amino acids about 2 to about 950 in
SEQ ID NO:2; a polypeptide comprising amino acids about 29 to about
950 in SEQ ID NO:2; a polypeptide comprising amino acids about 30
to about 950 in SEQ ID NO:2; a polypeptide comprising the
metalloprotease domain of METH1, amino acids 235 to 459 in SEQ ID
NO:2; a polypeptide comprising the disintegrin domain of METH1,
amino acids 460 to 544 in SEQ ID NO:2; a polypeptide comprising the
first TSP-like domain of METH1, amino acids 545 to 598 in SEQ ID
NO:2; a polypeptide comprising the second TSP-like domain of METH1,
amino acids 841 to 894 in SEQ ID NO:2; a polypeptide comprising the
third TSP-like domain of METH1, amino acids 895 to 934 in SEQ ID
NO:2; a polypeptide comprising amino acids 536 to 613 in SEQ ID
NO:2; a polypeptide comprising amino acids 549 to 563 in SEQ ID
NO:2; a polypeptide comprising amino acids 542 to 894 of SEQ ID
NO:2; and a polypeptide comprising amino acids 801 to 950 of SEQ ID
NO:2.
[0358] METH2 polypeptides which may be used to inhibit angiogenesis
in this manner include: the METH2 polypeptide encoded by the
deposited cDNA including the leader; the mature METH2 polypeptide
encoded by the deposited the cDNA minus the leader (i.e., the
mature protein); a polypeptide comprising amino acids about 1 to
about 890 in SEQ ID NO:4; a polypeptide comprising amino acids
about 2 to about 890 in SEQ ID NO:4; a polypeptide comprising amino
acids about 24 to about 890 in SEQ ID NO:4; a polypeptide
comprising amino acids about 112 to about 890 in SEQ ID NO:4; a
polypeptide comprising the metalloprotease domain of METH2, amino
acids 214 to 439 in SEQ ID NO:4; a polypeptide comprising the
disintegrin domain of METH2, amino acids 440 to 529 in SEQ ID NO:4;
a polypeptide comprising the first TSP-like domain of METH2, amino
acids 530 to 583 in SEQ ID NO:4; a polypeptide comprising the
second TSP-like domain of METH2, amino acids 837 to 890 in SEQ ID
NO:4; a polypeptide comprising amino acids 280 to 606 in SEQ ID
NO:4; and a polypeptide comprising amino acids 529 to 548 in SEQ ID
NO:4.
[0359] Also included are METH1 or METH2 proteins lacking TSP3; a
METH1 or METH2 protein lacking TSP2 and TSP3; a METH1 or METH2
protein lacking TSP3, TSP2, and TSP1; a METH1 or METH2 protein
lacking the cysteine-rich domain, TSP1, TSP2, and TSP3; a METH1 or
METH2 protein lacking the metalloprotease domain, the cysteine-rich
domain, TSP1, TSP2 and TSP3; and a METH1 or METH2 protein lacking
the prodomain, the metalloprotease domain, the cysteine-rich
domain, TSP1, TSP2, and TSP3. Finally, any combination of these
domains are also preferred. For example, the cysteine-rich domain
of METH1 may be combined with 1, 2, or 3 TSP domains of METH1. The
cysteine-rich domain of METH2 may be combined with 1, 2, or 3 TSP
domain of METH2. The metalloprotease domain and the cysteine-rich
domain of METH1 may be combined with 1, 2 or 3 TSP domains of
METH1. The metalloprotease domain and the cysteine-rich domain of
METH2 may be combined with 1, 2 or 3 TSP domains of METH2. The
prodomain, the metalloprotease domain, and the cysteine-rich domain
of METH1 may be combined with 1, 2 or 3 TSP domains of METH1. The
prodomain, the metalloprotease domain, and the cysteine-rich domain
of METH2 may be combined with 1, 2 or 3 TSP domains of METH2. The
signal sequence, the prodomain, the metalloprotease domain, and the
cysteine-rich domain of METH1 may be combined with 1, 2, or 3 TSP
domains of METH1. The signal sequence, the prodomain, the
metalloprotease domain, and the cysteine-rich domain of METH2 may
be combined with 1, 2, or 3 TSP domains of METH2.
[0360] As a general proposition, the total pharmaceutically
effective amount of METH1 or METH2 polypeptide administered
parenterally per dose will be in the range of about 1 .mu.g/kg/day
to 10 mg/kg/day of patient body weight, although, as noted above,
this will be subject to therapeutic discretion. More preferably,
this dose is at least 0.01 mg/kg/day, and most preferably for
humans between about 0.01 and 1 mg/kg/day for the polypeptide. If
given continuously, the METH1 or METH2 polypeptide is typically
administered at a dose rate of about 1 .mu.g/kg/hour to about 50
.mu.g/kg/hour, either by 1-4 injections per day or by continuous
subcutaneous infusions, for example, using a mini-pump. An
intravenous bag solution may also be employed.
[0361] Pharmaceutical compositions containing the METH1 or METH2 of
the invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
Gene Therapy Methods
[0362] Another aspect of the present invention is to gene therapy
methods for treating disorders, diseases and conditions. The gene
therapy methods relate to the introduction of nucleic acid (DNA,
RNA and antisense DNA or RNA) sequences into an animal to achieve
expression of the METH1 and/or METH2 polypeptide of the present
invention. This method requires a polynucleotide which codes for a
METH1 and/or METH2 polypeptide operatively linked to a promoter and
any other genetic elements necessary for the expression of the
polypeptide by the target tissue. Such gene therapy and delivery
techniques are known in the art, see, for example, WO90/11092,
which is herein incorporated by reference.
[0363] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) comprising a promoter operably
linked to a METH1 and/or METH2 polynucleotide ex vivo, with the
engineered cells then being provided to a patient to be treated
with the polypeptide. Such methods are well-known in the art. For
example, see Belldegrun, A., et al., J. Natl. Cancer Inst.
85:207-216 (1993); Ferrantini, M. et al., Cancer Research
53:1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:
4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229
(1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990);
Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996);
Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and
Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which
are herein incorporated by reference. In one embodiment, the cells
which are engineered are arterial cells. The arterial cells may be
reintroduced into the patient through direct injection to the
artery, the tissues surrounding the artery, or through catheter
injection.
[0364] As discussed in more detail below, the METH1 and/or METH2
polynucleotide constructs can be delivered by any method that
delivers injectable materials to the cells of an animal, such as,
injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver, and the like). The METH1 and/or METH2
polynucleotide constructs may be delivered in a pharmaceutically
acceptable liquid or aqueous carrier.
[0365] In one embodiment, the METH1 and/or METH2 polynucleotide is
delivered as a naked polynucleotide. The term "naked"
polynucleotide, DNA or RNA refers to sequences that are free from
any delivery vehicle that acts to assist, promote or facilitate
entry into the cell, including viral sequences, viral particles,
liposome formulations, lipofectin or precipitating agents and the
like. However, the METH1 and/or METH2 polynucleotides can also be
delivered in liposome formulations and lipofectin formulations and
the like can be prepared by methods well known to those skilled in
the art. Such methods are described, for example, in U.S. Pat. Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated
by reference.
[0366] The METH1 and/or METH2 polynucleotide vector constructs used
in the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Appropriate vectors include pWLNEO, pSV2CAT,
pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG
and pSVL available from Pharmacia; and pEF1/V5, pcDNA3.1, and
pRc/CMV2 available from Invitrogen. Other suitable vectors will be
readily apparent to the skilled artisan.
[0367] Any strong promoter known to those skilled in the art can be
used for driving the expression of METH1 and/or METH2 DNA. Suitable
promoters include adenoviral promoters, such as the adenoviral
major late promoter; or heterologous promoters, such as the
cytomegalovirus (CMV) promoter; the respiratory syncytial virus
(RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin
promoter; the ApoAI promoter; human globin promoters; viral
thymidine kinase promoters, such as the Herpes Simplex thymidine
kinase promoter; retroviral LTRs; the b-actin promoter; and human
growth hormone promoters. The promoter also may be the native
promoter for METH1 and/or METH2.
[0368] Unlike other gene therapy techniques, one major advantage of
introducing naked nucleic acid sequences into target cells is the
transitory nature of the polynucleotide synthesis in the cells.
Studies have shown that non-replicating DNA sequences can be
introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
[0369] The METH1 and/or METH2 polynucleotide construct can be
delivered to the interstitial space of tissues within the an
animal, including of muscle, skin, brain, lung, liver, spleen, bone
marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas,
kidney, gall bladder, stomach, intestine, testis, ovary, uterus,
rectum, nervous system, eye, gland, and connective tissue.
Interstitial space of the tissues comprises the intercellular,
fluid, mucopolysaccharide matrix among the reticular fibers of
organ tissues, elastic fibers in the walls of vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of
bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery
to the interstitial space of muscle tissue is preferred for the
reasons discussed below. They may be conveniently delivered by
injection into the tissues comprising these cells. They are
preferably delivered to and expressed in persistent, non-dividing
cells which are differentiated, although delivery and expression
may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts. In vivo muscle cells are particularly competent
in their ability to take up and express polynucleotides.
[0370] For the naked acid sequence injection, an effective dosage
amount of DNA or RNA will be in the range of from about 0.05 mg/kg
body weight to about 50 mg/kg body weight. Preferably the dosage
will be from about 0.005 mg/kg to about 20 mg/kg and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as
the artisan of ordinary skill will appreciate, this dosage will
vary according to the tissue site of injection. The appropriate and
effective dosage of nucleic acid sequence can readily be determined
by those of ordinary skill in the art and may depend on the
condition being treated and the route of administration.
[0371] The preferred route of administration is by the parenteral
route of injection into the interstitial space of tissues. However,
other parenteral routes may also be used, such as, inhalation of an
aerosol formulation particularly for delivery to lungs or bronchial
tissues, throat or mucous membranes of the nose. In addition, naked
METH1 and/or METH2 DNA constructs can be delivered to arteries
during angioplasty by the catheter used in the procedure.
[0372] The naked polynucleotides are delivered by any method known
in the art, including, but not limited to, direct needle injection
at the delivery site, intravenous injection, topical
administration, catheter infusion, and so-called "gene guns". These
delivery methods are known in the art.
[0373] The constructs may also be delivered with delivery vehicles
such as viral sequences, viral particles, liposome formulations,
lipofectin, precipitating agents, etc. Such methods of delivery are
known in the art.
[0374] In certain embodiments, the METH1 and/or METH2
polynucleotide constructs are complexed in a liposome preparation.
Liposomal preparations for use in the instant invention include
cationic (positively charged), anionic (negatively charged) and
neutral preparations. However, cationic liposomes are particularly
preferred because a tight charge complex can be formed between the
cationic liposome and the polyanionic nucleic acid. Cationic
liposomes have been shown to mediate intracellular delivery of
plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987)
84:7413-7416, which is herein incorporated by reference); mRNA
(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081,
which is herein incorporated by reference); and purified
transcription factors (Debs et al., J. Biol. Chem. (1990)
265:10189-10192, which is herein incorporated by reference), in
functional form.
[0375] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes
are particularly useful and are available under the trademark
Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner
et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is
herein incorporated by reference). Other commercially available
liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0376] Other cationic liposomes can be prepared from readily
available materials using techniques well known in the art. See,
e.g. PCT Publication No. WO 90/11092 (which is herein incorporated
by reference) for a description of the synthesis of DOTAP
(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Preparation of DOTMA liposomes is explained in the literature, see,
e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417,
which is herein incorporated by reference. Similar methods can be
used to prepare liposomes from other cationic lipid materials.
[0377] Similarly, anionic and neutral liposomes are readily
available, such as from Avanti Polar Lipids (Birmingham, Ala.), or
can be easily prepared using readily available materials. Such
materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphosphatidyl
ethanolamine (DOPE), among others. These materials can also be
mixed with the DOTMA and DOTAP starting materials in appropriate
ratios. Methods for making liposomes using these materials are well
known in the art.
[0378] For example, commercially dioleoylphosphatidyl choline
(DOPC), dioleoylphosphatidyl glycerol (DOPG), and
dioleoylphosphatidyl ethanolamine (DOPE) can be used in various
combinations to make conventional liposomes, with or without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be prepared by drying 50 mg each of DOPG and DOPC under a stream of
nitrogen gas into a sonication vial. The sample is placed under a
vacuum pump overnight and is hydrated the following day with
deionized water. The sample is then sonicated for 2 hours in a
capped vial, using a Heat Systems model 350 sonicator equipped with
an inverted cup (bath type) probe at the maximum setting while the
bath is circulated at 15EC. Alternatively, negatively charged
vesicles can be prepared without sonication to produce
multilamellar vesicles or by extrusion through nucleopore membranes
to produce unilamellar vesicles of discrete size. Other methods are
known and available to those of skill in the art.
[0379] The liposomes can comprise multilamellar vesicles (MLVs),
small unilamellar vesicles (SUVs), or large unilamellar vesicles
(LUVs), with SUVs being preferred. The various liposome-nucleic
acid complexes are prepared using methods well known in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983),
101:512-527, which is herein incorporated by reference. For
example, MLVs containing nucleic acid can be prepared by depositing
a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating with a solution of the material to be
encapsulated. SUVs are prepared by extended sonication of MLVs to
produce a homogeneous population of unilamellar liposomes. The
material to be entrapped is added to a suspension of preformed MLVs
and then sonicated. When using liposomes containing cationic
lipids, the dried lipid film is resuspended in an appropriate
solution such as sterile water or an isotonic buffer solution such
as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are
mixed directly with the DNA. The liposome and DNA form a very
stable complex due to binding of the positively charged liposomes
to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in
the art. Commonly used methods include Ca.sup.2+-EDTA chelation
(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al., Cell (1979) 17:77); ether injection (Deamer, D. and
Bangham, A., Biochim. Biophys. Acta (1976) 443:629; Ostro et al.,
Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc.
Natl. Acad. Sci. USA (1979) 76:3348); detergent dialysis (Enoch, H.
and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76:145);
and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
(1980) 255:10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl.
Acad. Sci. USA (1978) 75:145; Schaefer-Ridder et al., Science
(1982) 215:166), which are herein incorporated by reference.
[0380] Generally, the ratio of DNA to liposomes will be from about
10:1 to about 1:10. Preferably, the ration will be from about 5:1
to about 1:5. More preferably, the ratio will be about 3:1 to about
1:3. Still more preferably, the ratio will be about 1:1.
[0381] U.S. Pat. No. 5,676,954 (which is herein incorporated by
reference) reports on the injection of genetic material, complexed
with cationic liposomes carriers, into mice. U.S. Pat. Nos.
4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/29469
(which are herein incorporated by reference) provide cationic
lipids for use in transfecting DNA into cells and mammals. U.S.
Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international publication no. WO 94/29469 (which are herein
incorporated by reference) provide methods for delivering
DNA-cationic lipid complexes to mammals.
[0382] In certain embodiments, cells are engineered, ex vivo or in
vivo, using a retroviral particle containing RNA which comprises a
sequence encoding METH1 and/or METH2. Retroviruses from which the
retroviral plasmid vectors may be derived include, but are not
limited to, Moloney Murine Leukemia Virus, spleen necrosis virus,
Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,
gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[0383] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X,
VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy 1:5-14 (1990), which is
incorporated herein by reference in its entirety. The vector may
transduce the packaging cells through any means known in the art.
Such means include, but are not limited to, electroporation, the
use of liposomes, and CaPO.sub.4 precipitation. In one alternative,
the retroviral plasmid vector may be encapsulated into a liposome,
or coupled to a lipid, and then administered to a host.
[0384] The producer cell line generates infectious retroviral
vector particles which include polynucleotide encoding METH1 and/or
METH2. Such retroviral vector particles then may be employed, to
transduce eukaryotic cells, either in vitro or in vivo. The
transduced eukaryotic cells will express METH1 and/or METH2.
[0385] In certain other embodiments, cells are engineered, ex vivo
or in vivo, with METH1 and/or METH2 polynucleotide contained in an
adenovirus vector. Adenovirus can be manipulated such that it
encodes and expresses METH1 and/or METH2, and at the same time is
inactivated in terms of its ability to replicate in a normal lytic
viral life cycle. Adenovirus expression is achieved without
integration of the viral DNA into the host cell chromosome, thereby
alleviating concerns about insertional mutagenesis. Furthermore,
adenoviruses have been used as live enteric vaccines for many years
with an excellent safety profile (Schwartz, A. R. et al. (1974) Am.
Rev. Respir. Dis. 109:233-238). Finally, adenovirus mediated gene
transfer has been demonstrated in a number of instances including
transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton
rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld
et al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to establish adenovirus as a causative agent in human
cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl.
Acad. Sci. USA 76:6606).
[0386] Suitable adenoviral vectors useful in the present invention
are described, for example, in Kozarsky and Wilson, Curr. Opin.
Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155
(1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2
is useful and can be grown in human 293 cells. These cells contain
the E1 region of adenovirus and constitutively express E1a and E1b,
which complement the defective adenoviruses by providing the
products of the genes deleted from the vector. In addition to Ad2,
other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also
useful in the present invention.
[0387] Preferably, the adenoviruses used in the present invention
are replication deficient. Replication deficient adenoviruses
require the aid of a helper virus and/or packaging cell line to
form infectious particles. The resulting virus is capable of
infecting cells and can express a polynucleotide of interest which
is operably linked to a promoter, but cannot replicate in most
cells. Replication deficient adenoviruses may be deleted in one or
more of all or a portion of the following genes: E1a, E1b, E3, E4,
E2a, or L1 through L5.
[0388] In certain other embodiments, the cells are engineered, ex
vivo or in vivo, using an adeno-associated virus (AAV). AAVs are
naturally occurring defective viruses that require helper viruses
to produce infectious particles (Muzyczka, N., Curr. Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that may integrate its DNA into non-dividing cells. Vectors
containing as little as 300 base pairs of AAV can be packaged and
can integrate, but space for exogenous DNA is limited to about 4.5
kb. Methods for producing and using such AAVs are known in the art.
See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0389] For example, an appropriate AAV vector for use in the
present invention will include all the sequences necessary for DNA
replication, encapsidation, and host-cell integration. The METH1
and/or METH2 polynucleotide construct is inserted into the AAV
vector using standard cloning methods, such as those found in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press (1989). The recombinant AAV vector is then
transfected into packaging cells which are infected with a helper
virus, using any standard technique, including lipofection,
electroporation, calcium phosphate precipitation, etc. Appropriate
helper viruses include adenoviruses, cytomegaloviruses, vaccinia
viruses, or herpes viruses. Once the packaging cells are
transfected and infected, they will produce infectious AAV viral
particles which contain the METH1 and/or METH2 polynucleotide
construct. These viral particles are then used to transduce
eukaryotic cells, either ex vivo or in vivo. The transduced cells
will contain the METH1 and/or METH2 polynucleotide construct
integrated into its genome, and will express METH1 and/or
METH2.
[0390] Another method of gene therapy involves operably associating
heterologous control regions and endogenous polynucleotide
sequences (e.g. encoding METH1 and/or METH2) via homologous
recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24,
1997; International Publication No. WO 96/29411, published Sep. 26,
1996; International Publication No. WO 94/12650, published Aug. 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989). This method
involves the activation of a gene which is present in the target
cells, but which is not normally expressed in the cells, or is
expressed at a lower level than desired.
[0391] Polynucleotide constructs are made, using standard
techniques known in the art, which contain the promoter with
targeting sequences flanking the promoter. Suitable promoters are
described herein. The targeting sequence is sufficiently
complementary to an endogenous sequence to permit homologous
recombination of the promoter-targeting sequence with the
endogenous sequence. The targeting sequence will be sufficiently
near the 5' end of the METH1 and/or METH2 desired endogenous
polynucleotide sequence so the promoter will be operably linked to
the endogenous sequence upon homologous recombination.
[0392] The promoter and the targeting sequences can be amplified
using PCR. Preferably, the amplified promoter contains distinct
restriction enzyme sites on the 5' and 3' ends. Preferably, the 3'
end of the first targeting sequence contains the same restriction
enzyme site as the 5' end of the amplified promoter and the 5' end
of the second targeting sequence contains the same restriction site
as the 3' end of the amplified promoter. The amplified promoter and
targeting sequences are digested and ligated together.
[0393] The promoter-targeting sequence construct is delivered to
the cells, either as naked polynucleotide, or in conjunction with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, whole viruses, lipofection,
precipitating agents, etc., described in more detail above. The P
promoter-targeting sequence can be delivered by any method,
included direct needle injection, intravenous injection, topical
administration, catheter infusion, particle accelerators, etc. The
methods are described in more detail below.
[0394] The promoter-targeting sequence construct is taken up by
cells. Homologous recombination between the construct and the
endogenous sequence takes place, such that an endogenous METH1
and/or METH2 sequence is placed under the control of the promoter.
The promoter then drives the expression of the endogenous METH1
and/or METH2 sequence.
[0395] The polynucleotides encoding METH1 and/or METH2 may be
administered along with other polynucleotides encoding other
proteins. Such proteins include, but are not limited to, acidic and
basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, VEGF-E,
PIGF 1 and 2, epidermal growth factor alpha and beta,
platelet-derived endothelial cell growth factor, platelet-derived
growth factor alpha and beta, tumor necrosis factor alpha,
hepatocyte growth factor, insulin like growth factor, colony
stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide
synthase.
[0396] Preferably, the polynucleotide encoding METH1 and/or METH2
contains a secretory signal sequence that facilitates secretion of
the protein. Typically, the signal sequence is positioned in the
coding region of the polynucleotide to be expressed towards or at
the 5' end of the coding region. The signal sequence may be
homologous or heterologous to the polynucleotide of interest and
may be homologous or heterologous to the cells to be transfected.
Additionally, the signal sequence may be chemically synthesized
using methods known in the art.
[0397] Any mode of administration of any of the above-described
polynucleotides constructs can be used so long as the mode results
in the expression of one or more molecules in an amount sufficient
to provide a therapeutic effect. This includes direct needle
injection, systemic injection, catheter infusion, biolistic
injectors, particle accelerators (i.e., "gene guns"), gelfoam
sponge depots, other commercially available depot materials,
osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill) pharmaceutical formulations, and decanting or
topical applications during surgery. For example, direct injection
of naked calcium phosphate-precipitated plasmid into rat liver and
rat spleen or a protein-coated plasmid into the portal vein has
resulted in gene expression of the foreign gene in the rat livers
(Kaneda et al., Science 243:375 (1989)).
[0398] A preferred method of local administration is by direct
injection. Preferably, a recombinant molecule of the present
invention complexed with a delivery vehicle is administered by
direct injection into or locally within the area of arteries.
Administration of a composition locally within the area of arteries
refers to injecting the composition centimeters and preferably,
millimeters within arteries.
[0399] Another method of local administration is to contact a
polynucleotide construct of the present invention in or around a
surgical wound. For example, a patient can undergo surgery and the
polynucleotide construct can be coated on the surface of tissue
inside the wound or the construct can be injected into areas of
tissue inside the wound.
[0400] Therapeutic compositions useful in systemic administration
include recombinant molecules of the present invention complexed to
a targeted delivery vehicle of the present invention. Suitable
delivery vehicles for use with systemic administration comprise
liposomes comprising ligands for targeting the vehicle to a
particular site.
[0401] Preferred methods of systemic administration, include
intravenous injection, aerosol, oral and percutaneous (topical)
delivery. Intravenous injections can be performed using methods
standard in the art. Aerosol delivery can also be performed using
methods standard in the art (see, for example, Stribling et al.,
Proc. Natl. Acad. Sci. USA 189:11277-11281 (1992), which is
incorporated herein by reference). Oral delivery can be performed
by complexing a polynucleotide construct of the present invention
to a carrier capable of withstanding degradation by digestive
enzymes in the gut of an animal. Examples of such carriers, include
plastic capsules or tablets, such as those known in the art.
Topical delivery can be performed by mixing a polynucleotide
construct of the present invention with a lipophilic reagent (e.g.,
DMSO) that is capable of passing into the skin.
[0402] Determining an effective amount of substance to be delivered
can depend upon a number of factors including, for example, the
chemical structure and biological activity of the substance, the
age and weight of the animal, the precise condition requiring
treatment and its severity, and the route of administration. The
frequency of treatments depends upon a number of factors, such as
the amount of polynucleotide constructs administered per dose, as
well as the health and history of the subject. The precise amount,
number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0403] Therapeutic compositions of the present invention can be
administered to any animal, preferably to mammals and birds.
Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle, horses and pigs, with humans being particularly
preferred.
Chromosome Assays
[0404] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0405] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a METH1 or METH2
protein gene. This can be accomplished using a variety of well
known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose.
[0406] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes.
[0407] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
probes from the cDNA as short as 50 or 60 bp. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual Of Basic
Techniques, Pergamon Press, New York (1988).
[0408] Other mapping strategies that can similarly be used to map
to its chromosome include radiation hybrid mapping, prescreening
with labeled flow-sorted chromosomes and preselection by
hybridization to construct chromosome specific-cDNA libraries.
Radiation hybrid (RH) mapping relies upon fragmentation of human
chromosomes with X-rays, and retention of these random fragments in
Hamster A23 host cells. The DNAs for RH mapping are supplied by
Research Genetics (USA). Oligo pairs are designed from EST
sequences that will amplify products of between 80 bp and 300 bp.
The PCRs are performed on 93 human/hamster hybrid DNAs and the
results compared with a framework map
(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl; Gyapay et
al., Human Molecular Genetics 5:39-346 (1996)). RH mapping provides
greater precision than FISH and indicates clusters of genes as well
as disease locus/gene correlations.
[0409] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0410] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0411] The METH1 gene maps between STS markers D2111435 and
D2111442 which translates as 21q21. This is a similar chromosomal
location to amyloid precursor protein (APP). APP and METH1 are
approximately 3 million bases apart which is not a massive distance
in human genomics. The chromosomal location includes important
genes such as enterokinases (enzymes that activate trypsinogen by
converting it to trypsin) and genes responsible for Alzheimer's
disease.
[0412] The METH1 gene can be mapped to 21q21 using the following
oligos for radiation hybrid mapping:
TABLE-US-00004 5' primer: ACTGTGTGTGATCCGAG 3' primer:
GTTGGAAAGCATTGACG
[0413] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Identification and Cloning of METH1 and METH2
[0414] To search for novel genes with TSP-like domains, a large
human cDNA database consisting of approximately 900,000 expressed
sequence tags (ESTs) was screened for sequences homologous to the
second type I repeat of TSP1. Several ESTs were predicted to encode
proteins with TSP-like domains. Two cDNA clones originated from
human heart and lung libraries were further sequenced and chosen
for functional analysis.
[0415] The amino-terminal end of METH1 was obtained using 5' rapid
amplification of cDNA ends (RACE) PCR technique (Marathon cDNA
amplification kit, Clontech) according to manufacturer
instructions. The amino-terminal end of METH2 was obtained
partially through 5'RACE PCR and later confirmed and completed by
genomic screening. For the genomic screen, BAC clones (Genome
Systems) were initially identified by PCR. Positive BAC clones
containing 150-200 bp of sequence were subsequently subcloned into
pGEM vector as small fragments and sequenced.
[0416] Analysis and comparison of the deduced amino acid sequence
with the GenBank, EMBL and SwissProt databases suggested that these
genes belong to a new family of metalloproteases with homology to
the reprolysin family in their NH2-terminal end and with several
TSP-like motifs in the COOH-terminal end. These cDNAs were named
METH1 and METH2; ME, for metalloprotease and TH, for
thrombospondin. The mouse homologue of METH1 was identified and
named ADAMTS1 (Kuno, K., et al., J. Biol. Chem. 272:556-562
(1997)). Direct comparison of the human and mouse sequences
revealed a high level of conservation (83.4% amino acid identity).
Thus far no homologues for METH2 have been identified.
[0417] Interestingly, a recently identified protein named pNPI
(procollagen I N-proteinase; (Colidge, A., et al., Proc. Natl.
Acad. Sci. USA 94:2374-2379 (1997)) showed a striking sequence and
structural similarity to METH1 and METH2 (FIG. 3). As the novel
proteins described here, pNPI also contains metalloproteinase
(reprolysin subfamily) and TSP domains at the carboxy-terminal end.
Although the sequence for pNPI is of bovine origin, sequence
alignment revealed identical structural features. The amino acid
similarity between METH1 and METH2 is 51.7%, and between METH1 or
METH2 and pNPI the homology is lesser 33.9% and 36.3%,
respectively.
[0418] Sequence analysis showed that the ORF of METH1 and METH2
coded for proteins of 950 and 890 amino acids, respectively. In all
three proteins, the NH.sub.2 terminal end contains a putative
signal peptide followed by another putative transmembrane domain
around amino acid 300, deduced from the hydrophilicity plots. It is
not clear whether these proteins are bound to the membrane.
However, given preliminary data, it is more likely that this second
transmembrane domain will consist of a hydrophobic pocket and that
METH1, METH2 and pNPI are in fact secreted proteins. The
NH.sub.2-terminal end past the signal peptide has homology to the
superfamily of zinc metalloproteases and can be subdivided in a
prodomain, a metalloprotease domain, and a cysteine-rich
region.
[0419] The double underlined sequence in METH1 (amino acids
232-235) and METH2 (amino acids 211-214) in FIG. 3 localized at the
boundary between the prodomain and the metalloprotease domain, are
potential cleavage sites for mammalian subtilisins, such as furins
(Barr, 1991). Proteolytical processing occurs in SVMPs to yield
soluble metalloproteases and disintegrins (Bjarnason, J. B. &
Fox, J. W., Methods Enzymol. 248:345-368 (1995)) and has also been
detected in some ADAMs (reviewed by Wolsberg, T. G. & White, J.
M., Developmental Biology 180:389-401 (1996)). Proteolytical
processing occurs in both METH1 and 2 (see below). Additionally,
both METH1 and METH2 present a Zn.sup.2+-binding site (dotted line
in FIG. 3) that is presumed to be catalytically active due to the
conservation of certain functionally important amino acids
(Rawlings, N. D. & Barrett, A. J., Methods Enzymol. 248:183-228
(1995)) suggesting that these proteins may be active proteases.
[0420] Following the metalloprotease domain, there is a
cysteine-rich region which contains two putative disintegrin loops
(Wolsberg, T. G. & White, J. M., Developmental Biology
180:389-401 (1996)) (marked by arrows in FIG. 3). Disintegrin
domains are found within the superfamily of metalloproteases in
snake venom metalloproteases (SVMPs) and ADAMs (mammalian proteins
containing a disintegrin and a metalloprotease domain) and have a
possible function inhibiting binding of integrins to their ligands
in SVMPs. Conversely, the ADAM-disintegrin-like domain, as part of
membrane anchored proteins, may promote rather than disrupt,
cell-cell interactions (Wolsberg, T. G. & White, J. M.,
Developmental Biology 180:389-401 (1996)). The TSP-like domains are
located in the COOH-half of METH1 and METH2 proteins. METH1
contains two conserved TSP domains separated by a spacer region
with unknown function, and a subdomain with less homology, and only
5 cysteines, following the second anti-angiogenic region. METH2
contains two TSP domains separated by the spacer region. The
alignment of the TSP-like domains of METH1 and METH2 with those of
TSP1 and TSP2 are shown in FIG. 5. The homology varies between
19.2% to 52% amino acid similarity among all the TSP repeats. The
cysteines, numbered 1 to 6, and the tryptophans, labeled by
asterisks, are highly conserved.
[0421] Southern blot of human genomic DNA revealed the presence of
METH1 and METH2 in the genome. METH1 and METH2 probes revealed
bands of different size suggesting that they are transcribed from
different genes.
[0422] The consensus sequence for the type I repeats includes 16
residues with 6 perfectly conserved cysteines. Typically it begins
with the sequence motif WSXWS (SEQ ID NO: 82) that has also been
shown to bind to heparin (Guo, N., et al., J. Biol. Chem.
267:19349-19355 (1992)). The affinity of this region to heparin has
been proposed to the part of the anti-angiogenic activity of TSP-1
(Guo, N., et al., J. Peptide Res. 49 (1997)). Among the five
members of the TSP family of proteins, only TSP-1 and TSP-2 inhibit
angiogenesis and contain the type I repeats (Tolsma, S. S., et al.,
J. Cell. Biol 122:497-511 (1993); Kyriakides, T. R., et al., J.
Cell Biol. 140:419-430 (1998)). The type I or properdin repeats
were probably added to the precursor of TSP1 and 2 by exon
shuffling between 500 and 900 million years ago (Adams, J., et al.,
The Thrombospondin Gene Family, 1 Ed. Molecular Biology
Intelligence Unit (Springer, Ed.), R. G. Landes Company, Germany
(1995)). It is likely that the acquisition of this domain provided
the precursor of TSP1 and TSP2 with functions, such as regulation
of new vessel formation. More recently, BAI-1 (brain angiogenic
inhibitor-1), a protein isolated from a brain library for its
ability to be regulated by p53, has also been shown to contain the
type I repeat of TSP-1 and to provide anti-angiogenic potential to
this molecule (Nishimori, H., et al., Oncogene 15:2145-2150
(1997)). Nevertheless, it appears that additional sequences or
context are also important, since other proteins containing the
type I repeats appear not to have clear or more established
anti-angiogenic properties such as: properdin, F-spondin, and other
members of the complement family.
[0423] Because of the presence of TSP-repeats in METH1 and METH2,
along with their anti-angiogenic properties, these proteins were
originally considered members of the TSP superfamily. Nevertheless,
they have no additional homology to other TSPs, and in fact, the
similarity to TSP1 and TSP2 is restricted to the type I repeats.
Furthermore, the proteins also have strong sequence and structural
homology to members of the ADAM family. These features led Kuno and
colleagues to name ADAMTS to the mouse homolog of METH1 (Kuno, K.,
et al., J. Biol. Chem. 272:556-562 (1997)). The recent
identification of pNPI and its striking sequence homology to the
proteins here described, prompt all these three proteins to be
grouped in a subfamily named metallospondins. At this point, it is
not clear whether pNIP has anti-angiogenic properties or whether
METH1 and/or METH2 participate in the cleavage of the amino
terminal pro-peptide of .alpha.1(I) procollagen.
Example 2
Northern and Southern Blot Analysis
[0424] Total RNA was purified from cells by
guanidinium-isothiocyanate extraction, as previously described
(Chomczynski, P. & Sacchi, N., Anal. Biochem. 162:156-159
(1987)) Poly(A)+RNA was extracted using a Boehringer Mannheim (BMB,
Indianapolis, Ind.) kit according to the manufacturer conditions.
Other poly(A)+RNA blots were purchased from Clontech (Palo Alto,
Calif.). Pre-hybridization was performed in a solution containing:
50% formamide, 6.times.SSPE, 1.times.Denhardt's solution, 0.1% SDS
and 100 .mu.g/ml of heat denatured salmon sperm DNA for 12-18 h at
42.degree. C. Hybridization with labeled cDNA probes proceeded in
the same solution at 42.degree. C. for 12-18 h. TSP1 and METH1
probes corresponded to the entire human cDNAs. The METH2 probe
corresponded to a KpnI-EcoRI fragment from the human cDNA. A 1.3 Kb
PstI fragment of the glyceraldehyde-3-phosphate-dehydrogenase
(GPDH) was used to normalize for loading and transfer efficiency.
Membranes were exposed to Kodak Biomax MS film (Kodak, New Haven,
Conn.).
[0425] For Southern blots, human genomic DNA, purchased from
Promega (Madison, Wis.), was heated at 65.degree. C. for 10 min and
digested with EcoRI and PstI overnight at 37.degree. C. 5 .mu.g of
digested DNA was separated in a 1% agarose gel, transferred to a
nytran membrane and cross-linked by ultraviolet light. cDNA probes,
as well as, prehybridization and hybridization conditions were
identical to those described for Northern blots. Blots were washed
with high stringency (0.2.times.SSC, 0.2% SDS at 50.degree.
C.).
[0426] The expression patterns of METH1 and METH2 were examined in
both adult and embryonic tissues. Northern blot analysis was
performed under high-stringency conditions with blots that included
poly(A)+RNA from human tissues. METH1 and METH2 transcripts
revealed a single band of 4.6 and 3.7 Kb, respectively. Abundant
METH1 mRNA expression was observed in adrenal, heart, placenta,
followed by skeletal muscle, thyroid and stomach. From the
embryonic tissues analyzed, kidney showed the highest expression of
METH1 mRNA. Nevertheless, weaker expression of METH1 mRNA was seen
in all tissues analyzed. Distribution of METH2 mRNA was more
restricted and weaker than that of METH1. The highest expression
was seen in lung, both embryonic and adult. Interestingly, METH1
and METH2 expression do not appear to overlap. In combination, the
structural similarities and their pattern of expression suggest
functional redundancy yet different transcriptional regulation. The
expression levels of TSP1 transcripts in the same blots were also
analyzed, for purpose of comparison. TSP1 mRNA highest expression
was seen in the adult placenta and in all embryonic tissues
analyzed. In contrast to METH1 and METH2 we observed constant
levels of TSP1 transcript in all the other tissues examined.
[0427] The cell type distribution was also studied by Northern blot
analysis of poly(A)+RNA. METH1 mRNA was detectable, at low levels,
in dermal fibroblasts, vascular smooth muscle, endometrial stromal
cells, and in two cancer cell lines, HeLa and G631, an
adenocarcinoma and a melanoma, respectively. METH2 mRNA was
detected only on SW480, a colon carcinoma cell line, but no
expression was seen in any other of the cell lines or primary
strains analyzed.
[0428] The possibility that groups of angiogenic and
anti-angiogenic factors regulate vascular network formation in
specific organs has been a frequently discussed hypothesis likely
to be true, yet unproven. The expression patterns of METH1 and
METH2, which are clearly distinct and almost non-overlapping, were
puzzling, at least with concern to overall levels. TSP1 and TSP2
also share identical structure, high level of amino acid
similarity, yet their pattern of expression differs significantly
(Iruela-Arispe, M. L., Dev. Dyn. 197:40-56 (1993)). The differences
are likely based on dissimilar cis-acting elements in their
promoters and different regulatory mechanisms, as previously
suggested. Although the promoters for METH1 and 2 have not been
characterized, it is likely that they provide unique features for
the regulation of each gene. Nevertheless, the possibility that one
motif, the anti-angiogenic/type I repeat, with demonstrated
anti-angiogenic properties is present in several proteins with
different tissue specificities is appealing. Alternatively, the
small differences in sequence between closely related members of
the same family could possess significance that goes beyond
functional redundancy. In the case of TSP1 and TSP2, aside from the
striking structural similarities and perhaps having functionally
common anti-angiogenic properties, TSP1 and TSP2 also appear to
display functions of their own and not likely shared by their
similar relative. This became evident with the outcome of the two
knock-outs for these genes. TSP1 null animals exhibited primarily
lung disorders (Lawler, J., et al., J. Clin. Invest. 101:982-992
(1998)) and secondarily vascular abnormalities, but only under
specific pathological settings or on a restricted set of organs. In
contrast TSP2 knock-out mice exhibited unpredicted collagen
assembly anomalies, with carry-on consequences to the skin,
tendons, and bone (Kyriakides, T. R., et al., J. Cell Biol.
140:419-430 (1998)). In addition, these animals also appear to have
overall increase in capillary density in the dermis. It is not
understood how the resemblance between the newly described members
of the metallospondin family translate functionally. Clearly, pNIP
has been shown to display active proteolytic activity by cleaving
the N-terminus of type I procollagen (Colidge, A., et al., Proc.
Natl. Acad. Sci. USA 94:2374-2379 (1997)).
[0429] A second region of functional interest corresponds to the
disintegrin domain. This domain has been more fully characterized
in related members of the snake venom metalloproteases that have
been shown to bind to .alpha.IIb.beta.3 and inhibit platelet
interaction blocking coagulation (Pfaff, M., et al., Cell Adhes
Commun. 2:491-501 (1994); Usami, Y., et al., Biochem. Biophys. Res.
Commun. 201:331-339 (1994)). The disintegrin motif consists of a
thirteen to fifteen domain which frequently contain an RGD or a
negatively charged residue at the position of the aspartic acid.
The RGD, or equivalent, binds to integrins and serve as antagonist
or signaling ligands (Wolsberg, T. G. & White, J. M.,
Developmental Biology 180:389-401 (1996)). METH2, but not METH1,
has an RGD sequence located amino-terminal to the disintegrin
domain. In addition, both molecules present relatively high, but
not perfect, degree of conservation of cysteines within the
disintegrin motif. This appears to display an important role in the
tertiary structure of this region and its ability to interact with
integrins. In addition, some of these domains have been shown to
act as functional adhesion molecules, particularly those with
transmembrane regions (Wolsberg, T. G. & White, J. M.,
Developmental Biology 180:389-401 (1996)). It is unlikely that this
will be the case for METH1 and METH2, since both these proteins
appear to be secreted.
Example 3
Expression and Purification of Recombinant Proteins
[0430] Recombinant constructs for expression of His-tagged fusion
proteins were generated for expression in bacteria. METH1 nt
605-1839 (from ATG) was amplified by polymerase chain reaction
using primers containing BamHI and PstI sites and subcloned into
the pRSET vector (Invitrogen, Carlsbad, Calif.). The construct was
sequenced to verify frame and sequence fidelity and were then
transformed into BL21; DE3 E. Coli strain (Stratagene Cloning
Systems, La Jolla, Calif.). Purification was performed by affinity
chromatography on Ni-NTA columns. Recombinant protein was eluted
with 500 mM imidazole in PBS. Fractions containing recombinant
protein were dialyzed against phenol-red free DMEM and used to
generate antisera.
[0431] Antisera was generated by intramuscular injection of a 1:1
mixture of recombinant protein (500 .mu.g/ml) and Freud's adjuvant.
Eight animals, including five guinea pigs and three rabbits were
injected every 15 days for three cycles. After the third injection,
serum was evaluated for presence of anti-METH1 antibodies, only two
of the guinea pigs showed significant titers. The antibodies
recognized recombinant protein on Western blots, were able to
immunoprecipitate METH1 protein from cell extracts and recognize
the protein by immunocytochemistry. Pre-immune sera was always
included as control. One of the guinea pig antibodies was also able
to recognize METH2.
[0432] For mammalian expression, full-length METH1 and METH2 cDNA
were cloned into pcDNA3.1 expression vector (Invitrogen). The
vector is under the regulatory control of the CMV promoter. Cloning
was performed so that constructs contained their own termination
codons.
[0433] Recombinant protein was obtained by transient transfection
of the expression vectors in 293T cells using standard calcium
phosphate precipitation. Upon transfection, cells were incubated
for 6 to 16 h in serum-containing media and then switched to
serum-free media for 36 h for accumulation of recombinant protein.
As control, pcDNA3.1 vector alone was transiently transfected in
parallel plates. Purification of the protein included 30% ammonium
sulfate precipitation followed by dialysis on HS buffer (DB=10 mM
HEPES, 150 mM NaCl, 1 mM CaCl.sub.2 and 1 mM MgSO.sub.4). Samples
were then subjected to heparin-affinity chromatography. Elution
from heparin columns was achieved with HS buffer containing 550 mM
NaCl. Fractions were then loaded on 5-30% sucrose gradients and
spun at 48K. Separation on sucrose gradients was assessed by
Western blotting and purity was determined by Commmassie blue and
silver nitrate staining.
[0434] Generation of recombinant protein was initially done in
bacteria. A METH1 expression vector was generated containing an
amino terminal His Tag to aid on the purification. The resulting
protein coded for all METH1 translated sequence except the
prodomain. Affinity chromatography on Ni.sup.++-beads showed an
unique band of 68 kD. Isolation and purification was always
performed under denatured conditions and attempts to refold the
protein met with little success, probably due to a significant
number of intramolecular disulfide bonds associated with the large
number of cysteines. Nonetheless, the protein was used to generate
antibodies. From eight animals injected, only two were able to
mount an immune response and generate specific antibodies, possibly
due to the high conservation across species. Both antibodies
recognized recombinant METH1 protein before and after purification
on Ni.sup.++ columns. The antibody was also used to evaluate
expression of the protein on Western blots of cell lysates. A
single band of approximately 105-110 kD was detected in stromal
fibroblasts and smooth muscle cells.
[0435] To test the hypothesis that METH1 and METH2 could function
as regulators of angiogenesis, recombinant full length protein was
generated in mammalian cells. Evaluation of correct reading frame
and molecular weight was initially tested by in vitro translation.
Translation of the METH-1 open reading frame revealed a 110 kD
protein, slightly higher than the size predicted by translation of
the cDNA sequence. As previously indicated, there are two putative
glycosylation sites, the higher size of the protein is likely due
to addition of sugar residues. Similarly, METH2 was also slightly
higher than its predicted size, showing a 98 kD protein.
[0436] Recombinant proteins were isolated from 293T supernatants
under native conditions to preserve secondary structure. From
analysis of the deduced amino acid sequence and published
information on the murine homolog, ADAMTS, it was predicted that
both proteins could bind to heparin and used affinity
chromatography for purification. Both cell layer and conditioned
media of 293T cells transfected with METH1, METH2 and vector
control were used for purification. The molecular weight of METH1
and 2 were similar to those from the reticulocyte lysate. As
predicted, both proteins are secreted. Interestingly, the media
contains both full length (110 kD) and two processed forms of 85
and 67 kD for METH1, and 79 and 64 kD for METH2. The 85 and 79 kD
molecular weights agree with the predicted size for both proteins
after cleavage at the consensus sublisin site. However, a second
processing event must take place to generate the most abundant
fragments observed at 67 and 64 kD respectively. These forms are
stable after purification even in the absence of proteinase
inhibitors. For purification, proteins were initially concentrated
by ammonium sulfate precipitation, followed by dialysis. The
resulting protein suspension was then subjected to
heparin-sepharose columns. Recombinant METH1 and METH2 were eluted
with washing buffer containing 550 mM NaCl. Fractions contained
both pro-METH1, as well as the processed forms. Because it was
unclear whether processing was relevant for function of the
proteins, both forms were separated on sucrose gradients. Both
full-length and processed forms were used in angiogenesis
assays.
[0437] Recombinant constructs for expression of truncated fusion
proteins were as follows: (1) pRSET-METH1-Type I: METH1 nt
1605-1839 (from the start codon) was amplified by polymerase chain
reaction using the following primers: 5'-GCA TTT TGG ATC CGC CTT
TTC ATG-3' (SEQ ID NO:78) and 5'-GTT GTG TGC TGC AGA TTG TTC C-3'
(SEQ ID NO:79). The amplified fragment was then subcloned into the
BamHI and PstI sites of the pRSET vector; (2) pGEX-METH1-TSP was
generated by ligating the BamHI-EcoRI fragment from the
pRSET-METH1-TSP into the SmaI site of the pGEX-5.times. vector
(Pharmacia Biotech Inc., Piscataway, N.J.) by blunt-end ligation;
(3) pGEX-1.0-METH2: the fragment nt 838-1818 of METH2 cDNA (from
the start codon) was ligated into BamHI-EcoRI sites of pGEM-2TK.
The METH2 fragment was amplified by PCR using the following
primers: 5.quadrature.-GAAAAATGGGGATCCGAGGTG-3' (SEQ ID NO:80) and
5'-GCAGGAGAATTCCGTCCATG-3' (SEQ ID NO: 81) to generate BamHI and
EcoRI restriction sites; (4) pGEX-METH2-TSP: a 0.5 Kb XmaI-EcoRI
fragment isolated from pGEX-1,0-METH2 was subcloned into the XmaI
and EcoRI sites of pGEX-2TK vector. All constructs were sequenced
to verify sequence fidelity and correct open reading frame.
[0438] The recombinant proteins were named 6H-METH1, the
recombinant protein expressed with the plasmid pRSET-METH1-TSP,
GST-METH1, the protein expressed with the plasmid pGEX-METH1-TSP
and GST-METH2, the protein expressed with the plasmid
pGEX-METH2-TSP.
[0439] Expression plasmids were transformed into BL21:DE3 E. coli
strain (Stratagene Cloning Systems, La Jolla, Calif.) and fusion
proteins were induced following manufacturer recommendations.
Briefly, induced bacteria pellets were resuspended in PBS and
sonicated on ice for 1 min. The suspension was, subsequently,
incubated at RT for 20 min in the presence of 1% triton X-100 and
centrifuged at 4.degree. C. Histidine tagged fusion proteins were
then purified on Ni-NTA beads (Qiagen, Chatsworth, Calif.) by
incubating 20 ml of supernatant with 1 ml of beads (50% slurry) for
2 h at 4.degree. C. The suspension was transferred into a column
and washed with 10 columns volume of PBS containing 10 mM
imidazole, followed by 50 mM imidazole and finally 100 mM
imidazole. The protein was eluted with 500 mM imidazole in PBS.
Fractions containing the recombinant protein were dialyzed against
phenol-red free DMEM. Samples were centrifuged for 30 min at
4.degree. C., part of the protein was not soluble and was lost
during centrifugation. The supernatant was stored at -70.degree. C.
and used for proliferation, cornea pocket and chorioallantoic
membrane (CAM) assays.
[0440] For purification of GST-fusion proteins, the extract was
cleared by centrifugation and applied to a GST-affinity column
(Pharmacia). The column was washed with PBS-1% triton X-100 in the
presence of 0.1 mM reduced glutathione and, subsequently, with the
same buffer in the presence of 0.5 mM reduced glutathione. Fusion
proteins were eluted with 10 mM reduced glutathione in 50 mM
Tris-HCl, pH 7.5. Fractions containing the protein were dialyzed
against DMEM, stored at -70.degree. C. and used for proliferation,
cornea pocket and chorioallantoic membrane (CAM) assays.
[0441] Integrity and purity of recombinant proteins was analyzed in
12.5% or 15% acrylamide gels stained with Coomassie blue.
[0442] A recombinant GST fusion protein containing the first two
type I repeats of TSP was also dialyzed against DMEM before used in
functional assays. Intact TSP1 was purified from platelets as
previously described (Roberts, D. D., et al., J. Tissue Cult.
Methods 16:217-222 (1994)).
[0443] To test the hypothesis that METH1 and METH2 TSP domains
could function as regulators of angiogenesis recombinant fusion
proteins were generated in bacteria. The constructs included the
first TSP domain of METH1 or METH2. This domain is the most
conserved, 52% amino acid similarity with the second type I repeat
of TSP1, (this domain contains a putative binding site for CD36).
All recombinant proteins were isolated under native conditions to
preserve their secondary structure as much as possible. 6H-METH1
and GST-METH1 contained the first TSP-like domain of METH1 fused to
a histidine tag or a GST, respectively. METH1 recombinant protein
was made with two different tags because of purification and
structural advantages. The differences in size are due to the size
of the tag, 6 KDa the histidine and 27 KDa the GST. GST-METH2
contained the first TSP domain of METH2 also fused to a GST. A
fragment corresponding to the last two type I repeats of TSP1, also
fused to a GST, and intact TSP1 purified from platelets were used
as positive controls. In addition, GST alone was included in all
experiments as negative control.
Example 4
TSP Domains in METH1 and METH2 Disrupt Angiogenesis In Vivo
Cornea Pocket Assay
[0444] Swiss Webster females and males, were purchased from Charles
River (Boston, Mass.) and used between 8-10 weeks-old for
implantation of the pellets. Cornea pockets were performed as
described by Kenyon and colleagues (Kenyon, B. M., et al., Invest.
Opthalmol. Vis. Sci. 37:1625-1632 (1996)) with few modifications.
Briefly, a solution of 10 .mu.g of recombinant bFGF plus 5 mg of
sucralfate were mixed with 10 .mu.l of Hydron (200 mg/ml in
ethanol; New Brunswick, N.J.) and the recombinant protein of
interest (2 .mu.g). The suspension was then smeared onto a sterile
nylon mesh square (pore size 500 .mu.m; Tetko Inc., Briarcliff
Manor, N.Y.) and allowed to dry for 30 min. The fibers of the mesh
were pulled to produce pellets of 500 .mu.m.sup.3 that were stored
at -20.degree. C. Uniformly sized pellets were selected under a
microscope and used for the assays.
[0445] Mice were anesthetized with Avertin. An incision was made in
the cornea using a Nikon SMZ-U dissecting microscope with the aid
of a surgical blade. A single pellet was implanted into the pocket.
Five days after pellet implantation, corneal angiogenesis was
evaluated and photographed.
[0446] CAM Assay
[0447] Chorioallantoic membrane assays were performed on Leghorn
chicken embryos (SPAFAS, MA) at 12-14 days of embryonic
development. Matrigel (750 .mu.g/ml), VEGF (250 ng/mesh) and the
protein or peptide to be tested were mixed, placed onto nylon
meshes (pore size 250 .mu.m; Tetko Inc.) and incubated sequentially
at 37.degree. C. for 30 min and at 4.degree. C. for 2 h to induce
polymerization. A positive (matrigel and VEGF) and a negative (VEGF
alone) control were also prepared for each CAM. Polymerized meshes
were placed onto the third outer region of the CAM and incubated
for 24 h. To visualize vessels, 400 .mu.l of fluorescein
isothiocyanate dextran (10 mg/ml, SIGMA) was injected in the chick
blood stream. After 5-10 min incubation, the chick was topically
fixed with 3.7% formaldehyde for 5 min. The meshes were then
dissected and mounted onto slides. Fluorescence intensity was
analyzed with a computer-assisted image program (NIH Image
1.59).
[0448] Peptides used on these assays were synthesized by Chiron
(Raleigh, N.C.). Sequence corresponded to amino acids: P-TSP1,
430-447; P-METH1, 549-563; P-METH2, 529-548.
[0449] The evaluation of angiogenic or anti-angiogenic responses
relies heavily on the sensitivity and specificity of the assays
used to assess the response. To evaluate the anti-angiogenic
activity of these fragments in vivo, two popular and well-accepted
angiogenesis assays were used: the corneal pocket and the
chorioallantoic membrane. The visibility, accessibility, and
avascularity of the cornea are highly advantageous and facilitate
the visualization of the neovascular response and the topical
application of the test substances. A known amount of angiogenesis
factor(s) is implanted, as a pellet, in a pocket made in the cornea
eye. To test an angiogenesis inhibitor, the molecule is implanted
with the stimulator in the same pellet, and the response is
compared to the stimulator alone.
[0450] In these experiments, bFGF was used as the vascularization
stimulator. Pellets containing the recombinant protein were
implanted in mouse corneas and their ability to inhibit the
bFGF-induced angiogenic response was compared to that of controls.
When a bFGF pellet containing GST was implanted new capillary
vessels grew from the cornea limbus, across the cornea and into the
pellet within 5 days. In contrast, addition of GST-METH1 or
GST-METH2 to the bFGF pellets completely abolished blood vessel
growth. Table 4 contains a summary of the results obtained from 41
assays performed. Intact TSP1 purified from platelets and GST-TSP1
were used as positive controls. All assays were performed at
identical concentrations, suggesting that METH1 and METH2 have
similar potency to that of TSP1 in the inhibition of angiogenesis.
In addition, when half of the standard concentration was used, a
weak, however noticeable response was seen, indicating a
dose-dependent effect.
TABLE-US-00005 TABLE 4 Activity of METH1 and METH2 recombinant
proteins in the corneal pocket assay bFGF Pellets Vascularized
corneas/Total corneas Vehicle 5/5 TSP1 0/5 GST 11/11 GST-TSP1-TI
1/4 GST-METH1-TSP 0/8 GST-METH2-TSP 0/8
[0451] In the CAM assay, the angiogenic response is analyzed by
measuring the number of vessels that grow within a matrix polymer
containing the angiogenic growth factor. To determine whether
recombinant METH1 and METH2 proteins inhibited neovascularization
in the CAM assay induced by VEGF, a matrigel polymer containing
VEGF and the recombinant protein were implanted in the CAM.
Quantitative analysis of the experiments, which included three
different polymers per treatment are shown in FIG. 6A. Matrigels
polymers containing VEGF plus 5 .mu.g of GST-METH1 or GST-METH2
caused greater than 80% inhibition in blood vessel growth. A
similar potency was found using the GST recombinant protein derived
from the type I repeats of TSP1. Furthermore, the anti-angiogenic
effect of the TSP domains in METH1 and METH2 was dose-dependent
with a complete inhibition of blood vessel growth when 15 .mu.g/ml
of protein was used (FIGS. 6C and D). GST alone, at identical
concentrations, had no significant effect on VEGF-stimulated
angiogenesis. CAM assays performed with the unprocessed form of
METH-1 and METH2 provided similar results to the processed forms.
It was unclear whether processing is not required for function or
if the CAM tissue lead to processing of our proteins. Thus, the
intact protein was incubated with CAM tissue for 24 h and was
evaluated the protein on Western blots. The results demonstrate
that the CAM tissue was able to generate a 68 kD METH1 processed
protein.
[0452] Synthetic peptides from the second or the third type I
repeats of human TSP1 can mimic the anti-angiogenic effects of the
intact TSP1 (Tolsma, S. S., et al., J. Cell. Biol. 122:497-511
(1993)). In fact, a 19-residue polypeptide was shown to be
sufficient to block in vivo neovascularization in the rat cornea
and to inhibit the bFGF-induced migration of cultured endothelial
cells (Vogel, T., et al., J. Cell. Biochem. 53:74-84 (1993);
Tolsma, S. S., et al., J. Cell. Biol. 122:497-511 (1993)). To test
whether the same was true for the METH1 and METH2 TSP domains,
peptides derived from the same region were synthesized and their
anti-angiogenic activity was evaluated in the CAM assay. The
results are shown in FIG. 6B. Peptides derived from both the TSP
domain of METH1 and METH2 blocked VEGF-induced angiogenesis
similarly to that of TSP1. In contrast, scramble peptides had no
significant effects.
Example 5
Proliferation Assays
[0453] Human dermal endothelial cells (HDEC) were isolated and
grown on Vitrogen.TM. coated petri-dishes in EBM (Clonetics, San
Diego, Calif.) supplemented with 15% fetal calf serum, 25 .mu.g/ml
cAMP, and 1 .mu.g/ml of hydrocortisone-21-acetate and were used
from passages 3 to 6. Cells were made quiescent by incubation of
confluent monolayers with phenol red-free EBM containing 0.2% BSA
for 48 h. Human dermal fibroblasts were isolated from neonatal
foreskin and by enzymatic dissociation. Both fibroblasts and smooth
muscle cells were maintained in DMEM supplemented with 10% fetal
calf serum. Human mammary epithelial cells (HMEC) were purchased
from Clonetics and maintained in the recommended media (mammary
epithelial growth media, MEGM).
[0454] Quiescent human dermal endothelial cells, between passage 3
and 6, were plated on Vitrogen.TM. coated 24-well plates in EBM
supplemented with 0.2% BSA, 0.1% fetal calf serum and 1 ng/ml of
bFGF in the presence or absence of the recombinant protein and
incubated at 5% CO.sub.2 at 37.degree. C. for 48 h. For vascular
smooth muscle (VSM) and fibroblast proliferation assays, cells were
incubated under the same conditions but using DMEM instead of EBM.
Human mammary epithelial cells were incubated on their growth
media. A pulse of [.sup.3H]-Thymidine (1 .mu.Ci/.mu.l) was added
during the last 4 h prior harvesting. Cells were washed and fixed
in 10% TCA. Incorporation of [.sup.3H]-thymidine was determined by
scintillation counting, as previously described (Iruela-Arispe, M.
L. & Sage, E. H., J. Cell. Biochem. 52:414 (1993)).
[0455] Statistical analyses were done using In-Stat software (Graph
Pad Software) for Macintosh. Assuming normal distributions, data
were analyzed by one-way ANOVA, followed by either T-test Dunnett
test for comparisons between groups, or student-Newman-Kleus test
for multiple comparisons between groups.
[0456] To gain insight into the mechanism by which METH1 and METH2
inhibit neovascularization, the direct effect of the purified
recombinant fusion proteins on endothelial cell proliferation was
tested. Serum-starved endothelial cells were plated into growth
medium containing bFGF and FCS. Recombinant proteins (3 .mu.g/ml)
were added at the same time of plating. 40% (GST-METH1), 45%
(6H-GST) or 36% (GST-METH2) inhibition was observed, in contrast to
a non-significant effect when GST alone was added. The recombinant
protein from the type I repeats of TSP1 had similar inhibitory
effects. (FIG. 7A). Furthermore, suppression of proliferation
mediated by METH1 or METH2 was dose-dependent, as shown in FIG. 7E.
The inhibition was observed as early as one day after treatment and
the inhibitory effect was not toxic and reversible since the
removal of the recombinant protein and subsequent addition of
growth factor alone led to the resumption of endothelial cell
proliferation.
[0457] The cell specificity of the anti-proliferative effects for
METH1 and METH2 on the endothelium was evaluated by additional
proliferation assays on a variety of non-endothelial cells. No
significant inhibition of proliferation was seen on fibroblasts or
smooth muscle cell cultures. In contrast, a non significant, but
reproducible stimulation of proliferation for these two cell types
could be observed. This result rules out the presence of any
potential nonspecific inhibitor of cell growth in the recombinant
protein preparations. On mammary epithelial cell, however, METH1
and METH2 inhibited cell proliferation to the same degree as
endothelial cells. Interestingly, TSP1 also suppresses mammary
epithelial cell proliferation both in vitro and in a transgenic
model.
[0458] The possibility that METH1 and METH2 might act as
disintegrins is consistent with their anti-angiogenic properties.
Clearly blockade of .alpha.v.beta..sub.3 and .beta.1 integrins with
antibodies has been shown to inhibit neovascularization both during
development and in tumors (Brooks, P. C., et al., Cell 85:683-693
(1996); Brooks, P. C., et al., Cell 92:391-400 (1998); Senger, D.
R., et al., Proc. Natl. Acad. Sci. USA 94:13612-13617 (1997)).
Integrins are essential for the mediation of both proliferative and
migratory signals (Schwartz, M. A. & Ingber, D. E., Mol. Biol.
Cell 5:389-393 (1994)), therefore interference with those signals
can be highly deleterious to the angiogenic process. The angiogenic
functional assays were performed with recombinant protein
containing only the type I repeats in METH1 and METH2.
[0459] The mechanism of action of and METH1 and METH2 with regards
to their angio-inhibitory activity is not known. To date we have
evidence that these proteins are secreted and bind to endothelial
cells. Further investigations are guided towards the identification
of receptors and signal transduction mechanisms. A likely
hypothesis resulting from the lessons learned from TSP1 is that
both METH1 and METH2 bind to CD36. Recently, this scavenger
receptor has been implicated in the mediation of signals by which
TSP-1 exert its anti-angiogenic effects (Dawson, D. W., et al., J.
Cell. Biol. 138:707-717 (1997)). Both the CSVTCG (SEQ ID NO:83)
(Asch, A. S., et al., Nature 262:1436-1439 (1993); Catimel, B., et
al., Biochem. J. 284:231-236 (1992)) and the GCQXR (SEQ ID NO: 84)
sequences have been proposed as primary binding motifs to CD36
(Dawson, D. W., et al., J. Cell. Biol. 138:707-717 (1997)). METH1
and METH2 have almost entire conservation in both these regions. A
complementary and also likely occurrence is binding of METH1 and
METH2 to bFGF. Binding to heparin and bFGF has been proposed as
part of the anti-angiogenic activity of TSP1 (Guo, N., et al., J.
Peptide Res. 49 (1997)). This property appears to be mediated
through the WSXWS (SEQ ID NO:82) motif, also conserved in METH1 and
METH2. Future efforts will focus on the signals implicated in the
anti-angiogenic properties mediated by these novel proteins and on
their potential as proteases of the extracellular milieu.
Example 6
Isolation of the METH1 or METH2 cDNA Clone from the Deposited
Sample
[0460] Two approaches can be used to isolate METH1 or METH2 from
the deposited sample. First, the deposited clone is transformed
into a suitable host (such as XL-1 Blue (Stratagene)) using
techniques known to those of skill in the art, such as those
provided by the vector supplier or in related publications or
patents. The transformants are plated on 1.5% agar plates
(containing the appropriate selection agent, e.g., ampicillin) to a
density of about 150 transformants (colonies) per plate. A single
colony is then used to generate DNA using nucleic acid isolation
techniques well known to those skilled in the art. (e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989),
Cold Spring Harbor Laboratory Press.)
[0461] Alternatively, two primers of 17-20 nucleotide derived from
both ends of the SEQ ID NO:1 or SEQ ID NO:3 (i.e., within the
region of SEQ ID NO:1 or SEQ ID NO:3 bounded by the 5' NT and the
3' NT of the clone) are synthesized and used to amplify the METH1
or METH2 cDNA using the deposited cDNA plasmids as templates. The
polymerase chain reaction is carried out under routine conditions,
for instance, in 25 .mu.l of reaction mixture with 0.5 .mu.g of the
above cDNA template. A convenient reaction mixture is 1.5-5 mM
MgCl.sub.2, 0.01% (w/v) gelatin, 20 .mu.M each of dATP, dCTP, dGTP,
dTTP, 25 .mu.mol of each primer and 0.25 Unit of Taq polymerase.
Thirty five cycles of PCR (denaturation at 94.degree. C. for 1 min;
annealing at 55.degree. C. for 1 min; elongation at 72.degree. C.
for 1 min) are performed with a Perkin-Elmer Cetus automated
thermal cycler. The amplified product is analyzed by agarose gel
electrophoresis and the DNA band with expected molecular weight is
excised and purified. The PCR product is verified to be the
selected sequence by subcloning and sequencing the DNA product.
[0462] Several methods are available for the identification of the
5' or 3' non-coding portions of the METH1 or METH2 gene which may
not be present in the deposited clones. These methods include but
are not limited to, filter probing, clone enrichment using specific
probes, and protocols similar or identical to 5' and 3 "RACE"
protocols which are well known in the art. For instance, a method
similar to 5' RACE is available for generating the missing 5' end
of a desired full-length transcript. (Fromont-Racine et al.,
Nucleic Acids Res. 21(7):1683-1684 (1993).)
[0463] Briefly, a specific RNA oligonucleotide is ligated to the 5'
ends of a population of RNA presumably containing full-length gene
RNA transcripts. A primer set containing a primer specific to the
ligated RNA oligonucleotide and a primer specific to a known
sequence of the METH1 or METH2 gene of interest is used to PCR
amplify the 5' portion of the METH1 or METH2 full-length gene. This
amplified product may then be sequenced and used to generate the
full length gene.
[0464] This above method starts with total RNA isolated from the
desired source, although poly-A+ RNA can be used. The RNA
preparation can then be treated with phosphatase if necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may
interfere with the later RNA ligase step. The phosphatase should
then be inactivated and the RNA treated with tobacco acid
pyrophosphatase in order to remove the cap structure present at the
5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the 5' end of the cap cleaved RNA which can then be
ligated to an RNA oligonucleotide using T4 RNA ligase.
[0465] This modified RNA preparation is used as a template for
first strand cDNA synthesis using a gene specific oligonucleotide.
The first strand synthesis reaction is used as a template for PCR
amplification of the desired 5' end using a primer specific to the
ligated RNA oligonucleotide and a primer specific to the known
sequence of the gene of interest. The resultant product is then
sequenced and analyzed to confirm that the 5' end sequence belongs
to the METH1 or METH2 gene.
Example 7
Bacterial Expression of METH1 or METH2
[0466] A METH1 or METH2 polynucleotide encoding a METH1 or METH2
polypeptide invention is amplified using PCR oligonucleotide
primers corresponding to the 5' and 3' ends of the DNA sequence, as
outlined in Example 5, to synthesize insertion fragments. The
primers used to amplify the cDNA insert should preferably contain
restriction sites, such as BamHI and XbaI, at the 5' end of the
primers in order to clone the amplified product into the expression
vector. For example, BamHI and XbaI correspond to the restriction
enzyme sites on the bacterial expression vector pQE-9. (Qiagen,
Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic
resistance (Amp.sup.r), a bacterial origin of replication (ori), an
IPTG-regulatable promoter/operator (P/O), a ribosome binding site
(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning
sites.
[0467] The pQE-9 vector is digested with BamHI and XbaI and the
amplified fragment is ligated into the pQE-9 vector maintaining the
reading frame initiated at the bacterial RBS. The ligation mixture
is then used to transform the E. coli strain M15/rep4 (Qiagen,
Inc.) which contains multiple copies of the plasmid pREP4, which
expresses the lacI repressor and also confers kanamycin resistance
(Kan.sup.r). Transformants are identified by their ability to grow
on LB plates and ampicillin/kanamycin resistant colonies are
selected. Plasmid DNA is isolated and confirmed by restriction
analysis.
[0468] Clones containing the desired constructs are grown overnight
(O/N) in liquid culture in LB media supplemented with both Amp (100
ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a
large culture at a ratio of 1:100 to 1:250. The cells are grown to
an optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
(Isopropyl-B-D-thiogalacto pyranoside) is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/0 leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested
by centrifugation (20 mins at 6000.times.g). The cell pellet is
solubilized in the chaotropic agent 6 Molar Guanidine HCl by
stirring for 3-4 hours at 4.degree. C. The cell debris is removed
by centrifugation, and the supernatant containing the polypeptide
is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin column (available from QIAGEN, Inc., supra). Proteins with a
6.times.His tag bind to the Ni-NTA resin with high affinity and can
be purified in a simple one-step procedure (for details see: The
QIAexpressionist (1995) QIAGEN, Inc., supra). Briefly, the
supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8,
the column is first washed with 10 volumes of 6 M guanidine-HCl, pH
8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and
finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
[0469] The purified METH1 or METH2 protein is then renatured by
dialyzing it against phosphate-buffered saline (PBS) or 50 mM
Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the METH1
or METH2 protein can be successfully refolded while immobilized on
the Ni-NTA column. The recommended conditions are as follows:
renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20%
glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors.
The renaturation should be performed over a period of 1.5 hours or
more. After renaturation the proteins are eluted by the addition of
250 mM immidazole. Immidazole is removed by a final dialyzing step
against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl.
The purified METH1 or METH2 protein is stored at 4.degree. C. or
frozen at -80.degree. C.
[0470] In addition to the above expression vector, the present
invention further includes an expression vector comprising phage
operator and promoter elements operatively linked to a METH1 or
METH2 polynucleotide, called pHE4a. (ATCC.TM. Accession Number
209645, deposited Feb. 25, 1998.) This vector contains: 1) a
neomycinphosphotransferase gene as a selection marker, 2) an E.
coli origin of replication, 3) a T5 phage promoter sequence, 4) two
lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the
lactose operon repressor gene (lacIq). The origin of replication
(oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter
sequence and operator sequences are made synthetically.
[0471] DNA can be inserted into the pHEa by restricting the vector
with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted
product on a gel, and isolating the larger fragment (the stuffer
fragment should be about 310 base pairs). The DNA insert is
generated according to the PCR protocol described in Example 5,
using PCR primers having restriction sites for NdeI (5' primer) and
XbaI, BamHI, XhoI, or Asp718 (3' primer). The PCR insert is gel
purified and restricted with compatible enzymes. The insert and
vector are ligated according to standard protocols.
[0472] The engineered vector could easily be substituted in the
above protocol to express protein in a bacterial system.
Example 8
Purification of METH1 or METH2 Polypeptide from an Inclusion
Body
[0473] The following alternative method can be used to purify METH1
or METH2 polypeptide expressed in E coli when it is present in the
form of inclusion bodies. Unless otherwise specified, all of the
following steps are conducted at 4-10.degree. C.
[0474] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells harvested by continuous centrifugation at 15,000 rpm (Heraeus
Sepatech). On the basis of the expected yield of protein per unit
weight of cell paste and the amount of purified protein required,
an appropriate amount of cell paste, by weight, is suspended in a
buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The
cells are dispersed to a homogeneous suspension using a high shear
mixer.
[0475] The cells are then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0476] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the polypeptide containing supernatant is incubated at
4.degree. C. overnight to allow further GuHCl extraction.
[0477] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0478] To clarify the refolded polypeptide solution, a previously
prepared tangential filtration unit equipped with 0.16 um membrane
filter with appropriate surface area (e.g., Filtron), equilibrated
with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample
is loaded onto a cation exchange resin (e.g., Poros HS-50,
Perseptive Biosystems). The column is washed with 40 mM sodium
acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500
mM NaCl in the same buffer, in a stepwise manner. The absorbance at
280 nm of the effluent is continuously monitored. Fractions are
collected and further analyzed by SDS-PAGE.
[0479] Fractions containing the METH1 or METH2 polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the polypeptide (determined, for instance, by 16%
SDS-PAGE) are then pooled.
[0480] The resultant METH1 or METH2 polypeptide should exhibit
greater than 95% purity after the above refolding and purification
steps. No major contaminant bands should be observed from Coomassie
blue stained 16% SDS-PAGE gel when 5 ug of purified protein is
loaded. The purified METH1 or METH2 protein can also be tested for
endotoxin/LPS contamination, and typically the LPS content is less
than 0.1 ng/ml according to LAL assays.
Example 9
Cloning and Expression of METH1 or METH2 in a Baculovirus
Expression System
[0481] In this example, the plasmid shuttle vector pA2 is used to
insert METH1 or METH2 polynucleotide into a baculovirus to express
METH1 or METH2. This expression vector contains the strong
polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction
sites such as BamHI, Xba I and Asp718. The polyadenylation site of
the simian virus 40 ("SV40") is used for efficient polyadenylation.
For easy selection of recombinant virus, the plasmid contains the
beta-galactosidase gene from E. coli under control of a weak
Drosophila promoter in the same orientation, followed by the
polyadenylation signal of the polyhedrin gene. The inserted genes
are flanked on both sides by viral sequences for cell-mediated
homologous recombination with wild-type viral DNA to generate a
viable virus that expresses the cloned METH1 or METH2
polynucleotide.
[0482] Many other baculovirus vectors can be used in place of the
vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, in
Luckow et al., Virology 170:31-39 (1989).
[0483] Specifically, the METH1 or METH2 cDNA sequence contained in
the deposited clone, including the AUG initiation codon and any
naturally associated leader sequence, is amplified using the PCR
protocol described in Example 5. If the naturally occurring signal
sequence is used to produce the secreted protein, the pA2 vector
does not need a second signal peptide. Alternatively, the vector
can be modified (pA2 GP) to include a baculovirus leader sequence,
using the standard methods described in Summers et al., "A Manual
of Methods for Baculovirus Vectors and Insect Cell Culture
Procedures," Texas Agricultural Experimental Station Bulletin No.
1555 (1987).
[0484] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("Geneclean," BIO 101 Inc., La
Jolla, Calif.). The fragment then is digested with appropriate
restriction enzymes and again purified on a 1% agarose gel.
[0485] The plasmid is digested with the corresponding restriction
enzymes and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.).
[0486] The fragment and the dephosphorylated plasmid are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla,
Calif.) cells are transformed with the ligation mixture and spread
on culture plates. Bacteria containing the plasmid are identified
by digesting DNA from individual colonies and analyzing the
digestion product by gel electrophoresis. The sequence of the
cloned fragment is confirmed by DNA sequencing.
[0487] Five ug of a plasmid containing the polynucleotide is
co-transfected with 1.0 ug of a commercially available linearized
baculovirus DNA ("BaculoGold.sup.a baculovirus DNA", Pharmingen,
San Diego, Calif.), using the lipofection method described by
Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One
ug of BaculoGold.sup.a virus DNA and 5 ug of the plasmid are mixed
in a sterile well of a microtiter plate containing 50 ul of
serum-free Grace's medium (Life Technologies Inc., Gaithersburg,
Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are
added, mixed and incubated for 15 minutes at room temperature. Then
the transfection mixture is added drop-wise to Sf9 insect cells
(ATCC.TM. CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate is then incubated for 5
hours at 27.degree. C. The transfection solution is then removed
from the plate and 1 ml of Grace's insect medium supplemented with
10% fetal calf serum is added. Cultivation is then continued at
27.degree. C. for four days.
[0488] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg)
is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10.) After appropriate incubation, blue stained plaques are
picked with the tip of a micropipettor (e.g., Eppendorf). The agar
containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 ul of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at
4.degree. C.
[0489] To verify the expression of the polypeptide, Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus containing
the polynucleotide at a multiplicity of infection ("MOI") of about
2. If radiolabeled proteins are desired, 6 hours later the medium
is removed and is replaced with SF900 II medium minus methionine
and cysteine (available from Life Technologies Inc., Rockville,
Md.). After 42 hours, 5 uCi of .sup.35S-methionine and 5 uCi
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[0490] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the produced METH1 or METH2 protein.
Example 10
Expression of METH1 or METH2 in Mammalian Cells
[0491] METH1 or METH2 polypeptide can be expressed in a mammalian
cell. A typical mammalian expression vector contains a promoter
element, which mediates the initiation of transcription of mRNA, a
protein coding sequence, and signals required for the termination
of transcription and polyadenylation of the transcript. Additional
elements include enhancers, Kozak sequences and intervening
sequences flanked by donor and acceptor sites for RNA splicing.
Highly efficient transcription is achieved with the early and late
promoters from SV40, the long terminal repeats (LTRs) from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus (CMV). However, cellular elements can also be used
(e.g., the human actin promoter).
[0492] Suitable expression vectors for use in practicing the
present invention include, for example, vectors such as pSVL and
pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC.TM. 37152),
pSV2DHFR (ATCC.TM. 37146), pBC12MI (ATCC.TM. 67109), pCMVSport 2.0,
and pCMVSport 3.0. Mammalian host cells that could be used include,
human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells,
Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese
hamster ovary (CHO) cells.
[0493] Alternatively, METH1 or METH2 polypeptide can be expressed
in stable cell lines containing the METH1 or METH2 polynucleotide
integrated into a chromosome. The co-transfection with a selectable
marker such as DHFR, gpt, neomycin, hygromycin allows the
identification and isolation of the transfected cells.
[0494] The transfected METH1 or METH2 gene can also be amplified to
express large amounts of the encoded protein. The DHFR
(dihydrofolate reductase) marker is useful in developing cell lines
that carry several hundred or even several thousand copies of the
gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem.
253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et
Biophys. Acta 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,
Biotechnology 9:64-68 (1991).) Another useful selection marker is
the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.
227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175
(1992). Using these markers, the mammalian cells are grown in
selective medium and the cells with the highest resistance are
selected. These cell lines contain the amplified gene(s) integrated
into a chromosome. Chinese hamster ovary (CHO) and NSO cells are
often used for the production of proteins.
[0495] Derivatives of the plasmid pSV2-DHFR (ATCC.TM. Accession No.
37146), the expression vectors pC4 (ATCC.TM. Accession No. 209646)
and pC6 (ATCC.TM. Accession No. 209647) contain the strong promoter
(LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and
Cellular Biology, 438-447 (March, 1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985).) Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, XbaI and Asp718, facilitate the cloning of METH1 or METH2.
The vectors also contain the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene, and the mouse
DHFR gene under control of the SV40 early promoter.
[0496] If a naturally occurring signal sequence is used to produce
a secreted protein, the vector does not need a second signal
peptide. Alternatively, if a naturally occurring signal sequence is
not used, the vector can be modified to include a heterologous
signal sequence in an effort to secrete the protein from the cell.
(See, e.g., WO 96/34891.)
[0497] The amplified fragment is then digested with the appropriate
restriction enzyme and purified on a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla,
Calif.). The isolated fragment and the dephosphorylated vector are
then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells
are then transformed and bacteria are identified that contain the
fragment inserted into plasmid pC6 or pC4 using, for instance,
restriction enzyme analysis.
[0498] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC6 or
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSVneo using
lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a
dominant selectable marker, the neo gene from Tn5 encoding an
enzyme that confers resistance to a group of antibiotics including
G418. The cells are seeded in alpha minus MEM supplemented with 1
mg/ml G418. After 2 days, the cells are trypsinized and seeded in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM
supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml
G418. After about 10-14 days single clones are trypsinized and then
seeded in 6-well petri dishes or 10 ml flasks using different
concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM). Clones growing at the highest concentrations of methotrexate
are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained which grow
at a concentration of 100-200 uM. Expression of METH1 or METH2 is
analyzed, for instance, by SDS-PAGE and Western blot or by reversed
phase HPLC analysis.
Example 11
Construction of N-Terminal and/or C-Terminal Deletion Mutants
[0499] The following general approach may be used to clone a
N-terminal or C-terminal deletion METH1 or METH2 deletion mutant.
Generally, two oligonucleotide primers of about 15-25 nucleotides
are derived from the desired 5' and 3' positions of a
polynucleotide of SEQ ID NO:1 or SEQ ID NO:3. The 5' and 3'
positions of the primers are determined based on the desired METH1
or METH2 polynucleotide fragment. An initiation and stop codon are
added to the 5' and 3' primers respectively, if necessary, to
express the METH1 or METH2 polypeptide fragment encoded by the
polynucleotide fragment. Preferred METH1 or METH2 polynucleotide
fragments are those encoding the N-terminal and C-terminal deletion
mutants disclosed above in the "Polynucleotide and Polypeptide
Fragments" section of the Specification.
[0500] Additional nucleotides containing restriction sites to
facilitate cloning of the METH1 or METH2 polynucleotide fragment in
a desired vector may also be added to the 5' and 3' primer
sequences. The METH1 or METH2 polynucleotide fragment is amplified
from genomic DNA or from the deposited cDNA clone using the
appropriate PCR oligonucleotide primers and conditions discussed
herein or known in the art. The METH1 or METH2 polypeptide
fragments encoded by the METH1 or METH2 polynucleotide fragments of
the present invention may be expressed and purified in the same
general manner as the full length polypeptides, although routine
modifications may be necessary due to the differences in chemical
and physical properties between a particular fragment and full
length polypeptide.
[0501] As a means of exemplifying but not limiting the present
invention, the polynucleotide encoding the METH1 polypeptide
fragment R-235 to L-934 or the METH2 polypeptide fragment R-214 to
Q-836 is amplified and cloned as follows: A 5' primer is generated
comprising a restriction enzyme site followed by an initiation
codon in frame with the polynucleotide sequence encoding the
N-terminal portion of the polypeptide fragment beginning with R-235
or R-214, respectively. A complementary 3' primer is generated
comprising a restriction enzyme site followed by a stop codon in
frame with the polynucleotide sequence encoding C-terminal portion
of the METH1 or METH2 polypeptide fragment ending with L-934 or
Q-836, respectively.
[0502] The amplified polynucleotide fragment and the expression
vector are digested with restriction enzymes which recognize the
sites in the primers. The digested polynucleotides are then ligated
together. The METH1 or METH2 polynucleotide fragment is inserted
into the restricted expression vector, preferably in a manner which
places the METH1 or METH2 polypeptide fragment coding region
downstream from the promoter. The ligation mixture is transformed
into competent E. coli cells using standard procedures and as
described in the Examples herein. Plasmid DNA is isolated from
resistant colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
Example 12
Protein Fusions of METH1 or METH2
[0503] METH1 or METH2 polypeptides are preferably fused to other
proteins. These fusion proteins can be used for a variety of
applications. For example, fusion of METH1 or METH2 polypeptides to
His-tag, HA-tag, protein A, IgG domains, and maltose binding
protein facilitates purification. (See Example 7; see also EP A
394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly,
fusion to IgG-1, IgG-3, and albumin increases the halflife time in
vivo. Nuclear localization signals fused to METH1 or METH2
polypeptides can target the protein to a specific subcellular
localization, while covalent heterodimer or homodimers can increase
or decrease the activity of a fusion protein. Fusion proteins can
also create chimeric molecules having more than one function.
Finally, fusion proteins can increase solubility and/or stability
of the fused protein compared to the non-fused protein. All of the
types of fusion proteins described above can be made by modifying
the following protocol, which outlines the fusion of a polypeptide
to an IgG molecule, or the protocol described in Example 7.
[0504] Briefly, the human Fc portion of the IgG molecule can be PCR
amplified, using primers that span the 5' and 3' ends of the
sequence described below. These primers also should have convenient
restriction enzyme sites that will facilitate cloning into an
expression vector, preferably a mammalian expression vector.
[0505] For example, if pC4 (Accession No. 209646) is used, the
human Fc portion can be ligated into the BamHI cloning site. Note
that the 3' BamHI site should be destroyed. Next, the vector
containing the human Fc portion is re-restricted with BamHI,
linearizing the vector, and METH1 or METH2 polynucleotide, isolated
by the PCR protocol described in Example 5, is ligated into this
BamHI site. Note that the polynucleotide is cloned without a stop
codon, otherwise a fusion protein will not be produced.
[0506] If the naturally occurring signal sequence is used to
produce the secreted protein, pC4 does not need a second signal
peptide. Alternatively, if the naturally occurring signal sequence
is not used, the vector can be modified to include a heterologous
signal sequence. (See, e.g., WO 96/34891.)
TABLE-US-00006 Human IgG Fc region: (SEQ ID NO:85)
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC
CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG
TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG
GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA
ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC
ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG
GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT
GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG
TAAATGAGTGCGACGGCCGCGAC TCTAGAGGAT
Example 13
Production of an Antibody
[0507] The antibodies of the present invention can be prepared by a
variety of methods. (See, Current Protocols, Chapter 2.) For
example, cells expressing METH1 or METH2 is administered to an
animal to induce the production of sera containing polyclonal
antibodies. In a preferred method, a preparation of METH1 or METH2
protein is prepared and purified to render it substantially free of
natural contaminants. Such a preparation is then introduced into an
animal in order to produce polyclonal antisera of greater specific
activity.
[0508] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or protein binding fragments
thereof). Such monoclonal antibodies can be prepared using
hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981).) In
general, such procedures involve immunizing an animal (preferably a
mouse) with METH1 or METH2 polypeptide or, more preferably, with a
secreted METH1 or METH2 polypeptide-expressing cell. Such cells may
be cultured in any suitable tissue culture medium; however, it is
preferable to culture cells in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 .mu.l of
nonessential amino acids, about 1,000 U/ml of penicillin, and about
100 ug/ml of streptomycin.
[0509] The splenocytes of such mice are extracted and fused with a
suitable myeloma cell line. Any suitable myeloma cell line may be
employed in accordance with the present invention; however, it is
preferable to employ the parent myeloma cell line (SP2O), available
from the ATCC.TM.. After fusion, the resulting hybridoma cells are
selectively maintained in HAT medium, and then cloned by limiting
dilution as described by Wands et al. (Gastroenterology 80:225-232
(1981).) The hybridoma cells obtained through such a selection are
then assayed to identify clones which secrete antibodies capable of
binding the METH1 or METH2 polypeptide.
[0510] Alternatively, additional antibodies capable of binding to
METH1 or METH2 polypeptide can be produced in a two-step procedure
using anti-idiotypic antibodies. Such a method makes use of the
fact that antibodies are themselves antigens, and therefore, it is
possible to obtain an antibody which binds to a second antibody. In
accordance with this method, protein specific antibodies are used
to immunize an animal, preferably a mouse. The splenocytes of such
an animal are then used to produce hybridoma cells, and the
hybridoma cells are screened to identify clones which produce an
antibody whose ability to bind to the METH1 or METH2
protein-specific antibody can be blocked by METH1 or METH2. Such
antibodies comprise anti-idiotypic antibodies to the METH1 or METH2
protein-specific antibody and can be used to immunize an animal to
induce formation of further METH1 or METH2 protein-specific
antibodies.
[0511] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, secreted METH1 or METH2 protein-binding
fragments can be produced through the application of recombinant
DNA technology or through synthetic chemistry.
[0512] For in vivo use of antibodies in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art. (See, for review, Morrison, Science 229:1202 (1985); Oi et
al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No.
4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494;
Neuberger et al., WO 8601533; Robinson et al., WO 8702671;
Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature
314:268 (1985).)
[0513] Isolation of Antibody Fragments Directed Against METH1
and/or METH2 from a Library of scFvs.
[0514] Naturally occurring V-genes isolated from human PBLs are
constructed into a large library of antibody fragments which
contain reactivities against METH1 and/or METH2 to which the donor
may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793
incorporated herein in its entirety by reference).
[0515] Rescue of the Library. A library of scFvs is constructed
from the RNA of human PBLs as described in WO92/01047. To rescue
phage displaying antibody fragments, approximately 10.sup.9 E. coli
harbouring the phagemid are used to inoculate 50 ml of 2.times.TY
containing 1% glucose and 100 ug/ml of ampicillin
(2.times.TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five
ml of this culture is used to innoculate 50 ml of
2.times.TY-AMP-GLU, 2.times.10.sup.8 TU of delta gene 3 helper (M13
delta gene III, see WO92/01047) are added and the culture incubated
at 37.degree. C. for 45 minutes without shaking and then at
37.degree. C. for 45 minutes with shaking. The culture is
centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended
in 2 liters of 2.times.TY containing 100 ug/ml ampicillin and 50
ug/ml kanamycin and grown overnight. Phage are prepared as
described in WO92/01047.
[0516] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harbouring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8, 4000 revs/min for 10
min), resuspended in 300 ml 2.times.TY broth containing 100 ug
ampicillin/ml and 25 ug kanamycin/ml (2.times.TY-AMP-KAN) and grown
overnight, shaking at 37.degree. C. Phage particles are purified
and concentrated from the culture medium by two PEG-precipitations
(Sambrook et al., 1990), resuspended in 2 ml PBS and passed through
a 0.45 um filter (Minisart NML; Sartorius) to give a final
concentration of approximately 10.sup.13 transducing units/ml
(ampicillin-resistant clones).
[0517] Panning of the Library. Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100 ug/ml or 10 ug/ml of a
polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100 ug/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0518] Characterization of Binders. Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10 pg/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see e.g., WO92/01047) and then
by sequencing.
Example 14
Production of METH1 or METH2 Protein for High-Throughput Screening
Assays
[0519] The following protocol produces a supernatant containing
METH1 or METH2 polypeptide to be tested. This supernatant can then
be used in the Screening Assays described in Examples 16-23.
[0520] First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim)
stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or
magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml.
Add 200 ul of this solution to each well (24 well plates) and
incubate at RT for 20 minutes. Be sure to distribute the solution
over each well (note: a 12-channel pipetter may be used with tips
on every other channel). Aspirate off the Poly-D-Lysine solution
and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should
remain in the well until just prior to plating the cells and plates
may be poly-lysine coated in advance for up to two weeks.
[0521] Plate 293T cells (do not carry cells past P+20) at
2.times.10.sup.5 cells/well in 0.5 ml DMEM (Dulbecco's Modified
Eagle Medium) (with 4.5 G/L glucose and L-glutamine (12-604F
Biowhittaker))/10% heat inactivated FBS (14-503F
Biowhittaker)/1.times. Penstrep (17-602E Biowhittaker). Let the
cells grow overnight.
[0522] The next day, mix together in a sterile solution basin: 300
ul Lipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070
Gibco/BRL)/96-well plate. With a small volume multi-channel
pipetter, aliquot approximately 2 ug of an expression vector
containing a polynucleotide insert, produced by the methods
described in Examples 10-12, into an appropriately labeled 96-well
round bottom plate. With a multi-channel pipetter, add 50 ul of the
Lipofectamine/Optimem I mixture to each well. Pipette up and down
gently to mix. Incubate at RT 15-45 minutes. After about 20
minutes, use a multi-channel pipetter to add 150 ul Optimem I to
each well. As a control, one plate of vector DNA lacking an insert
should be transfected with each set of transfections.
[0523] Preferably, the transfection should be performed by
tag-teaming the following tasks. By tag-teaming, hands on time is
cut in half, and the cells do not spend too much time on PBS.
First, person A aspirates off the media from four 24-well plates of
cells, and then person B rinses each well with 0.5-1 ml PBS. Person
A then aspirates off PBS rinse, and person B, using a 12-channel
pipetter with tips on every other channel, adds the 200 ul of
DNA/Lipofectamine/Optimem I complex to the odd wells first, then to
the even wells, to each row on the 24-well plates. Incubate at
37.degree. C. for 6 hours.
[0524] While cells are incubating, prepare appropriate media,
either 1% BSA in DMEM with 1.times. penstrep, or HGS CHO-5 media
(116.6 mg/L of CaCl.sub.2 (anhyd); 0.00130 mg/L
CuSO.sub.4.5H.sub.2O; 0.050 mg/L of Fe(NO.sub.3).sub.3-9H.sub.2O;
0.417 mg/L of FeSO.sub.4-7H.sub.2O; 311.80 mg/L of Kcl; 28.64 mg/L
of MgCl.sub.2; 48.84 mg/L of MgSO.sub.4; 6995.50 mg/L of NaCl;
2400.0 mg/L of NaHCO.sub.3; 62.50 mg/L of
NaH.sub.2PO.sub.4--H.sub.2O; 71.02 mg/L of Na.sub.2HPO.sub.4;
0.4320 mg/L of ZnSO.sub.4-7H.sub.2O; 0.002 mg/L of Arachidonic
Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of
DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010
mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of
Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic
Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20
mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of
L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of
L-Asparagine-H.sub.2O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml
of L-Cystine-2HCL-H.sub.2O; 31.29 mg/ml of L-Cystine-2HCL; 7.35
mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml
of Glycine; 52.48 mg/ml of L-Histidine-HCL-H.sub.2O; 106.97 mg/ml
of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of
L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of
L-Phenylalanine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine;
101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79
mg/ml of L-Tyrosine-2Na-2H.sub.2O; and 99.65 mg/ml of L-Valine;
0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L
of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of
i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL;
0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L
of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin
B.sub.12; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine;
0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL;
55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM
of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of
Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of
Methyl-B-Cyclodextrin complexed with Oleic Acid; 10 mg/L of
Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust
osmolarity to 327 mOsm with 2 mm glutamine and 1.times. penstrep.
(BSA (81-068-3 Bayer) 100 gm dissolved in 1 L DMEM for a 10% BSA
stock solution). Filter the media and collect 50 ul for endotoxin
assay in 15 ml polystyrene conical.
[0525] The transfection reaction is terminated, preferably by
tag-teaming, at the end of the incubation period. Person A
aspirates off the transfection media, while person B adds 1.5 ml
appropriate media to each well. Incubate at 37.degree. C. for 45 or
72 hours depending on the media used: 1% BSA for 45 hours or CHO-5
for 72 hours.
[0526] On day four, using a 300 ul multichannel pipetter, aliquot
600 ul in one 1 ml deep well plate and the remaining supernatant
into a 2 ml deep well. The supernatants from each well can then be
used in the assays described in Examples 16-23.
[0527] It is specifically understood that when activity is obtained
in any of the assays described below using a supernatant, the
activity originates from either the METH1 or METH2 polypeptide
directly (e.g., as a secreted protein) or by METH1 or METH2
inducing expression of other proteins, which are then secreted into
the supernatant. Thus, the invention further provides a method of
identifying the protein in the supernatant characterized by an
activity in a particular assay.
Example 15
Construction of GAS Reporter Construct
[0528] One signal transduction pathway involved in the
differentiation and proliferation of cells is called the Jaks-STATs
pathway. Activated proteins in the Jaks-STATs pathway bind to gamma
activation site "GAS" elements or interferon-sensitive responsive
element ("ISRE"), located in the promoter of many genes. The
binding of a protein to these elements alter the expression of the
associated gene.
[0529] GAS and ISRE elements are recognized by a class of
transcription factors called Signal Transducers and Activators of
Transcription, or "STATs." There are six members of the STATs
family. Stat1 and Stat3 are present in many cell types, as is Stat2
(as response to IFN-alpha is widespread). Stat4 is more restricted
and is not in many cell types though it has been found in T helper
class I, cells after treatment with IL-12. Stat5 was originally
called mammary growth factor, but has been found at higher
concentrations in other cells including myeloid cells. It can be
activated in tissue culture cells by many cytokines.
[0530] The STATs are activated to translocate from the cytoplasm to
the nucleus upon tyrosine phosphorylation by a set of kinases known
as the Janus Kinase ("Jaks") family. Jaks represent a distinct
family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2,
and Jak3. These kinases display significant sequence similarity and
are generally catalytically inactive in resting cells.
[0531] The Jaks are activated by a wide range of receptors
summarized in the Table below. (Adapted from review by Schidler and
Darnell, Ann. Rev. Biochem. 64:621-51 (1995).) A cytokine receptor
family, capable of activating Jaks, is divided into two groups: (a)
Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9,
IL-1, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and
thrombopoietin; and (b) Class 2 includes IFN-a, IFN-g, and IL-10.
The Class 1 receptors share a conserved cysteine motif (a set of
four conserved cysteines and one tryptophan) and a WSXWS motif (a
membrane proxial region encoding Trp-Ser-Xxx-Trp-Ser (SEQ ID
NO:82)).
[0532] Thus, on binding of a ligand to a receptor, Jaks are
activated, which in turn activate STATs, which then translocate and
bind to GAS elements. This entire process is encompassed in the
Jaks-STATs signal transduction pathway.
[0533] Therefore, activation of the Jaks-STATs pathway, reflected
by the binding of the GAS or the ISRE element, can be used to
indicate proteins involved in the proliferation and differentiation
of cells. For example, growth factors and cytokines are known to
activate the Jaks-STATs pathway. (See Table below.) Thus, by using
GAS elements linked to reporter molecules, activators of the
Jaks-STATs pathway can be identified.
TABLE-US-00007 JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS (elements)
or ISRE IFN family IFN-a/B + + - - 1, 2, 3 ISRE IFN-g + + - 1 GAS
(IRF1 > Lys6 > IFP) Il-10 + ? ? - 1, 3 gp130 family IL-6
(Pleiotrophic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) Il-11
(Pleiotrophic) ? + ? ? 1, 3 OnM (Pleiotrophic) ? + + ? 1, 3 LIF
(Pleiotrophic) ? + + ? 1, 3 CNTF (Pleiotrophic) -/+ + + ? 1, 3
G-CSF (Pleiotrophic) ? + ? ? 1, 3 IL-12 (Pleiotrophic) + - + + 1, 3
g-C family IL-2 (lymphocytes) - + - + 1, 3, 5 GAS IL-4
(lymph/myeloid) - + - + 6 GAS (IRF1 = IFP >> Ly6) (IgH) IL-7
(lymphocytes) - + - + 5 GAS IL-9 (lymphocytes) - + - + 5 GAS IL-13
(lymphocyte) - + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3
(myeloid) - - + - 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid)
- - + - 5 GAS GM-CSF (myeloid) - - + - 5 GAS Growth hormone family
GH ? - + - 5 GAS (B-CAS > IRF1 = IFP >> Ly6) PRL ? +/- + -
1, 3, 5 EPO ? - + - 5 Receptor Tyrosine Kinases EGF ? + + - 1, 3
GAS (IRF1) PDGF ? + + - 1, 3 GAS (not IRF1) CSF-1 ? + + - 1, 3
[0534] To construct a synthetic GAS containing promoter element,
which is used in the Biological Assays described in Examples 16-17,
a PCR based strategy is employed to generate a GAS-SV40 promoter
sequence. The 5' primer contains four tandem copies of the GAS
binding site found in the IRF1 promoter and previously demonstrated
to bind STATs upon induction with a range of cytokines (Rothman et
al., Immunity 1:457-468 (1994).), although other GAS or ISRE
elements can be used instead. The 5' primer also contains 18 bp of
sequence complementary to the SV40 early promoter sequence and is
flanked with an XhoI site. The sequence of the 5' primer is:
TABLE-US-00008 (SEQ ID NO:86)
5':GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCC
CCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG:3'
[0535] The downstream primer is complementary to the SV40 promoter
and is flanked with a Hind III site:
TABLE-US-00009 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:
87)
[0536] PCR amplification is performed using the SV40 promoter
template present in the B-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI/Hind III
and subcloned into BLSK2-. (Stratagene.) Sequencing with forward
and reverse primers confirms that the insert contains the following
sequence:
TABLE-US-00010 (SEQ ID NO:88)
5':CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA
AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCA
TTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGG
CCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGA GGCCTAGGCTTTTGC
AAAAAGCTT:3'
[0537] With this GAS promoter element linked to the SV40 promoter,
a GAS:SEAP2 reporter construct is next engineered. Here, the
reporter molecule is a secreted alkaline phosphatase, or "SEAP."
Clearly, however, any reporter molecule can used be instead of
SEAP, in this or in any of the other Examples. Well known reporter
molecules that can be used instead of SEAP include chloramphenicol
acetyltransferase (CAT), luciferase, alkaline phosphatase,
.beta.-galactosidase, green fluorescent protein (GFP), or any
protein detectable by an antibody.
[0538] The above sequence confirmed synthetic GAS-SV40 promoter
element is subcloned into the pSEAP-Promoter vector obtained from
Clontech using HindIII and XhoI, effectively replacing the SV40
promoter with the amplified GAS:SV40 promoter element, to create
the GAS-SEAP vector. However, this vector does not contain a
neomycin resistance gene, and therefore, is not preferred for
mammalian expression systems.
[0539] Thus, in order to generate mammalian stable cell lines
expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed
from the GAS-SEAP vector using SalI and NotI, and inserted into a
backbone vector containing the neomycin resistance gene, such as
pGFP-1 (Clontech), using these restriction sites in the multiple
cloning site, to create the GAS-SEAP/Neo vector. Once this vector
is transfected into mammalian cells, this vector can then be used
as a reporter molecule for GAS binding as described in Examples
16-17.
[0540] Other constructs can be made using the above description and
replacing GAS with a different promoter sequence. For example,
construction of reporter molecules containing NFK-B and EGR
promoter sequences are described in Examples 18 and 19. However,
many other promoters can be substituted using the protocols
described in these Examples. For instance, SRE, IL-2, NFAT, or
Osteocalcin promoters can be substituted, alone or in combination
(e.g., GAS/NF-KB/EGR, GAS/NF-KB, I1-2/NFAT, or NF-KB/GAS).
Similarly, other cell lines can be used to test reporter construct
activity, such as HELA (epithelial), HUVEC (endothelial), Reh
(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or
Cardiomyocyte.
Example 16
High-Throughput Screening Assay for T-Cell Activity
[0541] The following protocol is used to assess T-cell activity of
METH1 or METH2 by determining whether METH1 or METH2 supernatant
proliferates and/or differentiates T-cells. T-cell activity is
assessed using the GAS/SEAP/Neo construct produced in Example 15.
Thus, factors that increase SEAP activity indicate the ability to
activate the Jaks-STATS signal transduction pathway. The T-cell
used in this assay is Jurkat T-cells (ATCC.TM. Accession No.
TIB-152), although Molt-3 cells (ATCC.TM. Accession No. CRL-1552)
and Molt-4 cells (ATCC.TM. Accession No. CRL-1582) cells can also
be used.
[0542] Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In
order to generate stable cell lines, approximately 2 million Jurkat
cells are transfected with the GAS-SEAP/neo vector using DMRIE-C
(Life Technologies) (transfection procedure described below). The
transfected cells are seeded to a density of approximately 20,000
cells per well and transfectants resistant to 1 mg/ml genticin
selected. Resistant colonies are expanded and then tested for their
response to increasing concentrations of interferon gamma. The dose
response of a selected clone is demonstrated.
[0543] Specifically, the following protocol will yield sufficient
cells for 75 wells containing 200 ul of cells. Thus, it is either
scaled up, or performed in multiple to generate sufficient cells
for multiple 96 well plates. Jurkat cells are maintained in
RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM (Life
Technologies) with 10 ug of plasmid DNA in a T25 flask. Add 2.5 ml
OPTI-MEM containing 50 ul of DMRIE-C and incubate at room
temperature for 15-45 mins.
[0544] During the incubation period, count cell concentration, spin
down the required number of cells (10.sup.7 per transfection), and
resuspend in OPTI-MEM to a final concentration of 10.sup.7
cells/ml. Then add 1 ml of 1.times.10.sup.7 cells in OPTI-MEM to
T25 flask and incubate at 37.degree. C. for 6 hrs. After the
incubation, add 10 ml of RPMI+15% serum.
[0545] The Jurkat:GAS-SEAP stable reporter lines are maintained in
RPMI+10% serum, 1 mg/m Genticin, and 1% Pen-Strep. These cells are
treated with supernatants containing METH1 or METH2 polypeptides or
METH1 or METH2 induced polypeptides as produced by the protocol
described in Example 14.
[0546] On the day of treatment with the supernatant, the cells
should be washed and resuspended in fresh RPMI+10% serum to a
density of 500,000 cells per ml. The exact number of cells required
will depend on the number of supernatants being screened. For one
96 well plate, approximately 10 million cells (for 10 plates, 100
million cells) are required.
[0547] Transfer the cells to a triangular reservoir boat, in order
to dispense the cells into a 96 well dish, using a 12 channel
pipette. Using a 12 channel pipette, transfer 200 ul of cells into
each well (therefore adding 100,000 cells per well).
[0548] After all the plates have been seeded, 50 ul of the
supernatants are transferred directly from the 96 well plate
containing the supernatants into each well using a 12 channel
pipette. In addition, a dose of exogenous interferon gamma (0.1,
1.0, 10 ng) is added to wells H9, H10, and H11 to serve as
additional positive controls for the assay.
[0549] The 96 well dishes containing Jurkat cells treated with
supernatants are placed in an incubator for 48 hrs (note: this time
is variable between 48-72 hrs). 35 ul samples from each well are
then transferred to an opaque 96 well plate using a 12 channel
pipette. The opaque plates should be covered (using sellophene
covers) and stored at -20.degree. C. until SEAP assays are
performed according to Example 20. The plates containing the
remaining treated cells are placed at 4.degree. C. and serve as a
source of material for repeating the assay on a specific well if
desired.
[0550] As a positive control, 100 Unit/ml interferon gamma can be
used which is known to activate Jurkat T cells. Over 30 fold
induction is typically observed in the positive control wells.
Example 17
High-Throughput Screening Assay Identifying Myeloid Activity
[0551] The following protocol is used to assess myeloid activity of
METH1 or METH2 by determining whether METH1 or METH2 proliferates
and/or differentiates myeloid cells. Myeloid cell activity is
assessed using the GAS/SEAP/Neo construct produced in Example 15.
Thus, factors that increase SEAP activity indicate the ability to
activate the Jaks-STATS signal transduction pathway. The myeloid
cell used in this assay is U937, a pre-monocyte cell line, although
TF-1, HL60, or KG1 can be used.
[0552] To transiently transfect U937 cells with the GAS/SEAP/Neo
construct produced in Example 15, a DEAE-Dextran method (Kharbanda
et. al., 1994, Cell Growth & Differentiation 5:259-265) is
used. First, harvest 2.times.10e.sup.7 U937 cells and wash with
PBS. The U937 cells are usually grown in RPMI 1640 medium
containing 10% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 mg/ml
streptomycin.
[0553] Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4)
buffer containing 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid
DNA, 140 mM NaCl, 5 mM KCl, 375 uM Na.sub.2HPO.sub.4.7H.sub.2O, 1
mM MgCl.sub.2, and 675 uM CaCl.sub.2. Incubate at 37.degree. C. for
45 min.
[0554] Wash the cells with RPMI 1640 medium containing 10% FBS and
then resuspend in 10 ml complete medium and incubate at 37.degree.
C. for 36 hr.
[0555] The GAS-SEAP/U937 stable cells are obtained by growing the
cells in 400 ug/ml G418. The G418-free medium is used for routine
growth but every one to two months, the cells should be re-grown in
400 ug/ml G418 for couple of passages.
[0556] These cells are tested by harvesting 1.times.10.sup.8 cells
(this is enough for ten 96-well plates assay) and wash with PBS.
Suspend the cells in 200 ml above described growth medium, with a
final density of 5.times.10.sup.5 cells/ml. Plate 200 ul cells per
well in the 96-well plate (or 1.times.10.sup.5 cells/well).
[0557] Add 50 .mu.l of the supernatant prepared by the protocol
described in Example 14. Incubate at 37.degree. C. for 48 to 72 hr.
As a positive control, 100 Unit/ml interferon gamma can be used
which is known to activate U937 cells. Over 30 fold induction is
typically observed in the positive control wells. SEAP assay the
supernatant according to the protocol described in Example 20.
Example 18
High-Throughput Screening Assay Identifying Neuronal Activity
[0558] When cells undergo differentiation and proliferation, a
group of genes are activated through many different signal
transduction pathways. One of these genes, EGR1 (early growth
response gene 1), is induced in various tissues and cell types upon
activation. The promoter of EGR1 is responsible for such induction.
Using the EGR1 promoter linked to reporter molecules, activation of
cells can be assessed by METH1 or METH2.
[0559] Particularly, the following protocol is used to assess
neuronal activity in PC12 cell lines. PC12 cells (rat
pheochromocytoma cells) are known to proliferate and/or
differentiate by activation with a number of mitogens, such as TPA
(tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF
(epidermal growth factor). The EGR1 gene expression is activated
during this treatment. Thus, by stably transfecting PC12 cells with
a construct containing an EGR promoter linked to SEAP reporter,
activation of PC12 cells by METH1 or METH2 can be assessed.
[0560] The EGR/SEAP reporter construct can be assembled by the
following protocol. The EGR-1 promoter sequence (-633 to +1)
(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified
from human genomic DNA using the following primers:
TABLE-US-00011 (SEQ ID NO:89) 5'
GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3' (SEQ ID NO:90) 5'
GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3'
[0561] Using the GAS:SEAP/Neo vector produced in Example 15, EGR1
amplified product can then be inserted into this vector. Linearize
the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII,
removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product
with these same enzymes. Ligate the vector and the EGR1
promoter.
[0562] To prepare 96 well-plates for cell culture, two mls of a
coating solution (1:30 dilution of collagen type I (Upstate Biotech
Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per
one 10 cm plate or 50 ml per well of the 96-well plate, and allowed
to air dry for 2 hr.
[0563] PC12 cells are routinely grown in RPMI-1640 medium (Bio
Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #
12449-78P), 5% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin and 100 .mu.g/ml
streptomycin on a precoated 10 cm tissue culture dish. One to four
split is done every three to four days. Cells are removed from the
plates by scraping and resuspended with pipetting up and down for
more than 15 times.
[0564] Transfect the EGR/SEAP/Neo construct into PC12 using the
Lipofectamine protocol described in Example 14. EGR-SEAP/PC12
stable cells are obtained by growing the cells in 300 ug/m G418.
The G418-free medium is used for routine growth but every one to
two months, the cells should be re-grown in 300 ug/ml G418 for
couple of passages.
[0565] To assay for neuronal activity, a 10 cm plate with cells
around 70 to 80% confluent is screened by removing the old medium.
Wash the cells once with PBS (Phosphate buffered saline). Then
starve the cells in low serum medium (RPMI-1640 containing 1% horse
serum and 0.5% FBS with antibiotics) overnight.
[0566] The next morning, remove the medium and wash the cells with
PBS. Scrape off the cells from the plate, suspend the cells well in
2 ml low serum medium. Count the cell number and add more low serum
medium to reach final cell density as 5.times.10.sup.5
cells/ml.
[0567] Add 200 .mu.l of the cell suspension to each well of 96-well
plate (equivalent to 1.times.10.sup.5 cells/well). Add 50 .mu.l
supernatant produced by Example 14, 37.degree. C. for 48 to 72 hr.
As a positive control, a growth factor known to activate PC12 cells
through EGR can be used, such as 50 ng/ul of Neuronal Growth Factor
(NGF). Over fifty-fold induction of SEAP is typically seen in the
positive control wells. SEAP assay the supernatant according to
Example 20.
Example 19
High-Throughput Screening Assay for T-Cell Activity
[0568] NF-KB (Nuclear Factor KB) is a transcription factor
activated by a wide variety of agents including the inflammatory
cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and
lymphotoxin-beta, by exposure to LPS or thrombin, and by expression
of certain viral gene products. As a transcription factor, NF-KB
regulates the expression of genes involved in immune cell
activation, control of apoptosis (NF-KB appears to shield cells
from apoptosis), B and T-cell development, anti-viral and
antimicrobial responses, and multiple stress responses.
[0569] In non-stimulated conditions, NF-KB is retained in the
cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB
is phosphorylated and degraded, causing NF-KB to shuttle to the
nucleus, thereby activating transcription of target genes. Target
genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and
class 1 MHC.
[0570] Due to its central role and ability to respond to a range of
stimuli, reporter constructs utilizing the NF-KB promoter element
are used to screen the supernatants produced in Example 14.
Activators or inhibitors of NF-KB would be useful in treating
diseases. For example, inhibitors of NF-KB could be used to treat
those diseases related to the acute or chronic activation of NF-KB,
such as rheumatoid arthritis.
[0571] To construct a vector containing the NF-KB promoter element,
a PCR based strategy is employed. The upstream primer contains four
tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID
NO:91), 18 bp of sequence complementary to the 5' end of the SV40
early promoter sequence, and is flanked with an XhoI site:
TABLE-US-00012 (SEQ ID NO:92)
5':GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGG
ACTTTCCATCCTGCCATCTCAATTAG:3'
[0572] The downstream primer is complementary to the 3' end of the
SV40 promoter and is flanked with a Hind III site:
TABLE-US-00013 5':GCGGCAAGCTTTTTGCAAAGCCTAGGC:3' (SEQ ID NO:93)
[0573] PCR amplification is performed using the SV40 promoter
template present in the pB-gal:promoter plasmid obtained from
Clontech. The resulting PCR fragment is digested with XhoI and Hind
III and subcloned into BLSK2-. (Stratagene) Sequencing with the T7
and T3 primers confirms the insert contains the following
sequence:
TABLE-US-00014 (SEQ ID NO:88)
5':CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTT
CCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCG
CCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGG
CTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTG
AGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGC AAAAAGCTT:3'
[0574] Next, replace the SV40 minimal promoter element present in
the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40
fragment using XhoI and HindIII. However, this vector does not
contain a neomycin resistance gene, and therefore, is not preferred
for mammalian expression systems.
[0575] In order to generate stable mammalian cell lines, the
NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP
vector using restriction enzymes SalI and NotI, and inserted into a
vector containing neomycin resistance. Particularly, the
NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),
replacing the GFP gene, after restricting pGFP-1 with SalI and
NotI.
[0576] Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat
T-cells are created and maintained according to the protocol
described in Example 16. Similarly, the method for assaying
supernatants with these stable Jurkat T-cells is also described in
Example 16. As a positive control, exogenous TNF alpha (0.1, 1, 10
ng) is added to wells H9, H10, and H11, with a 5-10 fold activation
typically observed.
Example 20
Assay for SEAP Activity
[0577] As a reporter molecule for the assays described in Examples
16-19, SEAP activity is assayed using the Tropix Phospho-light Kit
(Cat. BP-400) according to the following general procedure. The
Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction
Buffers used below.
[0578] Prime a dispenser with the 2.5.times. Dilution Buffer and
dispense 15 .mu.l of 2.5.times. dilution buffer into Optiplates
containing 35 .mu.l of a supernatant. Seal the plates with a
plastic sealer and incubate at 65.degree. C. for 30 min. Separate
the Optiplates to avoid uneven heating.
[0579] Cool the samples to room temperature for 15 minutes. Empty
the dispenser and prime with the Assay Buffer. Add 50 ml Assay
Buffer and incubate at room temperature 5 min. Empty the dispenser
and prime with the Reaction Buffer (see the table below). Add 50 ul
Reaction Buffer and incubate at room temperature for 20 minutes.
Since the intensity of the chemiluminescent signal is time
dependent, and it takes about 10 minutes to read 5 plates on
luminometer, one should treat 5 plates at each time and start the
second set 10 minutes later.
[0580] Read the relative light unit in the luminometer. Set H12 as
blank, and print the results. An increase in chemiluminescence
indicates reporter activity.
TABLE-US-00015 Reaction Buffer Formulation: # of plates Rxn buffer
diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14
80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110
5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140
7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170
8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200
10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44
230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255
12.75 50 260 13
Example 21
High-Throughput Screening Assay Identifying Changes in Small
Molecule Concentration and Membrane Permeability
[0581] Binding of a ligand to a receptor is known to alter
intracellular levels of small molecules, such as calcium,
potassium, sodium, and pH, as well as alter membrane potential.
These alterations can be measured in an assay to identify
supernatants which bind to receptors of a particular cell. Although
the following protocol describes an assay for calcium, this
protocol can easily be modified to detect changes in potassium,
sodium, pH, membrane potential, or any other small molecule which
is detectable by a fluorescent probe.
[0582] The following assay uses Fluorometric Imaging Plate Reader
("FLIPR") to measure changes in fluorescent molecules (Molecular
Probes) that bind small molecules. Clearly, any fluorescent
molecule detecting a small molecule can be used instead of the
calcium fluorescent molecule, fluo-3, used here.
[0583] For adherent cells, seed the cells at 10,000-20,000
cells/well in a Co-star black 96-well plate with clear bottom. The
plate is incubated in a CO.sub.2 incubator for 20 hours. The
adherent cells are washed two times in Biotek washer with 200 .mu.l
of HBSS (Hank's Balanced Salt Solution) leaving 100 ul of buffer
after the final wash.
[0584] A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic
acid DMSO. To load the cells with fluo-3, 50 .mu.l of 12 ug/ml
fluo-3 is added to each well. The plate is incubated at 37.degree.
C. in a CO.sub.2 incubator for 60 min. The plate is washed four
times in the Biotek washer with HBSS leaving 100 .mu.l of
buffer.
[0585] For non-adherent cells, the cells are spun down from culture
media. Cells are re-suspended to 2-5.times.10.sup.6 cells/ml with
HBSS in a 50-ml conical tube. 4 .mu.l of 1 mg/ml fluo-3 solution in
10% pluronic acid DMSO is added to each ml of cell suspension. The
tube is then placed in a 37.degree. C. water bath for 30-60 min.
The cells are washed twice with HBSS, resuspended to
1.times.10.sup.6 cells/ml, and dispensed into a microplate,
1001/well. The plate is centrifuged at 1000 rpm for 5 min. The
plate is then washed once in Denley CellWash with 200 ul, followed
by an aspiration step to 100 .mu.l final volume.
[0586] For a non-cell based assay, each well contains a fluorescent
molecule, such as fluo-3. The supernatant is added to the well, and
a change in fluorescence is detected.
[0587] To measure the fluorescence of intracellular calcium, the
FLIPR is set for the following parameters: (1) System gain is
300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is
F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 ul. Increased emission at 530 nm indicates an
extracellular signaling event caused by the a molecule, either
METH1 or METH2 or a molecule induced by METH1 or METH2, which has
resulted in an increase in the intracellular Ca.sup.++
concentration.
Example 22
High-Throughput Screening Assay Identifying Tyrosine Kinase
Activity
[0588] The Protein Tyrosine Kinases (PTK) represent a diverse group
of transmembrane and cytoplasmic kinases. Within the Receptor
Protein Tyrosine Kinase RPTK) group are receptors for a range of
mitogenic and metabolic growth factors including the PDGF, FGF,
EGF, NGF, HGF and Insulin receptor subfamilies. In addition there
are a large family of RPTKs for which the corresponding ligand is
unknown. Ligands for RPTKs include mainly secreted small proteins,
but also membrane-bound and extracellular matrix proteins.
[0589] Activation of RPTK by ligands involves ligand-mediated
receptor dimerization, resulting in transphosphorylation of the
receptor subunits and activation of the cytoplasmic tyrosine
kinases. The cytoplasmic tyrosine kinases include receptor
associated tyrosine kinases of the src-family (e.g., src, yes, lck,
lyn, fyn) and non-receptor linked and cytosolic protein tyrosine
kinases, such as the Jak family, members of which mediate signal
transduction triggered by the cytokine superfamily of receptors
(e.g., the Interleukins, Interferons, GM-CSF, and Leptin).
[0590] Because of the wide range of known factors capable of
stimulating tyrosine kinase activity, identifying whether METH1 or
METH2 or a molecule induced by METH1 or METH2 is capable of
activating tyrosine kinase signal transduction pathways is of
interest. Therefore, the following protocol is designed to identify
such molecules capable of activating the tyrosine kinase signal
transduction pathways.
[0591] Seed target cells (e.g., primary keratinocytes) at a density
of approximately 25,000 cells per well in a 96 well Loprodyne
Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.).
The plates are sterilized with two 30 minute rinses with 100%
ethanol, rinsed with water and dried overnight. Some plates are
coated for 2 hr with 100 ml of cell culture grade type I collagen
(50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can
be purchased from Sigma Chemicals (St. Louis, Mo.) or 10% Matrigel
purchased from Becton Dickinson (Bedford, Mass.), or calf serum,
rinsed with PBS and stored at 4.degree. C. Cell growth on these
plates is assayed by seeding 5,000 cells/well in growth medium and
indirect quantitation of cell number through use of alamarBlue as
described by the manufacturer Alamar Biosciences, Inc. (Sacramento,
Calif.) after 48 hr. Falcon plate covers #3071 from Becton
Dickinson (Bedford, Mass.) are used to cover the Loprodyne Silent
Screen Plates. Falcon Microtest III cell culture plates can also be
used in some proliferation experiments.
[0592] To prepare extracts, A431 cells are seeded onto the nylon
membranes of Loprodyne plates (20,000/200 ml/well) and cultured
overnight in complete medium. Cells are quiesced by incubation in
serum-free basal medium for 24 hr. After 5-20 minutes treatment
with EGF (60 ng/ml) or 50 ul of the supernatant produced in Example
14, the medium was removed and 100 ml of extraction buffer ((20 mM
HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4,
2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170)
obtained from Boehringer Mannheim (Indianapolis, Ind.) is added to
each well and the plate is shaken on a rotating shaker for 5
minutes at 4.degree. C. The plate is then placed in a vacuum
transfer manifold and the extract filtered through the 0.45 mm
membrane bottoms of each well using house vacuum. Extracts are
collected in a 96-well catch/assay plate in the bottom of the
vacuum manifold and immediately placed on ice. To obtain extracts
clarified by centrifugation, the content of each well, after
detergent solubilization for 5 minutes, is removed and centrifuged
for 15 minutes at 4.degree. C. at 16,000.times.g.
[0593] Test the filtered extracts for levels of tyrosine kinase
activity. Although many methods of detecting tyrosine kinase
activity are known, one method is described here.
[0594] Generally, the tyrosine kinase activity of a supernatant is
evaluated by determining its ability to phosphorylate a tyrosine
residue on a specific substrate (a biotinylated peptide).
Biotinylated peptides that can be used for this purpose include
PSK1 (corresponding to amino acids 6-20 of the cell division kinase
cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin).
Both peptides are substrates for a range of tyrosine kinases and
are available from Boehringer Mannheim.
[0595] The tyrosine kinase reaction is set up by adding the
following components in order. First, add 10 .mu.l of 5 .mu.M
Biotinylated Peptide, then 10 ul ATP/Mg.sup.2+ (5 mM ATP/50 mM
MgCl2), then 10 ul of 5.times. Assay Buffer (40 mM imidazole
hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100
mM MgCl.sub.2, 5 mM MnCl.sub.2, 0.5 mg/ml BSA), then 5 ul of Sodium
Vanadate (1 mM), and then 5 ul of water. Mix the components gently
and preincubate the reaction mix at 30.degree. C. for 2 min.
Initial the reaction by adding 10 .mu.l of the control enzyme or
the filtered supernatant.
[0596] The tyrosine kinase assay reaction is then terminated by
adding 10 .mu.l of 120 mm EDTA and place the reactions on ice.
[0597] Tyrosine kinase activity is determined by transferring 50
.mu.l aliquot of reaction mixture to a microtiter plate (MTP)
module and incubating at 37.degree. C. for 20 min. This allows the
streptavadin coated 96 well plate to associate with the
biotinylated peptide. Wash the MTP module with 300 .mu.l/well of
PBS four times. Next add 75 .mu.l of anti-phosphotyrosine antibody
conjugated to horse radish peroxidase (anti-P-Tyr-POD (0.5.mu./ml))
to each well and incubate at 37.degree. C. for one hour. Wash the
well as above.
[0598] Next add 100 .mu.l of peroxidase substrate solution
(Boehringer Mannheim) and incubate at room temperature for at least
5 mins (up to 30 min). Measure the absorbance of the sample at 405
nm by using ELISA reader. The level of bound peroxidase activity is
quantitated using an ELISA reader and reflects the level of
tyrosine kinase activity.
Example 23
High-Throughput Screening Assay Identifying Phosphorylation
Activity
[0599] As a potential alternative and/or compliment to the assay of
protein tyrosine kinase activity described in Example 22, an assay
which detects activation (phosphorylation) of major intracellular
signal transduction intermediates can also be used. For example, as
described below one particular assay can detect tyrosine
phosphorylation of the Erk-1 and Erk-2 kinases. However,
phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map
kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK,
Tec, and Janus, as well as any other phosphoserine,
phosphotyrosine, or phosphothreonine molecule, can be detected by
substituting these molecules for Erk-1 or Erk-2 in the following
assay.
[0600] Specifically, assay plates are made by coating the wells of
a 96-well ELISA plate with 0.1 ml of protein G (1 .mu.g/ml) for 2
hr at room temp, (RT). The plates are then rinsed with PBS and
blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are
then treated with 2 commercial monoclonal antibodies (100 ng/well)
against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology).
(To detect other molecules, this step can easily be modified by
substituting a monoclonal antibody detecting any of the above
described molecules.) After 3-5 rinses with PBS, the plates are
stored at 4.degree. C. until use.
[0601] A431 cells are seeded at 20,000/well in a 96-well Loprodyne
filterplate and cultured overnight in growth medium. The cells are
then starved for 48 hr in basal medium (DMEM) and then treated with
EGF (6 ng/well) or 50 .mu.l of the supernatants obtained in Example
14 for 5-20 minutes. The cells are then solubilized and extracts
filtered directly into the assay plate.
[0602] After incubation with the extract for 1 hr at RT, the wells
are again rinsed. As a positive control, a commercial preparation
of MAP kinase (10 ng/well) is used in place of A431 extract. Plates
are then treated with a commercial polyclonal (rabbit) antibody (1
.mu.g/ml) which specifically recognizes the phosphorylated epitope
of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is
biotinylated by standard procedures. The bound polyclonal antibody
is then quantitated by successive incubations with
Europium-streptavidin and Europium fluorescence enhancing reagent
in the Wallac DELFIA instrument (time-resolved fluorescence). An
increased fluorescent signal over background indicates a
phosphorylation by METH1 or METH2 or a molecule induced by METH1 or
METH2.
Example 24
Method of Determining Alterations in the METH1 or METH2 Gene
[0603] RNA isolated from entire families or individual patients
presenting with a phenotype of interest (such as a disease) is be
isolated. cDNA is then generated from these RNA samples using
protocols known in the art. (See, Sambrook.) The cDNA is then used
as a template for PCR, employing primers surrounding regions of
interest in SEQ ID NO:1. Suggested PCR conditions consist of 35
cycles at 95.degree. C. for 30 seconds; 60-120 seconds at
52-58.degree. C.; and 60-120 seconds at 70.degree. C., using buffer
solutions described in Sidransky, D. et al., Science 252:706
(1991).
[0604] PCR products are then sequenced using primers labeled at
their 5' end with T4 polynucleotide kinase, employing SequiTherm
Polymerase. (Epicentre Technologies). The intron-exon borders of
selected exons of METH1 or METH2 is also determined and genomic PCR
products analyzed to confirm the results. PCR products harboring
suspected mutations in METH1 or METH2 is then cloned and sequenced
to validate the results of the direct sequencing.
[0605] PCR products of METH1 or METH2 are cloned into T-tailed
vectors as described in Holton, T. A. and Graham, M. W., Nucleic
Acids Research 19:1156 (1991) and sequenced with T7 polymerase
(United States Biochemical). Affected individuals are identified by
mutations in METH1 or METH2 not present in unaffected
individuals.
[0606] Genomic rearrangements are also observed as a method of
determining alterations in the METH1 or METH2 gene. Isolated
genomic clones are nick-translated with digoxigenindeoxy-uridine
5'-triphosphate (Boehringer Manheim), and FISH performed as
described in Johnson, Cg. et al., Methods Cell Biol. 35:73-99
(1991). Hybridization with the labeled probe is carried out using a
vast excess of human cot-1 DNA for specific hybridization to the
METH1 or METH2 genomic locus.
[0607] Chromosomes are counterstained with
4,6-diamino-2-phenylindole and propidium iodide, producing a
combination of C- and R-bands. Aligned images for precise mapping
are obtained using a triple-band filter set (Chroma Technology,
Brattleboro, Vt.) in combination with a cooled charge-coupled
device camera (Photometrics, Tucson, Ariz.) and variable excitation
wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech. Appl.
8:75 (1991).) Image collection, analysis and chromosomal fractional
length measurements are performed using the ISee Graphical Program
System. (Inovision Corporation, Durham, N.C.) Chromosome
alterations of the genomic region of METH1 or METH2 (hybridized by
the probe) are identified as insertions, deletions, and
translocations. These METH1 or METH2 alterations are used as a
diagnostic marker for an associated disease.
Example 25
Method of Detecting Abnormal Levels of METH1 or METH2 in a
Biological Sample
[0608] METH1 or METH2 polypeptides can be detected in a biological
sample, and if an increased or decreased level of METH1 or METH2 is
detected, this polypeptide is a marker for a particular phenotype.
Methods of detection are numerous, and thus, it is understood that
one skilled in the art can modify the following assay to fit their
particular needs.
[0609] For example, antibody-sandwich ELISAs are used to detect
METH1 or METH2 in a sample, preferably a biological sample. Wells
of a microtiter plate are coated with specific antibodies to METH1
or METH2, at a final concentration of 0.2 to 10 .mu.g/ml. The
antibodies are either monoclonal or polyclonal and are produced by
the method described in Example 13. The wells are blocked so that
non-specific binding of METH1 or METH2 to the well is reduced.
[0610] The coated wells are then incubated for >2 hours at RT
with a sample containing METH1 or METH2. Preferably, serial
dilutions of the sample should be used to validate results. The
plates are then washed three times with deionized or distilled
water to remove unbounded METH1 or METH2.
[0611] Next, 50 .mu.l of specific antibody-alkaline phosphatase
conjugate, at a concentration of 25-400 ng, is added and incubated
for 2 hours at room temperature. The plates are again washed three
times with deionized or distilled water to remove unbounded
conjugate.
[0612] Add 75 .mu.l of 4-methylumbelliferyl phosphate (MUP) or
p-nitrophenyl phosphate (NPP) substrate solution to each well and
incubate 1 hour at room temperature. Measure the reaction by a
microtiter plate reader. Prepare a standard curve, using serial
dilutions of a control sample, and plot METH1 or METH2 polypeptide
concentration on the X-axis (log scale) and fluorescence or
absorbance of the Y-axis (linear scale). Interpolate the
concentration of the METH1 or METH2 in the sample using the
standard curve.
Example 26
Formulating a Polypeptide
[0613] The METH1 or METH2 composition will be formulated and dosed
in a fashion consistent with good medical practice, taking into
account the clinical condition of the individual patient
(especially the side effects of treatment with the METH1 or METH2
polypeptide alone), the site of delivery, the method of
administration, the scheduling of administration, and other factors
known to practitioners. The "effective amount" for purposes herein
is thus determined by such considerations.
[0614] As a general proposition, the total pharmaceutically
effective amount of METH1 or METH2 administered parenterally per
dose will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day
of patient body weight, although, as noted above, this will be
subject to therapeutic discretion. More preferably, this dose is at
least 0.01 mg/kg/day, and most preferably for humans between about
0.01 and 1 mg/kg/day for the hormone. If given continuously, METH1
or METH2 is typically administered at a dose rate of about 1
.mu.g/kg/hour to about 50 .mu.g/kg/hour, either by 1-4 injections
per day or by continuous subcutaneous infusions, for example, using
a mini-pump. An intravenous bag solution may also be employed. The
length of treatment needed to observe changes and the interval
following treatment for responses to occur appears to vary
depending on the desired effect.
[0615] Pharmaceutical compositions containing METH1 or METH2 are
administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, gels, drops or transdermal patch), bucally, or as an
oral or nasal spray. "Pharmaceutically acceptable carrier" refers
to a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0616] METH1 or METH2 is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or microcapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al.) or poly-D-(-)-3-hydroxybutyric
acid (EP 133,988). Sustained-release compositions also include
liposomally entrapped METH1 or METH2 polypeptides. Liposomes
containing the METH1 or METH2 are prepared by methods known per se:
DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA
82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA
77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949;
EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal secreted
polypeptide therapy.
[0617] For parenteral administration, in one embodiment, METH1 or
METH2 is formulated generally by mixing it at the desired degree of
purity, in a unit dosage injectable form (solution, suspension, or
emulsion), with a pharmaceutically acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. For example, the formulation preferably does not
include oxidizing agents and other compounds that are known to be
deleterious to polypeptides.
[0618] Generally, the formulations are prepared by contacting METH1
or METH2 uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[0619] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, manose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0620] METH1 or METH2 is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of polypeptide salts.
[0621] METH1 or METH2 used for therapeutic administration can be
sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic polypeptide compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0622] METH1 or METH2 polypeptides ordinarily will be stored in
unit or multi-dose containers, for example, sealed ampoules or
vials, as an aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
METH1 or METH2 polypeptide solution, and the resulting mixture is
lyophilized. The infusion solution is prepared by reconstituting
the lyophilized METH1 or METH2 polypeptide using bacteriostatic
Water-for-Injection.
[0623] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, METH1 or METH2 may be employed
in conjunction with other therapeutic compounds.
[0624] The compositions of the invention may be administered alone
or in combination with other therapeutic agents. Therapeutic agents
that may be administered in combination with the compositions of
the invention include, but are not limited to, other members of the
TNF family, chemotherapeutic agents, antibiotic, steroidal and
non-steroidal anti-inflammatories, conventional immunotherapeutic
agents, cytokines and/or growth factors. Combination may be
administered either concomitantly, e.g. as an admixture; separately
but simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered
together as a therapeutic mixture, and also procedures in which the
combined agents are administered separately but simultaneously,
e.g. as through separate intravenous lines into the same
individual. Administration "in combination" further includes the
separate administration of one of the compounds or agents given
first, followed by the second.
[0625] In one embodiment, the compositions of the invention are
administered in combination with other members of the TNF family.
TNF, TNF-related or TNF-like molecules that may be administered
with the compositions of the invention include, but are not limited
to, soluble forms of TNF-alpha, lymphotoxin alpha (LT-alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer
LT-alph2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3,
OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I
(International Publication No. WO 97/33899), endokine-alpha
(International Publication No. WO 98/07880), TR6 (International
Publication No. WO 98/30694), OPG, and neutrokine-alpha
(International Publication No. WO 98/18921), OX40, and nerve growth
factor (NGF) and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB,
TR2 (International Publication No. WO 98/34095), DR3 (International
Publication No. WO 97/33904), DR4 (International Publication No. WO
98/32856), TR5 (International Publication No. WO 98/30693), TR6
(International Publication No. WO 98/30694), TR7 (International
Publication No. WO 98/41629), TRANK, TR9 (International Publication
No. WO 98/56892), TR10 (International Publication No. WO 98/54202),
312C2 (International Publication No. WO 98/06842), and TR12, and
soluble forms of CD154, CD70 and CD153.
[0626] Conventional nonspecific immunosuppressive agents that may
be administered in combination with the compositions of the
invention include, but are not limited to, steroids, cyclosporine,
cyclosporine analogs, ayclophosphamide methylprednisone,
prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other
immunosuppressive agents that act by suppressing the function of
responding T cells.
[0627] In a further embodiment, the compositions of the invention
are administered in combination with an antibiotic agent.
Antibiotic agents that may be administered with the compositions of
the invention include, but are not limited to, tetracycline,
metronidazole, amoxicillin, beta-lactamases, aminoglycosides,
macrolides, quinolones, fluoroquinolones, cephalosporins,
erythromycin, ciprofloxacin, and streptomycin.
[0628] In an additional embodiment, the compositions of the
invention are administered alone or in combination with an
anti-inflammatory agent. Anti-inflammatory agents that may be
administered with the compositions of the invention include, but
are not limited to, glucocorticoids and the nonsteroidal
anti-inflammatories, aminoarylcarboxylic acid derivatives,
arylacetic acid derivatives, arylbutyric acid derivatives,
arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,
pyrazolones, salicylic acid derivatives, thiazinecarboxamides,
eacetamidocaproic acid, S-adenosylmethionine,
3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine,
bucolome, difenpiramide, ditazol, emorfazone, guaiazulene,
nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
[0629] In another embodiment, compositions of the invention are
administered in combination with a chemotherapeutic agent.
Chemotherapeutic agents that may be administered with the
compositions of the invention include, but are not limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin,
and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites
(e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon
alpha-2b, glutamic acid, plicamycin, mercaptopurine, and
6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU,
lomustine, CCNU, cytosine arabinoside, cyclophosphamide,
estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,
cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone, estramustine phosphate sodium, ethenyl
estradiol, estradiol, megestrol acetate, methyltestosterone,
diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone); nitrogen mustard derivatives (e.g., mephalen,
chorambucil, mechlorethamine (nitrogen mustard) and thiotepa);
steroids and combinations (e.g., bethamethasone sodium phosphate);
and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate, vinblastine sulfate, and etoposide).
[0630] In an additional embodiment, the compositions of the
invention are administered in combination with cytokines. Cytokines
that may be administered with the compositions of the invention
include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7,
IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and
TNF-alpha.
[0631] In an additional embodiment, the compositions of the
invention are administered in combination with angiogenic proteins.
Angiogenic proteins that may be administered with the compositions
of the invention include, but are not limited to, Glioma Derived
Growth Factor (GDGF), as disclosed in European Patent Number
EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed
in European Patent Number EP-682110; Platelet Derived Growth
Factor-B (PDGF-B), as disclosed in European Patent Number
EP-282317; Placental Growth Factor (PlGF), as disclosed in
International Publication Number WO 92/06194; Placental Growth
Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors,
4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as
disclosed in International Publication Number WO 90/13649; Vascular
Endothelial Growth Factor-A (VEGF-A), as disclosed in European
Patent Number EP-506477; Vascular Endothelial Growth Factor-2
(VEGF-2), as disclosed in International Publication Number WO
96/39515; Vascular Endothelial Growth Factor B-186 (VEGF-B 186), as
disclosed in International Publication Number WO 96/26736; Vascular
Endothelial Growth Factor-D (VEGF-D), as disclosed in International
Publication Number WO 98/02543; Vascular Endothelial Growth
Factor-D (VEGF-D), as disclosed in International Publication Number
WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as
disclosed in German Patent Number DE19639601. The above mentioned
references are incorporated herein by reference herein.
[0632] In an additional embodiment, the compositions of the
invention are administered in combination with Fibroblast Growth
Factors. Fibroblast Growth Factors that may be administered with
the compositions of the invention include, but are not limited to,
FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
[0633] In additional embodiments, the compositions of the invention
are administered in combination with other therapeutic or
prophylactic regimens, such as, for example, radiation therapy.
Example 27
Method of Treating Decreased Levels of METH1 or METH2
[0634] The present invention relates to a method for treating an
individual in need of a decreased level of METH1 or METH2 activity
in the body comprising, administering to such an individual a
composition comprising a therapeutically effective amount of METH1
or METH2 antagonist. Preferred antagonists for use in the present
invention are METH1 or METH2-specific antibodies.
[0635] Moreover, it will be appreciated that conditions caused by a
decrease in the standard or normal expression level of METH1 or
METH2 in an individual can be treated by administering METH1 or
METH2, preferably in the secreted form. Thus, the invention also
provides a method of treatment of an individual in need of an
increased level of METH1 or METH2 polypeptide comprising
administering to such an individual a pharmaceutical composition
comprising an amount of METH1 or METH2 to increase the activity
level of METH1 or METH2 in such an individual.
[0636] For example, a patient with decreased levels of METH1 or
METH2 polypeptide receives a daily dose 0.1-100 ug/kg of the
polypeptide for six consecutive days. Preferably, the polypeptide
is in the secreted form. The exact details of the dosing scheme,
based on administration and formulation, are provided in Example
26.
Example 28
Method of Treating Increased Levels of METH1 or METH2
[0637] The present invention also relates to a method for treating
an individual in need of an increased level of METH1 or METH2
activity in the body comprising administering to such an individual
a composition comprising a therapeutically effective amount of
METH1 or METH2 or an agonist thereof.
[0638] Antisense technology is used to inhibit production of METH1
or METH2. This technology is one example of a method of decreasing
levels of METH1 or METH2 polypeptide, preferably a secreted form,
due to a variety of etiologies, such as cancer.
[0639] For example, a patient diagnosed with abnormally increased
levels of METH1 or METH2 is administered intravenously antisense
polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21
days. This treatment is repeated after a 7-day rest period if the
treatment was well tolerated. The formulation of the antisense
polynucleotide is provided in Example 26.
Example 29
Method of Treatment Using Gene Therapy
Ex Vivo
[0640] One method of gene therapy transplants fibroblasts, which
are capable of expressing METH1 or METH2 polypeptides, onto a
patient. Generally, fibroblasts are obtained from a subject by skin
biopsy. The resulting tissue is placed in tissue-culture medium and
separated into small pieces. Small chunks of the tissue are placed
on a wet surface of a tissue culture flask, approximately ten
pieces are placed in each flask. The flask is turned upside down,
closed tight and left at room temperature over night. After 24
hours at room temperature, the flask is inverted and the chunks of
tissue remain fixed to the bottom of the flask and fresh media
(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin)
is added. The flasks are then incubated at 37.degree. C. for
approximately one week.
[0641] At this time, fresh media is added and subsequently changed
every several days. After an additional two weeks in culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and
scaled into larger flasks.
[0642] pMV-7 (Kirschmeier, P. T. et al., DNA 7:219-25 (1988)),
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0643] The cDNA encoding METH1 or METH2 can be amplified using PCR
primers which correspond to the 5' and 3' end sequences
respectively as set forth in Example 5. Preferably, the 5' primer
contains an EcoRI site and the 3' primer includes a HindIII site.
Equal quantities of the Moloney murine sarcoma virus linear
backbone and the amplified EcoRI and HindIII fragment are added
together, in the presence of T4 DNA ligase. The resulting mixture
is maintained under conditions appropriate for ligation of the two
fragments. The ligation mixture is then used to transform bacteria
HB101, which are then plated onto agar containing kanamycin for the
purpose of confirming that the vector contains properly inserted
METH1 or METH2.
[0644] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the METH1 or METH2 gene is
then added to the media and the packaging cells transduced with the
vector. The packaging cells now produce infectious viral particles
containing the METH1 or METH2 gene (the packaging cells are now
referred to as producer cells).
[0645] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his. Once the
fibroblasts have been efficiently infected, the fibroblasts are
analyzed to determine whether METH1 or METH2 protein is
produced.
[0646] The engineered fibroblasts are then transplanted onto the
host, either alone or after having been grown to confluence on
cytodex 3 microcarrier beads.
Example 30
Method of Treatment Using Gene Therapy
In Vivo
[0647] Another aspect of the present invention is using in vivo
gene therapy methods to treat disorders, diseases and conditions.
The gene therapy method relates to the introduction of naked
nucleic acid (DNA, RNA, and antisense DNA or RNA) METH1 or METH2
sequences into an animal to increase or decrease the expression of
the METH1 or METH2 polypeptide. The METH1 or METH2 polynucleotide
may be operatively linked to a promoter or any other genetic
elements necessary for the expression of the METH1 or METH2
polypeptide by the target tissue. Such gene therapy and delivery
techniques and methods are known in the art, see, for example, WO
90/11092, WO98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151,
5,580,859; Tabata, H. et al. (1997) Cardiovasc. Res. 35(3):470-479,
Chao, J. et al. (1997) Pharmacol Res. 35(6):517-522, Wolff, J. A.
(1997) Neuromuscul. Disord. 7(5):314-318, Schwartz, B. et al.
(1996) Gene Ther. 3(5):405-411, Tsurumi, Y. et al. (1996)
Circulation 94(12):3281-3290 (incorporated herein by
reference).
[0648] The METH1 or METH2 polynucleotide constructs may be
delivered by any method that delivers injectable materials to the
cells of an animal, such as, injection into the interstitial space
of tissues (heart, muscle, skin, lung, liver, intestine and the
like). The METH1 or METH2 polynucleotide constructs can be
delivered in a pharmaceutically acceptable liquid or aqueous
carrier.
[0649] The term "naked" polynucleotide, DNA or RNA, refers to
sequences that are free from any delivery vehicle that acts to
assist, promote, or facilitate entry into the cell, including viral
sequences, viral particles, liposome formulations, lipofectin or
precipitating agents and the like. However, the METH1 or METH2
polynucleotides may also be delivered in liposome formulations
(such as those taught in Felgner, P. L. et al. (1995) Ann. NY Acad.
Sci. 772:126-139 and Abdallah, B. et al. (1995) Biol. Cell
85(1):1-7) which can be prepared by methods well known to those
skilled in the art.
[0650] The METH1 or METH2 polynucleotide vector constructs used in
the gene therapy method are preferably constructs that will not
integrate into the host genome nor will they contain sequences that
allow for replication. Any strong promoter known to those skilled
in the art can be used for driving the expression of DNA. Unlike
other gene therapies techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory
nature of the polynucleotide synthesis in the cells. Studies have
shown that non-replicating DNA sequences can be introduced into
cells to provide production of the desired polypeptide for periods
of up to six months.
[0651] The METH1 or METH2 polynucleotide construct can be delivered
to the interstitial space of tissues within the an animal,
including of muscle, skin, brain, lung, liver, spleen, bone marrow,
thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney,
gall bladder, stomach, intestine, testis, ovary, uterus, rectum,
nervous system, eye, gland, and connective tissue. Interstitial
space of the tissues comprises the intercellular fluid,
mucopolysaccharide matrix among the reticular fibers of organ
tissues, elastic fibers in the walls of vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within
connective tissue ensheathing muscle cells or in the lacunae of
bone. It is similarly the space occupied by the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery
to the interstitial space of muscle tissue is preferred for the
reasons discussed below. They may be conveniently delivered by
injection into the tissues comprising these cells. They are
preferably delivered to and expressed in persistent, non-dividing
cells which are differentiated, although delivery and expression
may be achieved in non-differentiated or less completely
differentiated cells, such as, for example, stem cells of blood or
skin fibroblasts. In vivo muscle cells are particularly competent
in their ability to take up and express polynucleotides.
[0652] For the naked METH1 or METH2 polynucleotide injection, an
effective dosage amount of DNA or RNA will be in the range of from
about 0.0005 g/kg body weight to about 50 mg/kg body weight.
Preferably the dosage will be from about 0.005 mg/kg to about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg.
Of course, as the artisan of ordinary skill will appreciate, this
dosage will vary according to the tissue site of injection. The
appropriate and effective dosage of nucleic acid sequence can
readily be determined by those of ordinary skill in the art and may
depend on the condition being treated and the route of
administration. The preferred route of administration is by the
parenteral route of injection into the interstitial space of
tissues. However, other parenteral routes may also be used, such
as, inhalation of an aerosol formulation particularly for delivery
to lungs or bronchial tissues, throat or mucous membranes of the
nose. In addition, naked METH1 or METH2 polynucleotide constructs
can be delivered to arteries during angioplasty by the catheter
used in the procedure.
[0653] The dose response effects of injected METH1 or METH2
polynucleotide in muscle in vivo is determined as follows. Suitable
METH1 or METH2 template DNA for production of mRNA coding for METH1
or METH2 polypeptide is prepared in accordance with a standard
recombinant DNA methodology. The template DNA, which may be either
circular or linear, is either used as naked DNA or complexed with
liposomes. The quadriceps muscles of mice are then injected with
various amounts of the template DNA.
[0654] Five to six week old female and male Balb/C mice are
anesthetized by intraperitoneal injection with 0.3 ml of 2.5%
Avertin. A 1.5 cm incision is made on the anterior thigh, and the
quadriceps muscle is directly visualized. The METH1 or METH2
template DNA is injected in 0.1 ml of carrier in a 1 cc syringe
through a 27 gauge needle over one minute, approximately 0.5 cm
from the distal insertion site of the muscle into the knee and
about 0.2 cm deep. A suture is placed over the injection site for
future localization, and the skin is closed with stainless steel
clips.
[0655] After an appropriate incubation time (e.g., 7 days) muscle
extracts are prepared by excising the entire quadriceps. Every
fifth 15 um cross-section of the individual quadriceps muscles is
histochemically stained for METH1 or METH2 protein expression. A
time course for METH1 or METH2 protein expression may be done in a
similar fashion except that quadriceps from different mice are
harvested at different times. Persistence of METH1 or METH2 DNA in
muscle following injection may be determined by Southern blot
analysis after preparing total cellular DNA and HIRT supernatants
from injected and control mice. The results of the above
experimentation in mice can be use to extrapolate proper dosages
and other treatment parameters in humans and other animals using
METH1 or METH2 naked DNA.
Example 31
Gene Therapy Using Endogenous METH1 and/or METH2 Gene
[0656] Another method of gene therapy according to the present
invention involves operably associating the endogenous METH1 and/or
METH2 sequence with a promoter via homologous recombination as
described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24,
1997; International Publication No. WO 96/29411, published Sep. 26,
1996; International Publication No. WO 94/12650, published Aug. 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989). This method
involves the activation of a gene which is present in the target
cells, but which is not expressed in the cells, or is expressed at
a lower level than desired.
[0657] Polynucleotide constructs are made which contain a promoter
and targeting sequences, which are homologous to the 5' non-coding
sequence of endogenous METH1 and/or METH2, flanking the promoter.
The targeting sequence will be sufficiently near the 5' end of
METH1 and/or METH2 so the promoter will be operably linked to the
endogenous sequence upon homologous recombination. The promoter and
the targeting sequences can be amplified using PCR. Preferably, the
amplified promoter contains distinct restriction enzyme sites on
the 5' and 3' ends. Preferably, the 3' end of the first targeting
sequence contains the same restriction enzyme site as the 5' end of
the amplified promoter and the 5' end of the second targeting
sequence contains the same restriction site as the 3' end of the
amplified promoter.
[0658] The amplified promoter and the amplified targeting sequences
are digested with the appropriate restriction enzymes and
subsequently treated with calf intestinal phosphatase. The digested
promoter and digested targeting sequences are added together in the
presence of T4 DNA ligase. The resulting mixture is maintained
under conditions appropriate for ligation of the two fragments. The
construct is size fractionated on an agarose gel then purified by
phenol extraction and ethanol precipitation.
[0659] In this Example, the polynucleotide constructs are
administered as naked polynucleotides via electroporation. However,
the polynucleotide constructs may also be administered with
transfection-facilitating agents, such as liposomes, viral
sequences, viral particles, precipitating agents, etc. Such methods
of delivery are known in the art.
[0660] Once the cells are transfected, homologous recombination
will take place which results in the promoter being operably linked
to the endogenous METH1 and/or METH2 sequence. This results in the
expression of METH1 and/or METH2 in the cell. Expression may be
detected by immunological staining, or any other method known in
the art.
[0661] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in DMEM+10% fetal calf serum.
Exponentially growing or early stationary phase fibroblasts are
trypsinized and rinsed from the plastic surface with nutrient
medium. An aliquot of the cell suspension is removed for counting,
and the remaining cells are subjected to centrifugation. The
supernatant is aspirated and the pellet is resuspended in 5 ml of
electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl,
0.7 mM Na.sub.2 HPO.sub.4, 6 mM dextrose). The cells are
recentrifuged, the supernatant aspirated, and the cells resuspended
in electroporation buffer containing 1 mg/ml acetylated bovine
serum albumin. The final cell suspension contains approximately
3.times.10.sup.6 cells/ml. Electroporation should be performed
immediately following resuspension.
[0662] Plasmid DNA is prepared according to standard techniques.
For example, to construct a plasmid for targeting to the METH1
and/or METH2 locus, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is
digested with HindIII. The CMV promoter is amplified by PCR with an
XbaI site on the 5' end and a BamHI site on the 3' end. Two METH1
and/or METH2 non-coding sequences are amplified via PCR: one METH1
and/or METH2 non-coding sequence (METH1 and/or METH2 fragment 1) is
amplified with a HindIII site at the 5' end and an Xba site at the
3' end; the other METH1 and/or METH2 non-coding sequence (METH1
and/or METH2 fragment 2) is amplified with a BamHI site at the 5'
end and a HindIII site at the 3' end. The CMV promoter and METH1
and/or METH2 fragments are digested with the appropriate enzymes
(CMV promoter--XbaI and BamHI; METH1 and/or METH2 fragment 1--XbaI;
METH1 and/or METH2 fragment 2--BamHI) and ligated together. The
resulting ligation product is digested with HindIII, and ligated
with the HindIII-digested pUC18 plasmid.
[0663] Plasmid DNA is added to a sterile cuvette with a 0.4 cm
electrode gap (Bio-Rad). The final DNA concentration is generally
at least 120 .mu.g/ml. 0.5 ml of the cell suspension (containing
approximately 1.5.times.10.sup.6 cells) is then added to the
cuvette, and the cell suspension and DNA solutions are gently
mixed. Electroporation is performed with a Gene-Pulser apparatus
(Bio-Rad). Capacitance and voltage are set at 960 .mu.F and 250-300
V, respectively. As voltage increases, cell survival decreases, but
the percentage of surviving cells that stably incorporate the
introduced DNA into their genome increases dramatically. Given
these parameters, a pulse time of approximately 14-20 mSec should
be observed.
[0664] Electroporated cells are maintained at room temperature for
approximately 5 min, and the contents of the cuvette are then
gently removed with a sterile transfer pipette. The cells are added
directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf
serum) in a 10 cm dish and incubated at 37.degree. C. The following
day, the media is aspirated and replaced with 10 ml of fresh media
and incubated for a further 16-24 hours.
[0665] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product. The fibroblasts can then be introduced into a patient as
described above.
Example 32
METH1 and/or METH2 Transgenic Animals
[0666] The METH1 and/or METH2 polypeptides can also be expressed in
transgenic animals. Animals of any species, including, but not
limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs,
micro-pigs, goats, sheep, cows and non-human primates, e.g.,
baboons, monkeys, and chimpanzees may be used to generate
transgenic animals. In a specific embodiment, techniques described
herein or otherwise known in the art, are used to express
polypeptides of the invention in humans, as part of a gene therapy
protocol.
[0667] Any technique known in the art may be used to introduce the
transgene (i.e., polynucleotides of the invention) into animals to
produce the founder lines of transgenic animals. Such techniques
include, but are not limited to, pronuclear microinjection
(Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et
al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S.
Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into
germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA
82:6148-6152 (1985)), blastocysts or embryos; gene targeting in
embryonic stem cells (Thompson et al., Cell 56:313-321 (1989));
electroporation of cells or embryos (Lo, 1983, Mol. Cell. Biol.
3:1803-1814 (1983)); introduction of the polynucleotides of the
invention using a gene gun (see, e.g., Ulmer et al., Science
259:1745 (1993); introducing nucleic acid constructs into embryonic
pleuripotent stem cells and transferring the stem cells back into
the blastocyst; and sperm-mediated gene transfer (Lavitrano et al.,
Cell 57:717-723 (1989); etc. For a review of such techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989),
which is incorporated by reference herein in its entirety.
[0668] Any technique known in the art may be used to produce
transgenic clones containing polynucleotides of the invention, for
example, nuclear transfer into enucleated oocytes of nuclei from
cultured embryonic, fetal, or adult cells induced to quiescence
(Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-813 (1997)).
[0669] The present invention provides for transgenic animals that
carry the transgene in all their cells, as well as animals which
carry the transgene in some, but not all their cells, i.e., mosaic
animals or chimeric. The transgene may be integrated as a single
transgene or as multiple copies such as in concatamers, e.g.,
head-to-head tandems or head-to-tail tandems. The transgene may
also be selectively introduced into and activated in a particular
cell type by following, for example, the teaching of Lasko et al.
(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The
regulatory sequences required for such a cell-type specific
activation will depend upon the particular cell type of interest,
and will be apparent to those of skill in the art. When it is
desired that the polynucleotide transgene be integrated into the
chromosomal site of the endogenous gene, gene targeting is
preferred.
[0670] Briefly, when such a technique is to be utilized, vectors
containing some nucleotide sequences homologous to the endogenous
gene are designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and disrupting the
function of the nucleotide sequence of the endogenous gene. The
transgene may also be selectively introduced into a particular cell
type, thus inactivating the endogenous gene in only that cell type,
by following, for example, the teaching of Gu et al. (Gu et al.,
Science 265:103-106 (1994)). The regulatory sequences required for
such a cell-type specific inactivation will depend upon the
particular cell type of interest, and will be apparent to those of
skill in the art. The contents of each of the documents recited in
this paragraph is herein incorporated by reference in its
entirety.
[0671] Once transgenic animals have been generated, the expression
of the recombinant gene may be assayed utilizing standard
techniques. Initial screening may be accomplished by Southern blot
analysis or PCR techniques to analyze animal tissues to verify that
integration of the transgene has taken place. The level of mRNA
expression of the transgene in the tissues of the transgenic
animals may also be assessed using techniques which include, but
are not limited to, Northern blot analysis of tissue samples
obtained from the animal, in situ hybridization analysis, and
reverse transcriptase-PCR (rt-PCR). Samples of transgenic
gene-expressing tissue may also be evaluated immunocytochemically
or immunohistochemically using antibodies specific for the
transgene product.
[0672] Once the founder animals are produced, they may be bred,
inbred, outbred, or crossbred to produce colonies of the particular
animal. Examples of such breeding strategies include, but are not
limited to: outbreeding of founder animals with more than one
integration site in order to establish separate lines; inbreeding
of separate lines in order to produce compound transgenics that
express the transgene at higher levels because of the effects of
additive expression of each transgene; crossing of heterozygous
transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate
the need for screening of animals by DNA analysis; crossing of
separate homozygous lines to produce compound heterozygous or
homozygous lines; and breeding to place the transgene on a distinct
background that is appropriate for an experimental model of
interest.
[0673] Transgenic animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of METH1 and/or METH2 polypeptides,
studying conditions and/or disorders associated with aberrant METH1
and/or METH2 expression, and in screening for compounds effective
in ameliorating such conditions and/or disorders.
Example 33
METH1 and/or METH2 Knock-Out Animals
[0674] Endogenous METH1 and/or METH2 gene expression can also be
reduced by inactivating or "knocking out" the METH1 and/or METH2
gene and/or its promoter using targeted homologous recombination.
(E.g., see Smithies et al., Nature 317:230-234 (1985); Thomas &
Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321
(1989); each of which is incorporated by reference herein in its
entirety). For example, a mutant, non-functional polynucleotide of
the invention (or a completely unrelated DNA sequence) flanked by
DNA homologous to the endogenous polynucleotide sequence (either
the coding regions or regulatory regions of the gene) can be used,
with or without a selectable marker and/or a negative selectable
marker, to transfect cells that express polypeptides of the
invention in vivo. In another embodiment, techniques known in the
art are used to generate knockouts in cells that contain, but do
not express the gene of interest. Insertion of the DNA construct,
via targeted homologous recombination, results in inactivation of
the targeted gene. Such approaches are particularly suited in
research and agricultural fields where modifications to embryonic
stem cells can be used to generate animal offspring with an
inactive targeted gene (e.g., see Thomas & Capecchi 1987 and
Thompson 1989, supra). However this approach can be routinely
adapted for use in humans provided the recombinant DNA constructs
are directly administered or targeted to the required site in vivo
using appropriate viral vectors that will be apparent to those of
skill in the art.
[0675] In further embodiments of the invention, cells that are
genetically engineered to express the polypeptides of the
invention, or alternatively, that are genetically engineered not to
express the polypeptides of the invention (e.g., knockouts) are
administered to a patient in vivo. Such cells may be obtained from
the patient (i.e., animal, including human) or an MHC compatible
donor and can include, but are not limited to fibroblasts, bone
marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle
cells, endothelial cells etc. The cells are genetically engineered
in vitro using recombinant DNA techniques to introduce the coding
sequence of polypeptides of the invention into the cells, or
alternatively, to disrupt the coding sequence and/or endogenous
regulatory sequence associated with the polypeptides of the
invention, e.g., by transduction (using viral vectors, and
preferably vectors that integrate the transgene into the cell
genome) or transfection procedures, including, but not limited to,
the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc. The coding sequence of the polypeptides of the
invention can be placed under the control of a strong constitutive
or inducible promoter or promoter/enhancer to achieve expression,
and preferably secretion, of the et al. METH1 and/or METH2
polypeptides. The engineered cells which express and preferably
secrete the polypeptides of the invention can be introduced into
the patient systemically, e.g., in the circulation, or
intraperitoneally.
[0676] Alternatively, the cells can be incorporated into a matrix
and implanted in the body, e.g., genetically engineered fibroblasts
can be implanted as part of a skin graft; genetically engineered
endothelial cells can be implanted as part of a lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Pat. No.
5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each
of which is incorporated by reference herein in its entirety).
[0677] When the cells to be administered are non-autologous or
non-MHC compatible cells, they can be administered using well known
techniques which prevent the development of a host immune response
against the introduced cells. For example, the cells may be
introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular
environment, does not allow the introduced cells to be recognized
by the host immune system.
[0678] Knock-out animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating
the biological function of METH1 and/or METH2 polypeptides,
studying conditions and/or disorders associated with aberrant METH1
and/or METH2 expression, and in screening for compounds effective
in ameliorating such conditions and/or disorders.
Example 34
Assays Detecting Stimulation or Inhibition of B cell Proliferation
and Differentiation
[0679] Generation of functional humoral immune responses requires
both soluble and cognate signaling between B-lineage cells and
their microenvironment. Signals may impart a positive stimulus that
allows a B-lineage cell to continue its programmed development, or
a negative stimulus that instructs the cell to arrest its current
developmental pathway. To date, numerous stimulatory and inhibitory
signals have been found to influence B cell responsiveness
including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and
IL-15. Interestingly, these signals are by themselves weak
effectors but can, in combination with various co-stimulatory
proteins, induce activation, proliferation, differentiation,
homing, tolerance and death among B cell populations.
[0680] One of the best studied classes of B-cell co-stimulatory
proteins is the TNF-superfamily. Within this family CD40, CD27, and
CD30 along with their respective ligands CD154, CD70, and CD153
have been found to regulate a variety of immune responses. Assays
which allow for the detection and/or observation of the
proliferation and differentiation of these B-cell populations and
their precursors are valuable tools in determining the effects
various proteins may have on these B-cell populations in terms of
proliferation and differentiation. Listed below are two assays
designed to allow for the detection of the differentiation,
proliferation, or inhibition of B-cell populations and their
precursors.
[0681] In vitro Assay--Purified METH1 and/or METH2 protein, or
truncated forms thereof, is assessed for its ability to induce
activation, proliferation, differentiation or inhibition and/or
death in B-cell populations and their precursors. The activity of
METH1 and/or METH2 protein on purified human tonsillar B cells,
measured qualitatively over the dose range from 0.1 to 10,000
ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay
in which purified tonsillar B cells are cultured in the presence of
either formalin-fixed Staphylococcus aureus Cowan I (SAC) or
immobilized anti-human IgM antibody as the priming agent. Second
signals such as IL-2 and IL-15 synergize with SAC and IgM
crosslinking to elicit B cell proliferation as measured by
tritiated-thymidine incorporation. Novel synergizing agents can be
readily identified using this assay. The assay involves isolating
human tonsillar B cells by magnetic bead (MACS) depletion of
CD3-positive cells. The resulting cell population is greater than
95% B cells as assessed by expression of CD45R(B220).
[0682] Various dilutions of each sample are placed into individual
wells of a 96-well plate to which are added 10.sup.5 B-cells
suspended in culture medium (RPMI 1640 containing 10% FBS,
5.times.10.sup.-5M 2ME, 100 U/ml penicillin, 10 ug/ml streptomycin,
and 10.sup.-5 dilution of SAC) in a total volume of 150 ul.
Proliferation or inhibition is quantitated by a 20 h pulse (1
uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor
addition. The positive and negative controls are IL2 and medium
respectively.
[0683] In vivo Assay--BALB/c mice are injected (i.p.) twice per day
with buffer only, or 2 mg/Kg of METH1 and/or METH2 protein, or
truncated forms thereof. Mice receive this treatment for 4
consecutive days, at which time they are sacrificed and various
tissues and serum collected for analyses. Comparison of H&E
sections from normal and METH1 and/or METH2 protein-treated spleens
identify the results of the activity of METH1 and/or METH2 protein
on spleen cells, such as the diffusion of peri-arterial lymphatic
sheaths, and/or significant increases in the nucleated cellularity
of the red pulp regions, which may indicate the activation of the
differentiation and proliferation of B-cell populations.
Immunohistochemical studies using a B cell marker,
anti-CD45R(B220), are used to determine whether any physiological
changes to splenic cells, such as splenic disorganization, are due
to increased B-cell representation within loosely defined B-cell
zones that infiltrate established T-cell regions.
[0684] Flow cytometric analyses of the spleens from METH1 and/or
METH2 protein-treated mice is used to indicate whether METH1 and/or
METH2 protein specifically increases the proportion of ThB+,
CD45R(B220)dull B cells over that which is observed in control
mice.
[0685] Likewise, a predicted consequence of increased mature B-cell
representation in vivo is a relative increase in serum Ig titers.
Accordingly, serum IgM and IgA levels are compared between buffer
and METH1 and/or METH2 protein-treated mice.
[0686] The studies described in this example test activity in METH1
and/or METH2 protein. However, one skilled in the art could easily
modify the exemplified studies to test the activity of METH1 and/or
METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 35
T Cell Proliferation Assay
[0687] A CD3-induced proliferation assay is performed on PBMCs and
is measured by the uptake of .sup.3H-thymidine. The assay is
performed as follows. Ninety-six well plates are coated with 100
.mu.l/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched
control mAb (B33.1) overnight at 4.degree. C. (1 mg/ml in 0.05M
bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC
are isolated by F/H gradient centrifugation from human peripheral
blood and added to quadruplicate wells (5.times.10.sup.4/well) of
mAb coated plates in RPMI containing 10% FCS and P/S in the
presence of varying concentrations of et al. METH1 and/or METH2
protein (total volume 200 .mu.l). Relevant protein buffer and
medium alone are controls. After 48 hr. culture at 37.degree. C.,
plates are spun for 2 min. at 1000 rpm and 100 .mu.l of supernatant
is removed and stored -20.degree. C. for measurement of IL-2 (or
other cytokines) if effect on proliferation is observed. Wells are
supplemented with 100 .mu.l of medium containing 0.5 .mu.Ci of
.sup.3H-thymidine and cultured at 37.degree. C. for 18-24 hr. Wells
are harvested and incorporation of .sup.3H-thymidine used as a
measure of proliferation. Anti-CD3 alone is the positive control
for proliferation. IL-2 (100 U/ml) is also used as a control which
enhances proliferation. Control antibody which does not induce
proliferation of T cells is used as the negative control for the
effects of METH1 and/or METH2 proteins.
[0688] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 36
Effect of METH1 and/or METH2 on the Expression of MHC Class II,
Costimulatory and Adhesion Molecules and Cell Differentiation of
Monocytes and Monocyte-Derived Human Dendritic Cells
[0689] Dendritic cells are generated by the expansion of
proliferating precursors found in the peripheral blood: adherent
PBMC or elutriated monocytic fractions are cultured for 7-10 days
with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells
have the characteristic phenotype of immature cells (expression of
CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with
activating factors, such as TNF-.alpha., causes a rapid change in
surface phenotype (increased expression of MHC class I and II,
costimulatory and adhesion molecules, downregulation of
FC.gamma.RII, upregulation of CD83). These changes correlate with
increased antigen-presenting capacity and with functional
maturation of the dendritic cells.
[0690] FACS analysis of surface antigens is performed as follows.
Cells are treated 1-3 days with increasing concentrations of METH1
and/or METH2 or LPS (positive control), washed with PBS containing
1% BSA and 0.02 mM sodium azide, and then incubated with 1:20
dilution of appropriate FITC- or PE-labeled monoclonal antibodies
for 30 minutes at 4.degree. C. After an additional wash, the
labeled cells are analyzed by flow cytometry on a FACScan (Becton
Dickinson).
[0691] Effect on the production of cytokines. Cytokines generated
by dendritic cells, in particular IL-12, are important in the
initiation of T-cell dependent immune responses. IL-12 strongly
influences the development of Th1 helper T-cell immune response,
and induces cytotoxic T and NK cell function. An ELISA is used to
measure the IL-12 release as follows. Dendritic cells (10.sup.6/ml)
are treated with increasing concentrations of METH1 and/or METH2
for 24 hours. LPS (100 ng/ml) is added to the cell culture as
positive control. Supernatants from the cell cultures are then
collected and analyzed for IL-12 content using commercial ELISA kit
(e.g, R & D Systems (Minneapolis, Minn.)). The standard
protocols provided with the kits are used.
[0692] Effect on the expression of MHC Class II, costimulatory and
adhesion molecules. Three major families of cell surface antigens
can be identified on monocytes: adhesion molecules, molecules
involved in antigen presentation, and Fc receptor. Modulation of
the expression of MHC class II antigens and other costimulatory
molecules, such as B7 and ICAM-1, may result in changes in the
antigen presenting capacity of monocytes and ability to induce T
cell activation. Increase expression of Fc receptors may correlate
with improved monocyte cytotoxic activity, cytokine release and
phagocytosis.
[0693] FACS analysis is used to examine the surface antigens as
follows. Monocytes are treated 1-5 days with increasing
concentrations of METH1 and/or METH2 or LPS (positive control),
washed with PBS containing 1% BSA and 0.02 mM sodium azide, and
then incubated with 1:20 dilution of appropriate FITC- or
PE-labeled monoclonal antibodies for 30 minutes at 4.degree. C.
After an additional wash, the labeled cells are analyzed by flow
cytometry on a FACScan (Becton Dickinson).
[0694] Monocyte activation and/or increased survival. Assays for
molecules that activate (or alternatively, inactivate) monocytes
and/or increase monocyte survival (or alternatively, decrease
monocyte survival) are known in the art and may routinely be
applied to determine whether a molecule of the invention functions
as an inhibitor or activator of monocytes. METH1 and/or METH2,
agonists, or antagonists of METH1 and/or METH2 can be screened
using the three assays described below. For each of these assays,
Peripheral blood mononuclear cells (PBMC) are purified from single
donor leukopacks (American Red Cross, Baltimore, Md.) by
centrifugation through a Histopaque gradient (Sigma). Monocytes are
isolated from PBMC by counterflow centrifugal elutriation.
[0695] Monocyte Survival Assay. Human peripheral blood monocytes
progressively lose viability when cultured in absence of serum or
other stimuli. Their death results from internally regulated
process (apoptosis). Addition to the culture of activating factors,
such as TNF-alpha dramatically improves cell survival and prevents
DNA fragmentation.
[0696] Propidium iodide (PI) staining is used to measure apoptosis
as follows. Monocytes are cultured for 48 hours in polypropylene
tubes in serum-free medium (positive control), in the presence of
100 ng/ml TNF-alpha (negative control), and in the presence of
varying concentrations of the compound to be tested. Cells are
suspended at a concentration of 2.times.10.sup.6/ml in PBS
containing PI at a final concentration of 5 .mu.g/ml, and then
incubated at room temperature for 5 minutes before FACScan
analysis. PI uptake has been demonstrated to correlate with DNA
fragmentation in this experimental paradigm.
[0697] Effect on cytokine release. An important function of
monocytes/macrophages is their regulatory activity on other
cellular populations of the immune system through the release of
cytokines after stimulation. An ELISA to measure cytokine release
is performed as follows. Human monocytes are incubated at a density
of 5.times.10.sup.5 cells/ml with increasing concentrations of
METH1 and/or METH2 and under the same conditions, but in the
absence of METH1 and/or METH2. For IL-12 production, the cells are
primed overnight with IFN (100 U/ml) in presence of METH1 and/or
METH2. LPS (10 ng/ml) is then added. Conditioned media are
collected after 24 h and kept frozen until use. Measurement of
TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a
commercially available ELISA kit (e.g, R & D Systems
(Minneapolis, Minn.)) and applying the standard protocols provided
with the kit.
[0698] Oxidative burst. Purified monocytes are plated in 96-w plate
at 2-1.times.10.sup.5 cell/well. Increasing concentrations of METH1
and/or METH2 are added to the wells in a total volume of 0.2 ml
culture medium (RPMI 1640+10% FCS, glutamine and antibiotics).
After 3 days incubation, the plates are centrifuged and the medium
is removed from the wells. To the macrophage monolayers, 0.2 ml per
well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate
buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of
HRPO) is added, together with the stimulant (200 nM PMA). The
plates are incubated at 37.degree. C. for 2 hours and the reaction
is stopped by adding 20 .mu.l 1N NaOH per well. The absorbance is
read at 610 nm. To calculate the amount of H.sub.2O.sub.2 produced
by the macrophages, a standard curve of a H.sub.2O.sub.2 solution
of known molarity is performed for each experiment.
[0699] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 37
METH1 and/or METH2 Biological Effects
[0700] Astrocyte and Neuronal Assays. Recombinant METH1 and/or
METH2, expressed in Escherichia coli and purified as described
above, can be tested for activity in promoting the survival,
neurite outgrowth, or phenotypic differentiation of cortical
neuronal cells and for inducing the proliferation of glial
fibrillary acidic protein immunopositive cells, astrocytes. The
selection of cortical cells for the bioassay is based on the
prevalent expression of FGF-1 and FGF-2 in cortical structures and
on the previously reported enhancement of cortical neuronal
survival resulting from FGF-2 treatment. A thymidine incorporation
assay, for example, can be used to elucidate METH1 and/or METH2's
activity on these cells.
[0701] Moreover, previous reports describing the biological effects
of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro
have demonstrated increases in both neuron survival and neurite
outgrowth (Walicke, P. et al., "Fibroblast growth factor promotes
survival of dissociated hippocampal neurons and enhances neurite
extension." Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay
herein incorporated by reference in its entirety). However, reports
from experiments done on PC-12 cells suggest that these two
responses are not necessarily synonymous and may depend on not only
which FGF is being tested but also on which receptor(s) are
expressed on the target cells. Using the primary cortical neuronal
culture paradigm, the ability of METH1 and/or METH2 to induce
neurite outgrowth can be compared to the response achieved with
FGF-2 using, for example, a thymidine incorporation assay.
[0702] Fibroblast and endothelial cell assays. Human lung
fibroblasts are obtained from Clonetics (San Diego, Calif.) and
maintained in growth media from Clonetics. Dermal microvascular
endothelial cells are obtained from Cell Applications (San Diego,
Calif.). For proliferation assays, the human lung fibroblasts and
dermal microvascular endothelial cells can be cultured at 5,000
cells/well in a 96-well plate for one day in growth medium. The
cells are then incubated for one day in 0.1% BSA basal medium.
After replacing the medium with fresh 0.1% BSA medium, the cells
are incubated with the test proteins for 3 days. Alamar Blue
(Alamar Biosciences, Sacramento, Calif.) is added to each well to a
final concentration of 10%. The cells are incubated for 4 hr. Cell
viability is measured by reading in a CytoFluor fluorescence
reader. For the PGE.sub.2 assays, the human lung fibroblasts are
cultured at 5,000 cells/well in a 96-well plate for one day. After
a medium change to 0.1% BSA basal medium, the cells are incubated
with FGF-2 or METH1 and/or METH2 with or without IL-1.alpha. for 24
hours. The supernatants are collected and assayed for PGE.sub.2 by
EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human
lung fibroblasts are cultured at 5,000 cells/well in a 96-well
plate for one day. After a medium change to 0.1% BSA basal medium,
the cells are incubated with FGF-2 or METH1 and/or METH2 with or
without IL-1.alpha. for 24 hours. The supernatants are collected
and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).
[0703] Human lung fibroblasts are cultured with FGF-2 or METH1
and/or METH2 for 3 days in basal medium before the addition of
Alamar Blue to assess effects on growth of the fibroblasts. FGF-2
should show a stimulation at 10-2500 ng/ml which can be used to
compare stimulation with METH1 and/or METH2.
[0704] Parkinson Models. The loss of motor function in Parkinson's
disease is attributed to a deficiency of striatal dopamine
resulting from the degeneration of the nigrostriatal dopaminergic
projection neurons. An animal model for Parkinson's that has been
extensively characterized involves the systemic administration of
1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS,
MPTP is taken-up by astrocytes and catabolized by monoamine oxidase
B to 1-methyl-4-phenyl pyridine (MPP.sup.+) and released.
Subsequently, MPP.sup.+ is actively accumulated in dopaminergic
neurons by the high-affinity reuptake transporter for dopamine.
MPP.sup.+ is then concentrated in mitochondria by the
electrochemical gradient and selectively inhibits nicotidamide
adenine disphosphate: ubiquinone oxidoreductionase (complex I),
thereby interfering with electron transport and eventually
generating oxygen radicals.
[0705] It has been demonstrated in tissue culture paradigms that
FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic
neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's
group has demonstrated that administering FGF-2 in gel foam
implants in the striatum results in the near complete protection of
nigral dopaminergic neurons from the toxicity associated with MPTP
exposure (Otto and Unsicker, J. Neuroscience, 1990).
[0706] Based on the data with FGF-2, METH1 and/or METH2 can be
evaluated to determine whether it has an action similar to that of
FGF-2 in enhancing dopaminergic neuronal survival in vitro and it
can also be tested in vivo for protection of dopaminergic neurons
in the striatum from the damage associated with MPTP treatment. The
potential effect of METH1 and/or METH2 is first examined in vitro
in a dopaminergic neuronal cell culture paradigm. The cultures are
prepared by dissecting the midbrain floor plate from gestation day
14 Wistar rat embryos. The tissue is dissociated with trypsin and
seeded at a density of 200,000 cells/cm.sup.2 on
polyorthinine-laminin coated glass coverslips. The cells are
maintained in Dulbecco's Modified Eagle's medium and F12 medium
containing hormonal supplements (N1). The cultures are fixed with
paraformaldehyde after 8 days in vitro and are processed for
tyrosine hydroxylase, a specific marker for dopminergic neurons,
immunohistochemical staining. Dissociated cell cultures are
prepared from embryonic rats. The culture medium is changed every
third day and the factors are also added at that time.
[0707] Since the dopaminergic neurons are isolated from animals at
gestation day 14, a developmental time which is past the stage when
the dopaminergic precursor cells are proliferating, an increase in
the number of tyrosine hydroxylase immunopositive neurons would
represent an increase in the number of dopaminergic neurons
surviving in vitro. Therefore, if METH1 and/or METH2 acts to
prolong the survival of dopaminergic neurons, it would suggest that
METH1 and/or METH2 may be involved in Parkinson's Disease.
[0708] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 38
The Effect of METH1 and/or METH2 on the Growth of Vascular
Endothelial Cells
[0709] On day 1, human umbilical vein endothelial cells (HUVEC) are
seeded at 2-5.times.10.sup.4 cells/35 mm dish density in M199
medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin,
and 50 units/ml endothelial cell growth supplements (ECGS,
Biotechnique, Inc.). On day 2, the medium is replaced with M199
containing 10% FBS, 8 units/ml heparin. METH1 and/or METH2 protein
of SEQ ID NO. 2, and positive controls, such as VEGF and basic FGF
(bFGF) are added, at varying concentrations. On days 4 and 6, the
medium is replaced. On day 8, cell number is determined with a
Coulter Counter.
[0710] An increase in the number of HUVEC cells indicates that
METH1 and/or METH2 may proliferate vascular endothelial cells.
[0711] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 39
Stimulatory Effect of METH1 and/or METH2 on the Proliferation of
Vascular Endothelial Cells
[0712] For evaluation of mitogenic activity of growth factors, the
colorimetric MTS
(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)2H-tetrazolium) assay with the electron coupling reagent PMS
(phenazine methosulfate) is performed (CellTiter 96 AQ, Promega).
Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL
serum-supplemented medium and are allowed to attach overnight.
After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF,
VEGF.sub.165 or METH1 and/or METH2 in 0.5% FBS) with or without
Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS
mixture (1:0.05) are added per well and allowed to incubate for 1
hour at 37.degree. C. before measuring the absorbance at 490 nm in
an ELISA plate reader. Background absorbance from control wells
(some media, no cells) is subtracted, and seven wells are performed
in parallel for each condition. See, Leak et al. in vitro Cell.
Dev. Biol. 30A:512-518 (1994).
[0713] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 40
Inhibition of PDGF-Induced Vascular Smooth Muscle Cell
Proliferation Stimulatory Effect
[0714] HAoSMC proliferation can be measured, for example, by BrdUrd
incorporation. Briefly, subconfluent, quiescent cells grown on the
4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP.
Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd.
After 24 h, immunocytochemistry is performed by using BrdUrd
Staining Kit (Zymed Laboratories). In brief, the cells are
incubated with the biotinylated mouse anti-BrdUrd antibody at
4.degree. C. for 2 h after being exposed to denaturing solution and
then incubated with the streptavidin-peroxidase and
diaminobenzidine. After counterstaining with hematoxylin, the cells
are mounted for microscopic examination, and the BrdUrd-positive
cells are counted. The BrdUrd index is calculated as a percent of
the BrdUrd-positive cells to the total cell number. In addition,
the simultaneous detection of the BrdUrd staining (nucleus) and the
FITC uptake (cytoplasm) is performed for individual cells by the
concomitant use of bright field illumination and dark field-UV
fluorescent illumination. See, Hayashida et al., J. Biol. Chem.
6:271(36):21985-21992 (1996).
[0715] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 41
Stimulation of Endothelial Migration
[0716] This example will be used to explore the possibility that
METH1 and/or METH2 may stimulate lymphatic endothelial cell
migration.
[0717] Endothelial cell migration assays are performed using a 48
well microchemotaxis chamber (Neuroprobe Inc., Cabin John, M D;
Falk, W., et al., J. Immunological Methods 1980; 33:239-247).
Polyvinylpyrrolidone-free polycarbonate filters with a pore size of
8 um (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1%
gelatin for at least 6 hours at room temperature and dried under
sterile air. Test substances are diluted to appropriate
concentrations in M199 supplemented with 0.25% bovine serum albumin
(BSA), and 25 ul of the final dilution is placed in the lower
chamber of the modified Boyden apparatus. Subconfluent, early
passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for
the minimum time required to achieve cell detachment. After placing
the filter between lower and upper chamber, 2.5.times.10.sup.5
cells suspended in 50 ul M 199 containing 1% FBS are seeded in the
upper compartment. The apparatus is then incubated for 5 hours at
37.degree. C. in a humidified chamber with 5% CO2 to allow cell
migration. After the incubation period, the filter is removed and
the upper side of the filter with the non-migrated cells is scraped
with a rubber policeman. The filters are fixed with methanol and
stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park,
Ill.). Migration is quantified by counting cells of three random
high-power fields (40.times.) in each well, and all groups are
performed in quadruplicate.
[0718] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 42
Stimulation of Nitric Oxide Production by Endothelial Cells
[0719] Nitric oxide released by the vascular endothelium is
believed to be a mediator of vascular endothelium relaxation. Thus,
METH1 and/or METH2 activity can be assayed by determining nitric
oxide production by endothelial cells in response to METH1 and/or
METH2.
[0720] Nitric oxide is measured in 96-well plates of confluent
microvascular endothelial cells after 24 hours starvation and a
subsequent 4 hr exposure to various levels of a positive control
(such as VEGF-1) and METH1 and/or METH2. Nitric oxide in the medium
is determined by use of the Griess reagent to measure total nitrite
after reduction of nitric oxide-derived nitrate by nitrate
reductase. The effect of METH1 and/or METH2 on nitric oxide release
is examined on HUVEC.
[0721] Briefly, NO release from cultured HUVEC monolayer is
measured with a NO-specific polarographic electrode connected to a
NO meter (Iso-NO, World Precision Instruments Inc.) (1049).
Calibration of the NO elements is performed according to the
following equation:
2KNO.sub.2+2KI+2H.sub.2SO.sub.462NO+I.sub.2+2H.sub.2O+2K.sub.2SO.sub.4
The standard calibration curve is obtained by adding graded
concentrations of KNO.sub.2 (0, 5, 10, 25, 50, 100, 250, and 500
nmol/L) into the calibration solution containing KI and
H.sub.2SO.sub.4. The specificity of the Iso-NO electrode to NO is
previously determined by measurement of NO from authentic NO gas
(1050). The culture medium is removed and HUVECs are washed twice
with Dulbecco's phosphate buffered saline. The cells are then
bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well
plates, and the cell plates are kept on a slide warmer (Lab Line
Instruments Inc.) to maintain the temperature at 37.degree. C. The
NO sensor probe is inserted vertically into the wells, keeping the
tip of the electrode 2 mm under the surface of the solution, before
addition of the different conditions. S-nitroso acetyl penicillamin
(SNAP) is used as a positive control. The amount of released NO is
expressed as picomoles per 1.times.10.sup.6 endothelial cells. All
values reported are means of four to six measurements in each group
(number of cell culture wells). See, Leak et al. Biochem. and
Biophys. Res. Comm. 217:96-105 (1995).
[0722] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 43
Effect of METH1 and/or METH2 on Cord Formation in Angiogenesis
[0723] Another step in angiogenesis is cord formation, marked by
differentiation of endothelial cells. This bioassay measures the
ability of microvascular endothelial cells to form capillary-like
structures (hollow structures) when cultured in vitro.
[0724] CADMEC (microvascular endothelial cells) are purchased from
Cell Applications, Inc. as proliferating (passage 2) cells and are
cultured in Cell Applications CADMEC Growth Medium and used at
passage 5. For the in vitro angiogenesis assay, the wells of a
48-well cell culture plate are coated with Cell Applications'
Attachment Factor Medium (200 ml/well) for 30 min. at 37.degree. C.
CADMEC are seeded onto the coated wells at 7,500 cells/well and
cultured overnight in Growth Medium. The Growth Medium is then
replaced with 300 mg Cell Applications' Chord Formation Medium
containing control buffer or METH1 and/or METH2 (0.1 to 100 ng/ml)
and the cells are cultured for an additional 48 hr. The numbers and
lengths of the capillary-like chords are quantitated through use of
the Boeckeler VIA-170 video image analyzer. All assays are done in
triplicate.
[0725] Commercial (R&D) VEGF (50 ng/ml) is used as a positive
control. b-estradiol (1 ng/ml) is used as a negative control. The
appropriate buffer (without protein) is also utilized as a
control.
[0726] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 44
Rescue of Ischemia in Rabbit Lower Limb Model
[0727] To study the in vivo effects of METH1 and/or METH2 on
ischemia, a rabbit hindlimb ischemia model is created by surgical
removal of one femoral arteries as described previously (Takeshita,
S. et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the
femoral artery results in retrograde propagation of thrombus and
occlusion of the external iliac artery. Consequently, blood flow to
the ischemic limb is dependent upon collateral vessels originating
from the internal iliac artery (Takeshita, S. et al. Am J. Pathol
147:1649-1660 (1995)). An interval of 10 days is allowed for
post-operative recovery of rabbits and development of endogenous
collateral vessels. At 10 day post-operatively (day 0), after
performing a baseline angiogram, the internal iliac artery of the
ischemic limb is transfected with 500 mg naked METH1 and/or METH2
expression plasmid by arterial gene transfer technology using a
hydrogel-coated balloon catheter as described (Riessen, R. et al.
Hum Gene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin.
Invest. 90: 936-944 (1992)). When METH1 and/or METH2 is used in the
treatment, a single bolus of 500 mg METH1 and/or METH2 protein or
control is delivered into the internal iliac artery of the ischemic
limb over a period of 1 min. through an infusion catheter. On day
30, various parameters are measured in these rabbits: (a) BP
ratio--The blood pressure ratio of systolic pressure of the
ischemic limb to that of normal limb; (b) Blood Flow and Flow
Reserve--Resting FL: the blood flow during undilated condition and
Max FL: the blood flow during fully dilated condition (also an
indirect measure of the blood vessel amount) and Flow Reserve is
reflected by the ratio of max FL: resting FL; (c) Angiographic
Score--This is measured by the angiogram of collateral vessels. A
score is determined by the percentage of circles in an overlaying
grid that with crossing opacified arteries divided by the total
number m the rabbit thigh; (d) Capillary density--The number of
collateral capillaries determined in light microscopic sections
taken from hindlimbs.
[0728] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 45
Effect of METH1 and/or METH2 on Vasodilation
[0729] Since dilation of vascular endothelium is important in
reducing blood pressure, the ability of METH1 and/or METH2 to
affect the blood pressure in spontaneously hypertensive rats (SHR)
is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg)
of the METH1 and/or METH2 are administered to 13-14 week old
spontaneously hypertensive rats (SHR). Data are expressed as the
mean +/-SEM. Statistical analysis are performed with a paired
t-test and statistical significance is defined as p<0.05 vs. the
response to buffer alone.
[0730] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 46
Rat Ischemic Skin Flap Model
[0731] The evaluation parameters include skin blood flow, skin
temperature, and factor VIII immunohistochemistry or endothelial
alkaline phosphatase reaction. METH1 and/or METH2 expression,
during the skin ischemia, is studied using in situ
hybridization.
[0732] The study in this model is divided into three parts as
follows: [0733] a) Ischemic skin [0734] b) Ischemic skin wounds
[0735] c) Normal wounds
[0736] The experimental protocol includes:
TABLE-US-00016 a) Raising a 3 .times. 4 cm, single pedicle
full-thickness random skin flap (myocutaneous flap over the lower
back of the animal). b) An excisional wounding (4-6 mm in diameter)
in the ischemic skin (skin-flap). c) Topical treatment with METH1
and/or METH2 of the excisional wounds (day 0, 1, 2, 3, 4
post-wounding) at the following various dosage ranges: 1 mg to 100
mg. d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21
post-wounding for histological, immunohistochemical, and in situ
studies.
[0737] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 47
Peripheral Arterial Disease Model
[0738] Angiogenic therapy using METH1 and/or METH2 is a novel
therapeutic strategy to obtain restoration of blood flow around the
ischemia in case of peripheral arterial diseases. The experimental
protocol includes: [0739] a) One side of the femoral artery is
ligated to create ischemic muscle of the hindlimb, the other side
of hindlimb serves as a control. [0740] b) METH1 and/or METH2
protein, in a dosage range of 20 mg-500 mg, is delivered
intravenously and/or intramuscularly 3 times (perhaps more) per
week for 2-3 weeks. [0741] c) The ischemic muscle tissue is
collected after ligation of the femoral artery at 1, 2, and 3 weeks
for the analysis of METH1 and/or METH2 expression and histology.
Biopsy is also performed on the other side of normal muscle of the
contralateral hindlimb.
[0742] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 48
Ischemic Myocardial Disease Model
[0743] METH1 and/or METH2 is evaluated as a potent mitogen capable
of stimulating the development of collateral vessels, and
restructuring new vessels after coronary artery occlusion.
Alteration of METH1 and/or METH2 expression is investigated in
situ. The experimental protocol includes: [0744] a) The heart is
exposed through a left-side thoracotomy in the rat. Immediately,
the left coronary artery is occluded with a thin suture (6-0) and
the thorax is closed. [0745] b) METH1 and/or METH2 protein, in a
dosage range of 20 mg-500 mg, is delivered intravenously and/or
intramuscularly 3 times (perhaps more) per week for 2-4 weeks.
[0746] c) Thirty days after the surgery, the heart is removed and
cross-sectioned for morphometric and in situ analyzes.
[0747] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 49
Rat Corneal Wound Healing Model
[0748] This animal model shows the effect of METH1 and/or METH2 on
neovascularization. The experimental protocol includes: [0749] a)
Making a 1-1.5 mm long incision from the center of cornea into the
stromal layer. [0750] b) Inserting a spatula below the lip of the
incision facing the outer corner of the eye. [0751] c) Making a
pocket (its base is 1-1.5 mm form the edge of the eye). [0752] d)
Positioning a pellet, containing 50 ng-5 ug of METH1 and/or METH2,
within the pocket. [0753] e) METH1 and/or METH2 treatment can also
be applied topically to the corneal wounds in a dosage range of 20
mg-500 mg (daily treatment for five days).
[0754] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 50
Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models
[0755] A. Diabetic db+/db+ Mouse Model
[0756] To demonstrate that METH1 and/or METH2 has an effect on the
healing process, the genetically diabetic mouse model of wound
healing is used. The full thickness wound healing model in the
db+/db+ mouse is a well characterized, clinically relevant and
reproducible model of impaired wound healing. Healing of the
diabetic wound is dependent on formation of granulation tissue and
re-epithelialization rather than contraction (Gartner, M. H. et
al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J.
Pathol. 136:1235 (1990)).
[0757] The diabetic animals have many of the characteristic
features observed in Type II diabetes mellitus. Homozygous
(db+/db+) mice are obese in comparison to their normal heterozygous
(db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single
autosomal recessive mutation on chromosome 4 (db+) (Coleman et al.
Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show
polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+)
have elevated blood glucose, increased or normal insulin levels,
and suppressed cell-mediated immunity (Mandel et al., J. Immunol
120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.
51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55
(1985)). Peripheral neuropathy, myocardial complications, and
microvascular lesions, basement membrane thickening and glomerular
filtration abnormalities have been described in these animals
(Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et
al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest.
40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6
(1982)). These homozygous diabetic mice develop hyperglycemia that
is resistant to insulin analogous to human type II diabetes (Mandel
et al., J. Immunol 120:1375-1377 (1978)).
[0758] The characteristics observed in these animals suggests that
healing in this model may be similar to the healing observed in
human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246
(1990)).
[0759] Genetically diabetic female C57BL/KsJ (db+/db+) mice and
their non-diabetic (db+/+m) heterozygous littermates are used in
this study (Jackson Laboratories). The animals are purchased at 6
weeks of age and are 8 weeks old at the beginning of the study.
Animals are individually housed and received food and water ad
libitum. All manipulations are performed using aseptic techniques.
The experiments are conducted according to the rules and guidelines
of Human Genome Sciences, Inc. Institutional Animal Care and Use
Committee and the Guidelines for the Care and Use of Laboratory
Animals.
[0760] Wounding protocol is performed according to previously
reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med.
172:245-251 (1990)). Briefly, on the day of wounding, animals are
anesthetized with an intraperitoneal injection of Avertin (0.01
mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in
deionized water. The dorsal region of the animal is shaved and the
skin washed with 70% ethanol solution and iodine. The surgical area
is dried with sterile gauze prior to wounding. An 8 mm
full-thickness wound is then created using a Keyes tissue punch.
Immediately following wounding, the surrounding skin is gently
stretched to eliminate wound expansion. The wounds are left open
for the duration of the experiment. Application of the treatment is
given topically for 5 consecutive days commencing on the day of
wounding. Prior to treatment, wounds are gently cleansed with
sterile saline and gauze sponges.
[0761] Wounds are visually examined and photographed at a fixed
distance at the day of surgery and at two day intervals thereafter.
Wound closure is determined by daily measurement on days 1-5 and on
day 8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[0762] METH1 and/or METH2 is administered using at a range
different doses of METH1 and/or METH2, from 4 mg to 500 mg per
wound per day for 8 days in vehicle. Vehicle control groups
received 50 mL of vehicle solution.
[0763] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology and
immunohistochemistry. Tissue specimens are placed in 10% neutral
buffered formalin in tissue cassettes between biopsy sponges for
further processing.
[0764] Three groups of 10 animals each (5 diabetic and 5
non-diabetic controls) are evaluated: 1) Vehicle placebo control,
2) METH1 and/or 3) METH2.
[0765] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total square area of
the wound. Contraction is then estimated by establishing the
differences between the initial wound area (day 0) and that of post
treatment (day 8). The wound area on day 1 is 64 mm.sup.2, the
corresponding size of the dermal punch. Calculations are made using
the following formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[0766] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using a Reichert-Jung microtome. Routine
hematoxylin-eosin (H&E) staining is performed on cross-sections
of bisected wounds. Histologic examination of the wounds are used
to assess whether the healing process and the morphologic
appearance of the repaired skin is altered by treatment with METH1
and/or METH2. This assessment included verification of the presence
of cell accumulation, inflammatory cells, capillaries, fibroblasts,
re-epithelialization and epidermal maturity (Greenhalgh, D. G. et
al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer
is used by a blinded observer.
[0767] Tissue sections are also stained immunohistochemically with
a polyclonal rabbit anti-human keratin antibody using ABC Elite
detection system. Human skin is used as a positive tissue control
while non-immune IgG is used as a negative control. Keratinocyte
growth is determined by evaluating the extent of
reepithelialization of the wound using a calibrated lens
micrometer.
[0768] Proliferating cell nuclear antigen/cyclin (PCNA) in skin
specimens is demonstrated by using anti-PCNA:antibody (1:50) with
an ABC Elite detection system. Human colon cancer served as a
positive tissue control and human brain tissue is used as a
negative tissue control. Each specimen included a section with
omission of the primary antibody and substitution with non-immune
mouse IgG. Ranking of these sections is based on the extent of
proliferation on a scale of 0-8, the lower side of the scale
reflecting slight proliferation to the higher side reflecting
intense proliferation.
[0769] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[0770] B. Steroid Impaired Rat Model
[0771] The inhibition of wound healing by steroids has been well
documented in various in vitro and in vivo systems (Wahl, S. M.
Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid
Action: Basic and Clinical Aspects. 280-302 (1989); Wahl, S. M. et
al., J. Immunol. 115: 476-481 (1975); Werb, Z. et al., J. Exp. Med.
147:1684-1694 (1978)). Glucocorticoids retard wound healing by
inhibiting angiogenesis, decreasing vascular permeability (Ebert,
R. H., et al., An. Intern. Med. 37:701-705 (1952)), fibroblast
proliferation, and collagen synthesis (Beck, L. S. et al., Growth
Factors. 5: 295-304 (1991); Haynes, B. F. et al., J. Clin. Invest.
61: 703-797 (1978)) and producing a transient reduction of
circulating monocytes (Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989)). The systemic
administration of steroids to impaired wound healing is a well
establish phenomenon in rats (Beck, L. S. et al., Growth Factors.
5: 295-304 (1991); Haynes, B. F., et al., J. Clin. Invest. 61:
703-797 (1978); Wahl, S. M., "Glucocorticoids and wound healing",
In: Antiinflammatory Steroid Action: Basic and Clinical Aspects,
Academic Press, New York, pp. 280-302 (1989); Pierce, G. F. et al.,
Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).
[0772] To demonstrate that METH1 and/or METH2 has an effect on the
healing process, the effects of multiple topical applications of
METH1 and/or METH2 on full thickness excisional skin wounds in rats
in which healing has been impaired by the systemic administration
of methylprednisolone is assessed.
[0773] Young adult male Sprague Dawley rats weighing 250-300 g
(Charles River Laboratories) are used in this example. The animals
are purchased at 8 weeks of age and are 9 weeks old at the
beginning of the study. The healing response of rats is impaired by
the systemic administration of methylprednisolone (17 mg/kg/rat
intramuscularly) at the time of wounding. Animals are individually
housed and received food and water ad libitum. All manipulations
are performed using aseptic techniques. This study is conducted
according to the rules and guidelines of Human Genome Sciences,
Inc. Institutional Animal Care and Use Committee and the Guidelines
for the Care and Use of Laboratory Animals.
[0774] The wounding protocol is followed according to section A,
above. On the day of wounding, animals are anesthetized with an
intramuscular injection of ketamine (50 mg/kg) and xylazine (5
mg/kg). The dorsal region of the animal is shaved and the skin
washed with 70% ethanol and iodine solutions. The surgical area is
dried with sterile gauze prior to wounding. An 8 mm full-thickness
wound is created using a Keyes tissue punch. The wounds are left
open for the duration of the experiment. Applications of the
testing materials are given topically once a day for 7 consecutive
days commencing on the day of wounding and subsequent to
methylprednisolone administration. Prior to treatment, wounds are
gently cleansed with sterile saline and gauze sponges.
[0775] Wounds are visually examined and photographed at a fixed
distance at the day of wounding and at the end of treatment. Wound
closure is determined by daily measurement on days 1-5 and on day
8. Wounds are measured horizontally and vertically using a
calibrated Jameson caliper. Wounds are considered healed if
granulation tissue is no longer visible and the wound is covered by
a continuous epithelium.
[0776] METH1 and/or METH2 is administered using at a range
different doses of METH1 and/or METH2, from 4 mg to 500 mg per
wound per day for 8 days in vehicle. Vehicle control groups
received 50 mL of vehicle solution.
[0777] Animals are euthanized on day 8 with an intraperitoneal
injection of sodium pentobarbital (300 mg/kg). The wounds and
surrounding skin are then harvested for histology. Tissue specimens
are placed in 10% neutral buffered formalin in tissue cassettes
between biopsy sponges for further processing.
[0778] Four groups of 10 animals each (5 with methylprednisolone
and 5 without glucocorticoid) are evaluated: 1)
[0779] Untreated group 2) Vehicle placebo control 3) METH1 and 4)
METH2 treated groups.
[0780] Wound closure is analyzed by measuring the area in the
vertical and horizontal axis and obtaining the total area of the
wound. Closure is then estimated by establishing the differences
between the initial wound area (day 0) and that of post treatment
(day 8). The wound area on day 1 is 64 mm.sup.2, the corresponding
size of the dermal punch. Calculations are made using the following
formula:
[Open area on day 8]-[Open area on day 1]/[Open area on day 1]
[0781] Specimens are fixed in 10% buffered formalin and paraffin
embedded blocks are sectioned perpendicular to the wound surface (5
mm) and cut using an Olympus microtome. Routine hematoxylin-eosin
(H&E) staining is performed on cross-sections of bisected
wounds. Histologic examination of the wounds allows assessment of
whether the healing process and the morphologic appearance of the
repaired skin is improved by treatment with METH1 and/or METH2. A
calibrated lens micrometer is used by a blinded observer to
determine the distance of the wound gap.
[0782] Experimental data are analyzed using an unpaired t test. A p
value of <0.05 is considered significant.
[0783] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 51
Lymphadema Animal Model
[0784] The purpose of this experimental approach is to create an
appropriate and consistent lymphedema model for testing the
therapeutic effects of METH1 and/or METH2 in lymphangiogenesis and
re-establishment of the lymphatic circulatory system in the rat
hind limb. Effectiveness is measured by swelling volume of the
affected limb, quantification of the amount of lymphatic
vasculature, total blood plasma protein, and histopathology. Acute
lymphedema is observed for 7-10 days. Perhaps more importantly, the
chronic progress of the edema is followed for up to 3-4 weeks.
[0785] Prior to beginning surgery, blood sample is drawn for
protein concentration analysis. Male rats weighing approximately
.about.350 g are dosed with Pentobarbital. Subsequently, the right
legs are shaved from knee to hip. The shaved area is swabbed with
gauze soaked in 70% EtOH. Blood is drawn for serum total protein
testing. Circumference and volumetric measurements are made prior
to injecting dye into paws after marking 2 measurement levels (0.5
cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of
both right and left paws are injected with 0.05 ml of 1% Evan's
Blue. Circumference and volumetric measurements are then made
following injection of dye into paws.
[0786] Using the knee joint as a landmark, a mid-leg inguinal
incision is made circumferentially allowing the femoral vessels to
be located. Forceps and hemostats are used to dissect and separate
the skin flaps. After locating the femoral vessels, the lymphatic
vessel that runs along side and underneath the vessel(s) is
located. The main lymphatic vessels in this area are then
electrically coagulated or suture ligated.
[0787] Using a microscope, muscles in back of the leg (near the
semitendinosis and adductors) are bluntly dissected. The popliteal
lymph node is then located. The 2 proximal and 2 distal lymphatic
vessels and distal blood supply of the popliteal node are then and
ligated by suturing. The popliteal lymph node, and any accompanying
adipose tissue, is then removed by cutting connective tissues.
[0788] Care is taken to control any mild bleeding resulting from
this procedure. After lymphatics are occluded, the skin flaps are
sealed by using liquid skin (Vetbond) (AJ Buck). The separated skin
edges are sealed to the underlying muscle tissue while leaving a
gap of .about.0.5 cm around the leg. Skin also may be anchored by
suturing to underlying muscle when necessary.
[0789] To avoid infection, animals are housed individually with
mesh (no bedding). Recovering animals are checked daily through the
optimal edematous peak, which typically occurred by day 5-7. The
plateau edematous peak are then observed. To evaluate the intensity
of the lymphedema, the circumference and volumes of 2 designated
places on each paw before operation and daily for 7 days are
measured. The effect plasma proteins on lymphedema is determined
and whether protein analysis is a useful testing perimeter is also
investigated. The weights of both control and edematous limbs are
evaluated at 2 places. Analysis is performed in a blind manner.
[0790] Circumference Measurements: Under brief gas anesthetic to
prevent limb movement, a cloth tape is used to measure limb
circumference. Measurements are done at the ankle bone and dorsal
paw by 2 different people then those 2 readings are averaged.
Readings are taken from both control and edematous limbs.
[0791] Volumetric Measurements: On the day of surgery, animals are
anesthetized with Pentobarbital and are tested prior to surgery.
For daily volumetrics animals are under brief halothane anesthetic
(rapid immobilization and quick recovery), both legs are shaved and
equally marked using waterproof marker on legs. Legs are first
dipped in water, then dipped into instrument to each marked level
then measured by Buxco edema software (Chen/Victor). Data is
recorded by one person, while the other is dipping the limb to
marked area.
[0792] Blood-plasma protein measurements: Blood is drawn, spun, and
serum separated prior to surgery and then at conclusion for total
protein and Ca.sup.2+ comparison.
[0793] Limb Weight Comparison: After drawing blood, the animal is
prepared for tissue collection. The limbs are amputated using a
quillitine, then both experimental and control legs are cut at the
ligature and weighed. A second weighing is done as the
tibio-cacaneal joint is disarticulated and the foot is weighed.
[0794] Histological Preparations: The transverse muscle located
behind the knee (popliteal) area is dissected and arranged in a
metal mold, filled with freezeGel, dipped into cold methylbutane,
placed into labeled sample bags at -80EC until sectioning. Upon
sectioning, the muscle is observed under fluorescent microscopy for
lymphatics.
[0795] The studies described in this example tested activity in
METH1 and/or METH2 protein. However, one skilled in the art could
easily modify the exemplified studies to test the activity of METH1
and/or METH2 polynucleotides (e.g., gene therapy), agonists, and/or
antagonists of METH1 and/or METH2.
Example 52
Generation of Constructs and Expression of METH1
[0796] Two constructs having either a Flag peptide sequence or a
human IgG1 Fc domain fused to the full-length METH1 gene at its
C-terminus were generated, using methods well known in the art. The
construct names, pFlag-CMV-5a:METH1 (ID 822) and pC4Fc:METH1 (ID
821) were assigned.
[0797] The following primers were used for pFlag-CMV-5a:METH1:
TABLE-US-00017 5': AAGAATGCGGCCGCAGCCACCATGGGGAACGCGGAGCGGGCTCC 3':
GATCGCGGTACCACTGCATTCTGCCATTGTGCAAAAGTCTATG
[0798] METH1 was amplified using the indicated primers, and
digested with Asp718. The vector pFLAGCMV-5a was also digested with
Asp718. The resulting restriction products were ligated
together.
[0799] The following primers were used for pC4Fc:METH1:
TABLE-US-00018 5': GATCTATGATCAGCCACCATGGGGAACGCGGAGCGGGCTCC 3':
GACTGCTCTAGAACTGCATTCTGCCATTGTGCAAAAGTCTATG
[0800] METH1 was amplified using the indicated primers, and
digested with BcII and Xba. The vector pC4Fc was also digested with
BcII and Xba. The resulting restriction products were ligated
together.
[0801] Constructs pA2gp:METH1(H542-Q894).Fc and
pA2gp:METH1(H542-Q894) can also be made.
[0802] Also, pC4Fc:Methyl.M1-P799 can be made using the following
primers:
TABLE-US-00019 5' primer: GATCTA TGATCA
GCCACCATGGGGAACGCGGAGCGGGCTCC 3' primer: GCGTGC TCTAGA
AGGGCTAAAGCTGCGAATTC
[0803] METH1 is amplified using the indicated primers, and digested
with BclI and Xba. The vector pC4Fc was also digested with BclI and
Xba, and ligated to the digested METH1 fragment. [0804]
pFLAG-CMV-1:Methyl.F236-E614 can be made using the following
primers:
TABLE-US-00020 [0804] 5' primer: GTACCC AAGCTT TTTGTGTCCAGTCACCGC
3' primer: GCGTGC TCTAGA TTACTCGTTGTGTGCTTCAC
[0805] METH1 is amplified using the indicated primers and digested
with Hind III and Xba. The vector pFLAG-CMV-1 is also digested with
Hind III and Xba and ligated to the digested METH1 fragment.
[0806] The constructs were made in order to confirm the
anti-angiogenesis activity of METH1. The full length METH1 gene was
PCR cloned into pC4Fc and pFlagCMV5a vectors. Both pC4Fc:METH1 and
pFLAGCMV5a:METH1 were obtained and the sequence confirmed.
[0807] Transient transfections on 293T cells were done using
lipofectamine plus (LTI) reagent and held for production under
serum-free conditions. Western analysis was done with either
anti-huFc Ab or anti-Flag M2 Ab. METH1-Fc conditioned media showed
at least five bands with varying degree of intensity. Their
estimated MWs are 130-140 kD (weak), 110-120 kD (weak), 52 kD
(strong), 45-48 kD (strong) and 32-35 kD (strongest). Two weaker
bands at about 60 and 90 kD were also detectable. METH1-Flag
conditioned medium revealed three major bands with equal intensity.
They are about 100-110 kD, 70-80 kD and 22 kD. Transient
transfection of METH1-Fc in 293T cells. A second batch of METH1-Fc
protein was produced in medium with 1% serum as described
above.
[0808] 5.5 day conditioned medium from transiently transfected
cells was run on a Protein A column and eluted. The fractions
containing protein were examined by SDS-PAGE under reduced and
non-reduced conditions and stained with Coomassie Blue. A second
gel was also prepared for N-terminal sequence analysis.
[0809] 197 .mu.g of protein were recovered which demonstrated 4
bands under reducing conditions. Three of the bands were strong,
one was weak. N-terminal sequencing of the bands suggested that 2
of the bands contained proteins with a blocked N-termini. Of the 2
bands giving sequence, one was an Fc-derived fragment, the other a
cleavage product of the METH1.Fc fusion starting at L800
(containing two of the thrombospondin-like domains). This suggests
that METH1 is processed with at least 2 cleavage sites (possibly
more) since only the C-terminal fragments still linked to the Fc
fragment would be purified on the Protein A column.
[0810] The transfected 293T cells were conditioned in medium
containing 1% dialyzed, low IgG, fetal calf serum to attempt to
decrease the proteolysis of the recombinant secreted protein. The
purification and analysis was as described above. The yield of
protein was significantly higher than the first batch, possibly
reflecting the effect of the serum in the medium. While some
processing may have been slowed by the serum, the majority of the
protein remained approximately 31 kD on a reducing gel.
[0811] N-terminal sequencing of resolved bands under reducing
conditions indicated the protein is processed at L800 of the 950
residue METH1 orf, with other possible cleavage occurring
N-terminal to this site. The observed cleavage site was considered
unusual since it followed a Pro. A total of 197.4 .mu.g of protein
was isolated (HG12100-D293T1). Analysis of flag protein
(pFlag-CMV-5a:METH1), consisting of at least three bands on the
Western blot (120, 97 and 21 Kd) indicated that only one band (21
kd) could be confirmed as METH1 and the other bands were of
non-METH1 origin.
[0812] Since sequencing of the purified METH1 Fc protein suggested
an unusual cleavage site, a second batch of METH1 Fc was prepared
with cells grown in 1% FBS, to possibly inhibit undesirable
processing. A preliminary assessment of the product suggests that
no difference in processing resulted from the change in medium, but
protein yields were increased.
[0813] Functional assays of the initial Fc and Flag protein
supernatants performed included proliferation of Human
Microvascular Endothelial Cells (HMVECs) and in vitro cord
formation using Bovine aortic endothelial cells (BAECs). The
proliferation assay indicated increased rates of HMVEC
proliferation in response to both culture supernatants, which may
be attributable to high background stimulation from the conditioned
medium. Cord formation assays of both the Fc and Flag supernatants
indicated inhibition of cord formation relative to a
medium/collagen control in two independent experiments.
Example 53
In Vitro Activity of METH1
Proliferation
[0814] HMVECs were used in an alamar blue assay to determine if
METH1 supernatants have functional anti-angiogenic activity,
detectable by an inhibition of EC proliferation. FGF-2 was used as
the primary stimulus for proliferation and culture supernatants
were used at a 1:4 final dilution. The proliferation assays
indicated significantly increased rates of HMVEC proliferation in
response to both culture supernatants, which may be attributable to
high background stimulation from the conditioned medium. This
problem should be reduced or eliminated by the use of purified
proteins.
Cord Formation
[0815] The addition of soluble type I collagen to endothelial cells
and the appropriate growth factors will induce the production of
tube-like structures or cords of endothelial cells in culture which
involves both the migration of endothelial cells and the selective
deletion (apoptosis) of cells not involved in these structures.
Bovine aortic endothelial cells (BAECs) were used to detect
inhibition of stable cord formation when cultured with METH1-Fc and
METH1-Flag containing culture supernatants at a 1:4 dilution.
Qualitative assessment of the cord formation indicated inhibition
with both of the tested supernatants relative to the
collagen-treated control. However, a non-matched conditioned medium
control also generated inhibition of cord formation, suggesting
that non-specific cellular toxicity might also contribute to the
observed inhibition.
[0816] The studies described in this example tested activity in
METH1 protein. However, one skilled in the art could easily modify
the exemplified studies to test the activity of METH2 polypeptides,
METH1 and/or METH2 polynucleotides (e.g., gene therapy), agonists,
and/or antagonists of METH1 and/or METH2.
[0817] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0818] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0819] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
12613261DNAHomo sapiensCDS(1)..(2853)misc_feature(3095)n is any
nucleic acid 1atg ggg aac gcg gag cgg gct ccg ggg tct cgg agc ttt
ggg ccc gta 48Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe
Gly Pro Val1 5 10 15ccc acg ctg ctg ctg ctc gcc gcg gcg cta ctg gcc
gtg tcg gac gca 96Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala
Val Ser Asp Ala 20 25 30ctc ggg cgc ccc tcc gag gag gac gag gag cta
gtg gtg ccg gag ctg 144Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu
Val Val Pro Glu Leu 35 40 45gag cgc gcc ccg gga cac ggg acc acg cgc
ctc cgc ctg cac gcc ttt 192Glu Arg Ala Pro Gly His Gly Thr Thr Arg
Leu Arg Leu His Ala Phe 50 55 60gac cag cag ctg gat ctg gag ctg cgg
ccc gac agc agc ttt ttg gcg 240Asp Gln Gln Leu Asp Leu Glu Leu Arg
Pro Asp Ser Ser Phe Leu Ala65 70 75 80ccc ggc ttc acg ctc cag aac
gtg ggg cgc aaa tcc ggg tcc gag acg 288Pro Gly Phe Thr Leu Gln Asn
Val Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95ccg ctt ccg gaa acc gac
ctg gcg cac tgc ttc tac tcc ggc acc gtg 336Pro Leu Pro Glu Thr Asp
Leu Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110aat ggc gat ccc
agc tcg gct gcc gcc ctc agc ctc tgc gag ggc gtg 384Asn Gly Asp Pro
Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125cgc ggc
gcc ttc tac ctg ctg ggg gag gcg tat ttc atc cag ccg ctg 432Arg Gly
Ala Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu 130 135
140ccc gcc gcc agc gag cgc ctc gcc acc gcc gcc cca ggg gag aag ccg
480Pro Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys
Pro145 150 155 160ccg gca cca cta cag ttc cac ctc ctg cgg cgg aat
cgg cag ggc gac 528Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn
Arg Gln Gly Asp 165 170 175gta ggc ggc acg tgc ggg gtc gtg gac gac
gag ccc cgg ccg act ggg 576Val Gly Gly Thr Cys Gly Val Val Asp Asp
Glu Pro Arg Pro Thr Gly 180 185 190aaa gcg gag acc gaa gac gag gac
gaa ggg act gag ggc gag gac gaa 624Lys Ala Glu Thr Glu Asp Glu Asp
Glu Gly Thr Glu Gly Glu Asp Glu 195 200 205ggg cct cag tgg tcg ccg
cag gac ccg gca ctg caa ggc gta gga cag 672Gly Pro Gln Trp Ser Pro
Gln Asp Pro Ala Leu Gln Gly Val Gly Gln 210 215 220ccc aca gga act
gga agc ata aga aag aag cga ttt gtg tcc agt cac 720Pro Thr Gly Thr
Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His225 230 235 240cgc
tat gtg gaa acc atg ctt gtg gca gac cag tcg atg gca gaa ttc 768Arg
Tyr Val Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe 245 250
255cac ggc agt ggt cta aag cat tac ctt ctc acg ttg ttt tcg gtg gca
816His Gly Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala
260 265 270gcc aga ttg tac aaa cac ccc agc att cgt aat tca gtt agc
ctg gtg 864Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser
Leu Val 275 280 285gtg gtg aag atc ttg gtc atc cac gat gaa cag aag
ggg ccg gaa gtg 912Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys
Gly Pro Glu Val 290 295 300acc tcc aat gct gcc ctc act ctg cgg aac
ttt tgc aac tgg cag aag 960Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn
Phe Cys Asn Trp Gln Lys305 310 315 320cag cac aac cca ccc agt gac
cgg gat gca gag cac tat gac aca gca 1008Gln His Asn Pro Pro Ser Asp
Arg Asp Ala Glu His Tyr Asp Thr Ala 325 330 335att ctt ttc acc aga
cag gac ttg tgt ggg tcc cag aca tgt gat act 1056Ile Leu Phe Thr Arg
Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350ctt ggg atg
gct gat gtt gga act gtg tgt gat ccg agc aga agc tgc 1104Leu Gly Met
Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365tcc
gtc ata gaa gat gat ggt tta caa gct gcc ttc acc aca gcc cat 1152Ser
Val Ile Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His 370 375
380gaa tta ggc cac gtg ttt aac atg cca cat gat gat gca aag cag tgt
1200Glu Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln
Cys385 390 395 400gcc agc ctt aat ggt gtg aac cag gat tcc cac atg
atg gcg tca atg 1248Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met
Met Ala Ser Met 405 410 415ctt tcc aac ctg gac cac agc cag cct tgg
tct cct tgc agt gcc tac 1296Leu Ser Asn Leu Asp His Ser Gln Pro Trp
Ser Pro Cys Ser Ala Tyr 420 425 430atg att aca tca ttt ctg gat aat
ggt cat ggg gaa tgt ttg atg gac 1344Met Ile Thr Ser Phe Leu Asp Asn
Gly His Gly Glu Cys Leu Met Asp 435 440 445aag cct cag aat ccc ata
cag ctc cca ggc gat ctc cct ggc acc tcg 1392Lys Pro Gln Asn Pro Ile
Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser 450 455 460tac gat gcc aac
cgg cag tgc cag ttt aca ttt ggg gag gac tcc aaa 1440Tyr Asp Ala Asn
Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser Lys465 470 475 480cac
tgc cct gat gca gcc agc aca tgt agc acc ttg tgg tgt acc ggc 1488His
Cys Pro Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485 490
495acc tct ggt ggg gtg ctg gtg tgt caa acc aaa cac ttc ccg tgg gcg
1536Thr Ser Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp Ala
500 505 510gat ggc acc agc tgt gga gaa ggg aaa tgg tgt atc aac ggc
aag tgt 1584Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly
Lys Cys 515 520 525gtg aac aaa acc gac aga aag cat ttt gat acg cct
ttt cat gga agc 1632Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro
Phe His Gly Ser 530 535 540tgg gga atg tgg ggg cct tgg gga gac tgt
tcg aga acg tgc ggt gga 1680Trp Gly Met Trp Gly Pro Trp Gly Asp Cys
Ser Arg Thr Cys Gly Gly545 550 555 560gga gtc cag tac acg atg agg
gaa tgt gac aac cca gtc cca aag aat 1728Gly Val Gln Tyr Thr Met Arg
Glu Cys Asp Asn Pro Val Pro Lys Asn 565 570 575gga ggg aag tac tgt
gaa ggc aaa cga gtg cgc tac aga tcc tgt aac 1776Gly Gly Lys Tyr Cys
Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn 580 585 590ctt gag gac
tgt cca gac aat aat gga aaa acc ttt aga gag gaa caa 1824Leu Glu Asp
Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu Gln 595 600 605tgt
gaa gca cac aac gag ttt tca aaa gct tcc ttt ggg agt ggg cct 1872Cys
Glu Ala His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly Pro 610 615
620gcg gtg gaa tgg att ccc aag tac gct ggc gtc tca cca aag gac agg
1920Ala Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp
Arg625 630 635 640tgc aag ctc atc tgc caa gcc aaa ggc att ggc tac
ttc ttc gtt ttg 1968Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr
Phe Phe Val Leu 645 650 655cag ccc aag gtt gta gat ggt act cca tgt
agc cca gat tcc acc tct 2016Gln Pro Lys Val Val Asp Gly Thr Pro Cys
Ser Pro Asp Ser Thr Ser 660 665 670gtc tgt gtg caa gga cag tgt gta
aaa gct ggt tgt gat cgc atc ata 2064Val Cys Val Gln Gly Gln Cys Val
Lys Ala Gly Cys Asp Arg Ile Ile 675 680 685gac tcc aaa aag aag ttt
gat aaa tgt ggt gtt tgc ggg gga aat gga 2112Asp Ser Lys Lys Lys Phe
Asp Lys Cys Gly Val Cys Gly Gly Asn Gly 690 695 700tct act tgt aaa
aaa ata tca gga tca gtt act agt gca aaa cct gga 2160Ser Thr Cys Lys
Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro Gly705 710 715 720tat
cat gat atc atc aca att cca act gga gcc acc aac atc gaa gtg 2208Tyr
His Asp Ile Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu Val 725 730
735aaa cag cgg aac cag agg gga tcc agg aac aat ggc agc ttt ctt gcc
2256Lys Gln Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser Phe Leu Ala
740 745 750atc aaa gct gct gat ggc aca tat att ctt aat ggt gac tac
act ttg 2304Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asp Tyr
Thr Leu 755 760 765tcc acc tta gag caa gac att atg tac aaa ggt gtt
gtc ttg agg tac 2352Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val
Val Leu Arg Tyr 770 775 780agc ggc tcc tct gcg gca ttg gaa aga att
cgc agc ttt agc cct ctc 2400Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile
Arg Ser Phe Ser Pro Leu785 790 795 800aaa gag ccc ttg acc atc cag
gtt ctt act gtg ggc aat gcc ctt cga 2448Lys Glu Pro Leu Thr Ile Gln
Val Leu Thr Val Gly Asn Ala Leu Arg 805 810 815cct aaa att aaa tac
acc tac ttc gta aag aag aag aag gaa tct ttc 2496Pro Lys Ile Lys Tyr
Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser Phe 820 825 830aat gct atc
ccc act ttt tca gca tgg gtc att gaa gag tgg ggc gaa 2544Asn Ala Ile
Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu 835 840 845tgt
tct aag tca tgt gaa ttg ggt tgg cag aga aga ctg gta gaa tgc 2592Cys
Ser Lys Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu Cys 850 855
860cga gac att aat gga cag cct gct tcc gag tgt gca aag gaa gtg aag
2640Arg Asp Ile Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val
Lys865 870 875 880cca gcc agc acc aga cct tgt gca gac cat ccc tgc
ccc cag tgg cag 2688Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys
Pro Gln Trp Gln 885 890 895ctg ggg gag tgg tca tca tgt tct aag acc
tgt ggg aag ggt tac aaa 2736Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr
Cys Gly Lys Gly Tyr Lys 900 905 910aaa aga agc ttg aag tgt ctg tcc
cat gat gga ggg gtg tta tct cat 2784Lys Arg Ser Leu Lys Cys Leu Ser
His Asp Gly Gly Val Leu Ser His 915 920 925gag agc tgt gat cct tta
aag aaa cct aaa cat ttc ata gac ttt tgc 2832Glu Ser Cys Asp Pro Leu
Lys Lys Pro Lys His Phe Ile Asp Phe Cys 930 935 940aca atg gca gaa
tgc agt taa gtggtttaag tggtgttagc tttgaggcaa 2883Thr Met Ala Glu
Cys Ser945 950ggcaaagtga ggaagggctg gtgcagggaa agcaagaagg
ctggagggat ccagcgtatc 2943ttgccagtaa ccagtgaggt gtatcagtaa
ggtgggatta tgggggtaga tagaaaagga 3003gttgaatcat cagagtaaac
tgccagttgc aaatttgata ggatagttag tgaggattat 3063taacctctga
gcagtgatat agcataataa anccccgggc attattatta ttatttcttt
3123tgttacatct attacaagtt tagaaaaaac aaagcaattg tcaaaaaaaa
aaaaaaaaaa 3183aaaaaaaaaa aaagggcggc cgctctagag gatccctcga
ggggcccaag cttacgcgtg 3243catgntgtca tnagtctn 32612950PRTHomo
sapiens 2Met Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly
Pro Val1 5 10 15Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val
Ser Asp Ala 20 25 30Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val
Val Pro Glu Leu 35 40 45Glu Arg Ala Pro Gly His Gly Thr Thr Arg Leu
Arg Leu His Ala Phe 50 55 60Asp Gln Gln Leu Asp Leu Glu Leu Arg Pro
Asp Ser Ser Phe Leu Ala65 70 75 80Pro Gly Phe Thr Leu Gln Asn Val
Gly Arg Lys Ser Gly Ser Glu Thr 85 90 95Pro Leu Pro Glu Thr Asp Leu
Ala His Cys Phe Tyr Ser Gly Thr Val 100 105 110Asn Gly Asp Pro Ser
Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly Val 115 120 125Arg Gly Ala
Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro Leu 130 135 140Pro
Ala Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys Pro145 150
155 160Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly
Asp 165 170 175Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg
Pro Thr Gly 180 185 190Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr
Glu Gly Glu Asp Glu 195 200 205Gly Pro Gln Trp Ser Pro Gln Asp Pro
Ala Leu Gln Gly Val Gly Gln 210 215 220Pro Thr Gly Thr Gly Ser Ile
Arg Lys Lys Arg Phe Val Ser Ser His225 230 235 240Arg Tyr Val Glu
Thr Met Leu Val Ala Asp Gln Ser Met Ala Glu Phe 245 250 255His Gly
Ser Gly Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val Ala 260 265
270Ala Arg Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu Val
275 280 285Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro
Glu Val 290 295 300Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys
Asn Trp Gln Lys305 310 315 320Gln His Asn Pro Pro Ser Asp Arg Asp
Ala Glu His Tyr Asp Thr Ala 325 330 335Ile Leu Phe Thr Arg Gln Asp
Leu Cys Gly Ser Gln Thr Cys Asp Thr 340 345 350Leu Gly Met Ala Asp
Val Gly Thr Val Cys Asp Pro Ser Arg Ser Cys 355 360 365Ser Val Ile
Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr Thr Ala His 370 375 380Glu
Leu Gly His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln Cys385 390
395 400Ala Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser
Met 405 410 415Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys
Ser Ala Tyr 420 425 430Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly
Glu Cys Leu Met Asp 435 440 445Lys Pro Gln Asn Pro Ile Gln Leu Pro
Gly Asp Leu Pro Gly Thr Ser 450 455 460Tyr Asp Ala Asn Arg Gln Cys
Gln Phe Thr Phe Gly Glu Asp Ser Lys465 470 475 480His Cys Pro Asp
Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys Thr Gly 485 490 495Thr Ser
Gly Gly Val Leu Val Cys Gln Thr Lys His Phe Pro Trp Ala 500 505
510Asp Gly Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys Cys
515 520 525Val Asn Lys Thr Asp Arg Lys His Phe Asp Thr Pro Phe His
Gly Ser 530 535 540Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg
Thr Cys Gly Gly545 550 555 560Gly Val Gln Tyr Thr Met Arg Glu Cys
Asp Asn Pro Val Pro Lys Asn 565 570 575Gly Gly Lys Tyr Cys Glu Gly
Lys Arg Val Arg Tyr Arg Ser Cys Asn 580 585 590Leu Glu Asp Cys Pro
Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu Gln 595 600 605Cys Glu Ala
His Asn Glu Phe Ser Lys Ala Ser Phe Gly Ser Gly Pro 610 615 620Ala
Val Glu Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp Arg625 630
635 640Cys Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val
Leu 645 650 655Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp
Ser Thr Ser 660 665 670Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly
Cys Asp Arg Ile Ile 675 680 685Asp Ser Lys Lys Lys Phe Asp Lys Cys
Gly Val Cys Gly Gly Asn Gly 690 695 700Ser Thr Cys Lys Lys Ile Ser
Gly Ser Val Thr Ser Ala Lys Pro Gly705 710 715 720Tyr His Asp Ile
Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile Glu Val 725 730 735Lys Gln
Arg Asn Gln Arg Gly Ser Arg Asn Asn Gly Ser Phe Leu Ala 740 745
750Ile Lys Ala Ala Asp Gly Thr Tyr Ile Leu Asn Gly Asp Tyr Thr Leu
755 760 765Ser Thr Leu Glu Gln Asp Ile Met Tyr Lys Gly Val Val Leu
Arg Tyr 770 775 780Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser
Phe Ser Pro Leu785 790 795 800Lys Glu Pro Leu Thr Ile Gln Val Leu
Thr Val Gly Asn Ala Leu Arg 805 810 815Pro Lys Ile Lys Tyr Thr Tyr
Phe Val Lys Lys Lys Lys Glu Ser Phe 820 825 830Asn Ala Ile Pro Thr
Phe Ser Ala Trp Val Ile Glu Glu Trp Gly Glu 835 840 845Cys Ser Lys
Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu Val Glu Cys 850 855 860Arg
Asp Ile
Asn Gly Gln Pro Ala Ser Glu Cys Ala Lys Glu Val Lys865 870 875
880Pro Ala Ser Thr Arg Pro Cys Ala Asp His Pro Cys Pro Gln Trp Gln
885 890 895Leu Gly Glu Trp Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly
Tyr Lys 900 905 910Lys Arg Ser Leu Lys Cys Leu Ser His Asp Gly Gly
Val Leu Ser His 915 920 925Glu Ser Cys Asp Pro Leu Lys Lys Pro Lys
His Phe Ile Asp Phe Cys 930 935 940Thr Met Ala Glu Cys Ser945
95033008DNAHomo sapiensCDS(1)..(2670)Misc_feature(2887)N is any
nucleic acid 3atg ttc ccc gcc ccc gcc gcc ccc cgg tgg ctt ccg ttc
ctg ctg ctg 48Met Phe Pro Ala Pro Ala Ala Pro Arg Trp Leu Pro Phe
Leu Leu Leu1 5 10 15ctg ctg ctg ctg ctg ctg ccg ctg gcc cgc ggc gcc
ccg gcc cgg ccc 96Leu Leu Leu Leu Leu Leu Pro Leu Ala Arg Gly Ala
Pro Ala Arg Pro 20 25 30gca gcc ggg ggg cag gcc tcg gag ctg gtg gtg
ccc acg cgg ttg ccc 144Ala Ala Gly Gly Gln Ala Ser Glu Leu Val Val
Pro Thr Arg Leu Pro 35 40 45ggc agc gcg ggc gag ctc gcg ctc cac ctg
tcc gcc ttc ggc aag ggc 192Gly Ser Ala Gly Glu Leu Ala Leu His Leu
Ser Ala Phe Gly Lys Gly 50 55 60ttc gtg ttg cgc ctg gcg ccc gac gac
agc ttc ctg gcg ccc gag ttc 240Phe Val Leu Arg Leu Ala Pro Asp Asp
Ser Phe Leu Ala Pro Glu Phe65 70 75 80aag atc gag cgc ctc ggg ggc
tcc ggc cgg gcg acc ggg ggc gag cgg 288Lys Ile Glu Arg Leu Gly Gly
Ser Gly Arg Ala Thr Gly Gly Glu Arg 85 90 95ggg ctg cgc ggc tgt ttt
ttt tcc ggc acc gtg aat ggg gag ccc gag 336Gly Leu Arg Gly Cys Phe
Phe Ser Gly Thr Val Asn Gly Glu Pro Glu 100 105 110tcg ctg gcg gcg
gtc agc ctg tgc cgc ggg ctg agc ggc tcc ttc ctg 384Ser Leu Ala Ala
Val Ser Leu Cys Arg Gly Leu Ser Gly Ser Phe Leu 115 120 125ctg gac
ggc gag gag ttc acc atc cag ccg cag ggc gcg ggg ggc tcc 432Leu Asp
Gly Glu Glu Phe Thr Ile Gln Pro Gln Gly Ala Gly Gly Ser 130 135
140ctg gct cag ccg cac cgc ctg cag cgc tgg ggt ccc gcc gga gcc cgc
480Leu Ala Gln Pro His Arg Leu Gln Arg Trp Gly Pro Ala Gly Ala
Arg145 150 155 160ccc ctc ccg cga gga ccc gag tgg gag gtg gag acg
gga gag ggt cag 528Pro Leu Pro Arg Gly Pro Glu Trp Glu Val Glu Thr
Gly Glu Gly Gln 165 170 175agg cag gag aga gga gac cac cag gag gac
agc gag gag gag agc caa 576Arg Gln Glu Arg Gly Asp His Gln Glu Asp
Ser Glu Glu Glu Ser Gln 180 185 190gaa gag gag gca gaa ggc gct agc
gag ccg cca ccg ccc ctg ggg gcc 624Glu Glu Glu Ala Glu Gly Ala Ser
Glu Pro Pro Pro Pro Leu Gly Ala 195 200 205acg agt agg acc aag cgg
ttt gtg tct gag gcg cgc ttc gtg gag acg 672Thr Ser Arg Thr Lys Arg
Phe Val Ser Glu Ala Arg Phe Val Glu Thr 210 215 220ctg ctg gtg gcc
gat gcg tcc atg gct gcc ttc tac ggg gcc gac ctg 720Leu Leu Val Ala
Asp Ala Ser Met Ala Ala Phe Tyr Gly Ala Asp Leu225 230 235 240cag
aac cac atc ctg acg tta atg tct gtg gca gcc cga atc tac aag 768Gln
Asn His Ile Leu Thr Leu Met Ser Val Ala Ala Arg Ile Tyr Lys 245 250
255cac ccc agc atc aag aat tcc atc aac ctg atg gtg gta aaa gtg ctg
816His Pro Ser Ile Lys Asn Ser Ile Asn Leu Met Val Val Lys Val Leu
260 265 270atc gta gaa gat gaa aaa tgg ggc cca gag gtg tcc gac aat
ggg ggg 864Ile Val Glu Asp Glu Lys Trp Gly Pro Glu Val Ser Asp Asn
Gly Gly 275 280 285ctt aca ctg cgt aac ttc tgc aac tgg cag cgg cgt
ttc aac cag ccc 912Leu Thr Leu Arg Asn Phe Cys Asn Trp Gln Arg Arg
Phe Asn Gln Pro 290 295 300agc gac cgc cac cca gag cac tac gac acg
gcc atc ctg ctc acc aga 960Ser Asp Arg His Pro Glu His Tyr Asp Thr
Ala Ile Leu Leu Thr Arg305 310 315 320cag aac ttc tgt ggg cag gag
ggg ctg tgt gac acc ctg ggt gtg gca 1008Gln Asn Phe Cys Gly Gln Glu
Gly Leu Cys Asp Thr Leu Gly Val Ala 325 330 335gac atc ggg acc att
tgt gac ccc aac aaa agc tgc tcc gtg atc gag 1056Asp Ile Gly Thr Ile
Cys Asp Pro Asn Lys Ser Cys Ser Val Ile Glu 340 345 350gat gag ggg
ctc cag gcg gcc cac acc ctg gcc cat gaa cta ggg cac 1104Asp Glu Gly
Leu Gln Ala Ala His Thr Leu Ala His Glu Leu Gly His 355 360 365gtc
ctc agc atg ccc cac gac gac tcc aag ccc tgc aca cgg ctc ttc 1152Val
Leu Ser Met Pro His Asp Asp Ser Lys Pro Cys Thr Arg Leu Phe 370 375
380ggg ccc atg ggc aag cac cac gtg atg gca ccg ctg ttc gtc cac ctg
1200Gly Pro Met Gly Lys His His Val Met Ala Pro Leu Phe Val His
Leu385 390 395 400aac cag acg ctg ccc tgg tcc ccc tgc agc gcc atg
tat ctc aca gag 1248Asn Gln Thr Leu Pro Trp Ser Pro Cys Ser Ala Met
Tyr Leu Thr Glu 405 410 415ctt ctg gac ggc ggg cac gga gac tgt ctc
ctg gat gcc cct ggt gcg 1296Leu Leu Asp Gly Gly His Gly Asp Cys Leu
Leu Asp Ala Pro Gly Ala 420 425 430gcc ctg ccc ctc ccc aca ggc ctc
ccg ggc cgc atg gcc ctg tac cag 1344Ala Leu Pro Leu Pro Thr Gly Leu
Pro Gly Arg Met Ala Leu Tyr Gln 435 440 445ctg gac cag cag tgc agg
cag atc ttt ggg ccg gat ttc cgc cac tgc 1392Leu Asp Gln Gln Cys Arg
Gln Ile Phe Gly Pro Asp Phe Arg His Cys 450 455 460ccc aac acc tct
gct cag gac gtc tgc gcc cag ctt tgg tgc cac act 1440Pro Asn Thr Ser
Ala Gln Asp Val Cys Ala Gln Leu Trp Cys His Thr465 470 475 480gat
ggg gct gag ccc ctg tgc cac acg aag aat ggc agc ctg ccc tgg 1488Asp
Gly Ala Glu Pro Leu Cys His Thr Lys Asn Gly Ser Leu Pro Trp 485 490
495gct gac ggc acg ccg tgc ggg cct ggg cac ctc tgc tca gaa ggc agc
1536Ala Asp Gly Thr Pro Cys Gly Pro Gly His Leu Cys Ser Glu Gly Ser
500 505 510tgt cta cct gag gag gaa gtg gag agg ccc aag ccc gtg gta
gat gga 1584Cys Leu Pro Glu Glu Glu Val Glu Arg Pro Lys Pro Val Val
Asp Gly 515 520 525ggc tgg gca ccg tgg gga ccc tgg gga gaa tgt tct
cgg acc tgt gga 1632Gly Trp Ala Pro Trp Gly Pro Trp Gly Glu Cys Ser
Arg Thr Cys Gly 530 535 540gga gga gta cag ttt tca cac cgt gag tgc
aag gac ccc gag cct cag 1680Gly Gly Val Gln Phe Ser His Arg Glu Cys
Lys Asp Pro Glu Pro Gln545 550 555 560aat gga gga aga tac tgc ctg
ggt cgg aga gcc aag tac cag tca tgc 1728Asn Gly Gly Arg Tyr Cys Leu
Gly Arg Arg Ala Lys Tyr Gln Ser Cys 565 570 575cac acg gag gaa tgc
ccc cct gac ggg aaa agc ttc agg gag cag cag 1776His Thr Glu Glu Cys
Pro Pro Asp Gly Lys Ser Phe Arg Glu Gln Gln 580 585 590tgt gag aag
tat aat gcc tac aat tac act gac atg gac ggg aat ctc 1824Cys Glu Lys
Tyr Asn Ala Tyr Asn Tyr Thr Asp Met Asp Gly Asn Leu 595 600 605ctg
cag tgg gtc ccc aag tat gct ggg gtg tcc ccc cgg gac cgc tgc 1872Leu
Gln Trp Val Pro Lys Tyr Ala Gly Val Ser Pro Arg Asp Arg Cys 610 615
620aag ttg ttc tgc cga gcc cgg ggg agg agc gag ttc aaa gtg ttc gag
1920Lys Leu Phe Cys Arg Ala Arg Gly Arg Ser Glu Phe Lys Val Phe
Glu625 630 635 640gcc aag gtg att gat ggc acc ctg tgt ggg cca gaa
aca ctg gcc atc 1968Ala Lys Val Ile Asp Gly Thr Leu Cys Gly Pro Glu
Thr Leu Ala Ile 645 650 655tgt gtc cgt ggc cag tgt gtc aag gcc ggc
tgt gac cat gtg gtg gac 2016Cys Val Arg Gly Gln Cys Val Lys Ala Gly
Cys Asp His Val Val Asp 660 665 670tcg cct cgg aag ctg gac aaa tgc
ggg gtg tgt ggg ggc aaa ggc aac 2064Ser Pro Arg Lys Leu Asp Lys Cys
Gly Val Cys Gly Gly Lys Gly Asn 675 680 685tcc tgc agg aag gtc tcc
ggg tcc ctc acc ccc acc aat tat ggc tac 2112Ser Cys Arg Lys Val Ser
Gly Ser Leu Thr Pro Thr Asn Tyr Gly Tyr 690 695 700aat gac att gtc
acc atc cca gct ggt gcc act aat att gac gtg aag 2160Asn Asp Ile Val
Thr Ile Pro Ala Gly Ala Thr Asn Ile Asp Val Lys705 710 715 720cag
cgg agc cac ccg ggt gtg cag aac gat ggg aac tac ctg gcg ctg 2208Gln
Arg Ser His Pro Gly Val Gln Asn Asp Gly Asn Tyr Leu Ala Leu 725 730
735aag acg gct gat ggg cag tac ctg ctc aac ggc aac ctg gcc atc tct
2256Lys Thr Ala Asp Gly Gln Tyr Leu Leu Asn Gly Asn Leu Ala Ile Ser
740 745 750gcc ata gag cag gac atc ttg gtg aag ggg acc atc ctg aag
tac agc 2304Ala Ile Glu Gln Asp Ile Leu Val Lys Gly Thr Ile Leu Lys
Tyr Ser 755 760 765ggc tcc atc gcc acc ctg gag cgc ctg cag agc ttc
cgg ccc ttg cca 2352Gly Ser Ile Ala Thr Leu Glu Arg Leu Gln Ser Phe
Arg Pro Leu Pro 770 775 780gag cct ctg aca gtg cag ctc ctg aca gtc
cct ggc gag gtc ttc ccc 2400Glu Pro Leu Thr Val Gln Leu Leu Thr Val
Pro Gly Glu Val Phe Pro785 790 795 800cca aaa gtc aaa tac acc ttc
ttt gtt cct aat gac gtg gac ttt agc 2448Pro Lys Val Lys Tyr Thr Phe
Phe Val Pro Asn Asp Val Asp Phe Ser 805 810 815atg cag agc agc aaa
gag aga gca acc acc aac atc atc cag ccg ctg 2496Met Gln Ser Ser Lys
Glu Arg Ala Thr Thr Asn Ile Ile Gln Pro Leu 820 825 830ctc cac gca
cag tgg gtg ctg ggg gac tgg tct gag tgc tct agc acc 2544Leu His Ala
Gln Trp Val Leu Gly Asp Trp Ser Glu Cys Ser Ser Thr 835 840 845tgc
ggg gcc ggc tgg cag agg cga act gta gag tgc agg gac ccc tcc 2592Cys
Gly Ala Gly Trp Gln Arg Arg Thr Val Glu Cys Arg Asp Pro Ser 850 855
860ggc cag gcc tct gcc acc tgc aac aag gct ctg aaa ccc gag gat gcc
2640Gly Gln Ala Ser Ala Thr Cys Asn Lys Ala Leu Lys Pro Glu Asp
Ala865 870 875 880aag ccc tgc gaa agc cag ctg tgc ccc ctg
tgattcaggg gggcaggggc 2690Lys Pro Cys Glu Ser Gln Leu Cys Pro Leu
885 890cagtcttgtg ctcctggaca tgcggtactg aggtgcagac aaggtctcca
ctgtggtgac 2750tgggtccctt ggccatatca aggcagcacg gcccacccag
gcctcccatt gccgcaaccc 2810ctccagtact gcacaaattc ctaaggggga
agagaaaagg tatggggcgg caaaacctat 2870catcaactgt ccawtgnaat
ggaacttgct cgggttcaat taaaggcata agttaaagta 2930aattcattat
gatcaacaga cctcacntca tctgttgcan gatacaacta ntaaaaaaaa
2990aaaaaaaaaa aaaaaaaa 30084890PRTHomo sapiens 4Met Phe Pro Ala
Pro Ala Ala Pro Arg Trp Leu Pro Phe Leu Leu Leu1 5 10 15Leu Leu Leu
Leu Leu Leu Pro Leu Ala Arg Gly Ala Pro Ala Arg Pro 20 25 30Ala Ala
Gly Gly Gln Ala Ser Glu Leu Val Val Pro Thr Arg Leu Pro 35 40 45Gly
Ser Ala Gly Glu Leu Ala Leu His Leu Ser Ala Phe Gly Lys Gly 50 55
60Phe Val Leu Arg Leu Ala Pro Asp Asp Ser Phe Leu Ala Pro Glu Phe65
70 75 80Lys Ile Glu Arg Leu Gly Gly Ser Gly Arg Ala Thr Gly Gly Glu
Arg 85 90 95Gly Leu Arg Gly Cys Phe Phe Ser Gly Thr Val Asn Gly Glu
Pro Glu 100 105 110Ser Leu Ala Ala Val Ser Leu Cys Arg Gly Leu Ser
Gly Ser Phe Leu 115 120 125Leu Asp Gly Glu Glu Phe Thr Ile Gln Pro
Gln Gly Ala Gly Gly Ser 130 135 140Leu Ala Gln Pro His Arg Leu Gln
Arg Trp Gly Pro Ala Gly Ala Arg145 150 155 160Pro Leu Pro Arg Gly
Pro Glu Trp Glu Val Glu Thr Gly Glu Gly Gln 165 170 175Arg Gln Glu
Arg Gly Asp His Gln Glu Asp Ser Glu Glu Glu Ser Gln 180 185 190Glu
Glu Glu Ala Glu Gly Ala Ser Glu Pro Pro Pro Pro Leu Gly Ala 195 200
205Thr Ser Arg Thr Lys Arg Phe Val Ser Glu Ala Arg Phe Val Glu Thr
210 215 220Leu Leu Val Ala Asp Ala Ser Met Ala Ala Phe Tyr Gly Ala
Asp Leu225 230 235 240Gln Asn His Ile Leu Thr Leu Met Ser Val Ala
Ala Arg Ile Tyr Lys 245 250 255His Pro Ser Ile Lys Asn Ser Ile Asn
Leu Met Val Val Lys Val Leu 260 265 270Ile Val Glu Asp Glu Lys Trp
Gly Pro Glu Val Ser Asp Asn Gly Gly 275 280 285Leu Thr Leu Arg Asn
Phe Cys Asn Trp Gln Arg Arg Phe Asn Gln Pro 290 295 300Ser Asp Arg
His Pro Glu His Tyr Asp Thr Ala Ile Leu Leu Thr Arg305 310 315
320Gln Asn Phe Cys Gly Gln Glu Gly Leu Cys Asp Thr Leu Gly Val Ala
325 330 335Asp Ile Gly Thr Ile Cys Asp Pro Asn Lys Ser Cys Ser Val
Ile Glu 340 345 350Asp Glu Gly Leu Gln Ala Ala His Thr Leu Ala His
Glu Leu Gly His 355 360 365Val Leu Ser Met Pro His Asp Asp Ser Lys
Pro Cys Thr Arg Leu Phe 370 375 380Gly Pro Met Gly Lys His His Val
Met Ala Pro Leu Phe Val His Leu385 390 395 400Asn Gln Thr Leu Pro
Trp Ser Pro Cys Ser Ala Met Tyr Leu Thr Glu 405 410 415Leu Leu Asp
Gly Gly His Gly Asp Cys Leu Leu Asp Ala Pro Gly Ala 420 425 430Ala
Leu Pro Leu Pro Thr Gly Leu Pro Gly Arg Met Ala Leu Tyr Gln 435 440
445Leu Asp Gln Gln Cys Arg Gln Ile Phe Gly Pro Asp Phe Arg His Cys
450 455 460Pro Asn Thr Ser Ala Gln Asp Val Cys Ala Gln Leu Trp Cys
His Thr465 470 475 480Asp Gly Ala Glu Pro Leu Cys His Thr Lys Asn
Gly Ser Leu Pro Trp 485 490 495Ala Asp Gly Thr Pro Cys Gly Pro Gly
His Leu Cys Ser Glu Gly Ser 500 505 510Cys Leu Pro Glu Glu Glu Val
Glu Arg Pro Lys Pro Val Val Asp Gly 515 520 525Gly Trp Ala Pro Trp
Gly Pro Trp Gly Glu Cys Ser Arg Thr Cys Gly 530 535 540Gly Gly Val
Gln Phe Ser His Arg Glu Cys Lys Asp Pro Glu Pro Gln545 550 555
560Asn Gly Gly Arg Tyr Cys Leu Gly Arg Arg Ala Lys Tyr Gln Ser Cys
565 570 575His Thr Glu Glu Cys Pro Pro Asp Gly Lys Ser Phe Arg Glu
Gln Gln 580 585 590Cys Glu Lys Tyr Asn Ala Tyr Asn Tyr Thr Asp Met
Asp Gly Asn Leu 595 600 605Leu Gln Trp Val Pro Lys Tyr Ala Gly Val
Ser Pro Arg Asp Arg Cys 610 615 620Lys Leu Phe Cys Arg Ala Arg Gly
Arg Ser Glu Phe Lys Val Phe Glu625 630 635 640Ala Lys Val Ile Asp
Gly Thr Leu Cys Gly Pro Glu Thr Leu Ala Ile 645 650 655Cys Val Arg
Gly Gln Cys Val Lys Ala Gly Cys Asp His Val Val Asp 660 665 670Ser
Pro Arg Lys Leu Asp Lys Cys Gly Val Cys Gly Gly Lys Gly Asn 675 680
685Ser Cys Arg Lys Val Ser Gly Ser Leu Thr Pro Thr Asn Tyr Gly Tyr
690 695 700Asn Asp Ile Val Thr Ile Pro Ala Gly Ala Thr Asn Ile Asp
Val Lys705 710 715 720Gln Arg Ser His Pro Gly Val Gln Asn Asp Gly
Asn Tyr Leu Ala Leu 725 730 735Lys Thr Ala Asp Gly Gln Tyr Leu Leu
Asn Gly Asn Leu Ala Ile Ser 740 745 750Ala Ile Glu Gln Asp Ile Leu
Val Lys Gly Thr Ile Leu Lys Tyr Ser 755 760 765Gly Ser Ile Ala Thr
Leu Glu Arg Leu Gln Ser Phe Arg Pro Leu Pro 770 775 780Glu Pro Leu
Thr Val Gln Leu Leu Thr Val Pro Gly Glu Val Phe Pro785 790 795
800Pro Lys Val Lys Tyr Thr Phe Phe Val Pro Asn Asp Val Asp Phe Ser
805 810 815Met Gln Ser Ser Lys Glu Arg Ala Thr Thr Asn Ile Ile Gln
Pro Leu 820 825 830Leu His Ala Gln Trp Val Leu Gly Asp Trp Ser Glu
Cys Ser Ser Thr 835 840 845Cys Gly Ala Gly Trp Gln Arg Arg Thr Val
Glu Cys Arg Asp Pro Ser 850 855 860Gly Gln Ala Ser Ala Thr Cys Asn
Lys Ala Leu Lys Pro Glu Asp Ala865 870 875 880Lys Pro Cys Glu Ser
Gln Leu Cys Pro Leu
885 89051203PRTBos taurus 5Met Asp Pro Pro Ala Gly Ala Ala Gly Arg
Leu Leu Cys Pro Ala Leu1 5 10 15Leu Leu Leu Leu Leu Leu Pro Leu Pro
Ala Asp Ala Arg Leu Ala Ala 20 25 30Ala Ala Ala Asp Pro Pro Gly Gly
Pro Gln Gly His Gly Ala Glu Arg 35 40 45Ile Leu Ala Val Pro Val Arg
Thr Asp Ala Gln Gly Arg Leu Val Ser 50 55 60His Val Val Ser Ala Ala
Thr Ala Pro Ala Gly Val Arg Thr Arg Arg65 70 75 80Ala Ala Pro Ala
Gln Ile Pro Gly Leu Ser Gly Gly Ser Glu Glu Asp 85 90 95Pro Gly Gly
Arg Leu Phe Tyr Asn Val Thr Val Phe Gly Arg Asp Leu 100 105 110His
Leu Arg Leu Arg Pro Asn Ala Arg Leu Val Ala Pro Gly Ala Thr 115 120
125Val Glu Trp Gln Gly Glu Ser Gly Ala Thr Arg Val Glu Pro Leu Leu
130 135 140Gly Thr Cys Leu Tyr Val Gly Asp Val Ala Gly Leu Ala Glu
Ser Ser145 150 155 160Ser Val Ala Leu Ser Asn Cys Asp Gly Leu Ala
Gly Leu Ile Arg Met 165 170 175Glu Glu Glu Glu Phe Phe Ile Glu Pro
Leu Glu Lys Gly Leu Ala Ala 180 185 190Lys Glu Ala Glu Gln Gly Arg
Val His Val Val Tyr His Arg Pro Thr 195 200 205Thr Ser Arg Pro Pro
Pro Leu Gly Gln Ala Leu Asp Thr Gly Ile Ser 210 215 220Ala Asp Ser
Leu Asp Ser Leu Ser Arg Ala Leu Gly Val Leu Glu Glu225 230 235
240Arg Val Asn Ser Ser Arg Arg Arg Met Arg Arg His Ala Ala Asp Asp
245 250 255Asp Tyr Asn Ile Glu Val Leu Leu Gly Val Asp Asp Ser Val
Val Gln 260 265 270Phe His Gly Thr Glu His Val Gln Lys Tyr Leu Leu
Thr Leu Met Asn 275 280 285Ile Val Asn Glu Ile Tyr His Asp Glu Ser
Leu Gly Ala His Ile Asn 290 295 300Val Val Leu Val Arg Ile Ile Leu
Leu Ser Tyr Gly Lys Ser Met Ser305 310 315 320Leu Ile Glu Ile Gly
Asn Pro Ser Gln Ser Leu Glu Asn Val Cys Arg 325 330 335Trp Ala Tyr
Leu Gln Gln Lys Pro Asp Thr Asp His Asp Glu Tyr His 340 345 350Asp
His Ala Ile Phe Leu Thr Arg Gln Asp Phe Gly Pro Ser Gly Met 355 360
365Gln Gly Tyr Ala Pro Val Thr Gly Met Cys His Pro Val Arg Ser Cys
370 375 380Thr Leu Asn His Glu Asp Gly Phe Ser Ser Ala Phe Val Val
Ala His385 390 395 400Glu Thr Gly His Val Leu Gly Met Glu His Asp
Gly Gln Gly Asn Arg 405 410 415Cys Gly Asp Glu Val Arg Leu Gly Ser
Ile Met Ala Pro Leu Val Gln 420 425 430Ala Ala Phe His Arg Phe His
Trp Ser Arg Cys Ser Gln Gln Glu Leu 435 440 445Ser Arg Tyr Leu His
Ser Tyr Asp Cys Leu Arg Asp Asp Pro Phe Thr 450 455 460His Asp Trp
Pro Ala Leu Pro Gln Leu Pro Gly Leu His Tyr Ser Met465 470 475
480Asn Glu Gln Cys Arg Phe Asp Phe Gly Leu Gly Tyr Met Met Cys Thr
485 490 495Ala Phe Arg Thr Phe Asp Pro Cys Lys Gln Leu Trp Cys Ser
His Pro 500 505 510Asp Asn Pro Tyr Phe Cys Lys Thr Lys Lys Gly Pro
Pro Leu Asp Gly 515 520 525Thr Met Cys Ala Pro Gly Lys His Cys Phe
Lys Gly His Cys Ile Trp 530 535 540Leu Thr Pro Asp Ile Leu Lys Arg
Asp Gly Asn Trp Gly Ala Trp Ser545 550 555 560Pro Phe Gly Ser Cys
Ser Arg Thr Cys Gly Thr Gly Val Lys Phe Arg 565 570 575Thr Arg Gln
Cys Asp Asn Pro His Pro Ala Asn Gly Gly Arg Thr Cys 580 585 590Ser
Gly Leu Ala Tyr Asp Phe Gln Leu Cys Asn Ser Gln Asp Cys Pro 595 600
605Asp Ala Leu Ala Asp Phe Arg Glu Glu Gln Cys Arg Gln Trp Asp Leu
610 615 620Tyr Phe Glu His Gly Asp Ala Gln His His Trp Leu Pro His
Glu His625 630 635 640Arg Asp Ala Lys Glu Arg Cys His Leu Tyr Cys
Glu Ser Lys Glu Thr 645 650 655Gly Glu Val Val Ser Met Lys Arg Met
Val His Asp Gly Thr Arg Cys 660 665 670Ser Tyr Lys Asp Ala Phe Ser
Leu Cys Val Arg Gly Asp Cys Arg Lys 675 680 685Val Gly Cys Asp Gly
Val Ile Gly Ser Ser Lys Gln Glu Asp Lys Cys 690 695 700Gly Val Cys
Gly Gly Asp Asn Ser His Cys Lys Val Val Lys Gly Thr705 710 715
720Phe Ser Arg Ser Pro Lys Lys Leu Gly Tyr Ile Lys Met Phe Glu Ile
725 730 735Pro Ala Gly Ala Arg His Leu Leu Ile Gln Glu Ala Asp Thr
Thr Ser 740 745 750His His Leu Ala Val Lys Asn Leu Glu Thr Gly Lys
Phe Ile Leu Asn 755 760 765Glu Glu Asn Asp Val Asp Pro Asn Ser Lys
Thr Phe Ile Ala Met Gly 770 775 780Val Glu Trp Glu Tyr Arg Asp Glu
Asp Gly Arg Glu Thr Leu Gln Thr785 790 795 800Met Gly Pro Leu His
Gly Thr Ile Thr Val Leu Val Ile Pro Glu Gly 805 810 815Asp Ala Arg
Ile Ser Leu Thr Tyr Lys Tyr Met Ile His Glu Asp Ser 820 825 830Leu
Asn Val Asp Asp Asn Asn Val Leu Glu Asp Asp Ser Val Gly Tyr 835 840
845Glu Trp Ala Leu Lys Lys Trp Ser Pro Cys Ser Lys Pro Cys Gly Gly
850 855 860Gly Ser Gln Phe Thr Lys Tyr Gly Cys Arg Arg Arg Leu Asp
His Lys865 870 875 880Met Val His Arg Gly Phe Cys Asp Ser Val Ser
Lys Pro Lys Ala Ile 885 890 895Arg Arg Thr Cys Asn Pro Gln Glu Cys
Ser Gln Pro Val Trp Val Thr 900 905 910Gly Glu Trp Glu Pro Cys Ser
Arg Ser Cys Gly Arg Thr Gly Met Gln 915 920 925Val Arg Ser Val Arg
Cys Val Gln Pro Leu His Asn Asn Thr Thr Arg 930 935 940Ser Val His
Thr Lys His Cys Asn Asp Ala Arg Pro Glu Gly Arg Arg945 950 955
960Ala Cys Asn Arg Glu Leu Cys Pro Gly Arg Trp Arg Ala Gly Ser Trp
965 970 975Ser Gln Cys Ser Val Thr Cys Gly Asn Gly Thr Gln Glu Arg
Pro Val 980 985 990Leu Cys Arg Thr Ala Asp Asp Ser Phe Gly Val Cys
Arg Glu Glu Arg 995 1000 1005Pro Glu Thr Ala Arg Ile Cys Arg Leu
Gly Pro Cys Pro Arg Asn Thr 1010 1015 1020Ser Asp Pro Ser Lys Lys
Ser Tyr Val Val Gln Trp Leu Ser Arg Pro1025 1030 1035 1040Asp Pro
Asn Ser Pro Val Gln Glu Thr Ser Ser Lys Gly Arg Cys Gln 1045 1050
1055Gly Asp Lys Ser Val Phe Cys Arg Met Glu Val Leu Ser Arg Tyr Cys
1060 1065 1070Ser Ile Pro Gly Tyr Asn Lys Leu Cys Cys Lys Ser Cys
Asn Pro His 1075 1080 1085Asp Asn Leu Thr Asp Val Asp Asp Arg Ala
Glu Pro Pro Ser Gly Lys 1090 1095 1100His Asn Asp Ile Glu Glu Leu
Met Pro Thr Leu Ser Val Pro Thr Leu1105 1110 1115 1120Val Met Glu
Val Gln Pro Pro Pro Gly Ile Pro Leu Glu Val Pro Leu 1125 1130
1135Asn Thr Ser Ser Thr Asn Ala Thr Glu Asp His Pro Glu Thr Asn Ala
1140 1145 1150Val Asp Val Pro Tyr Lys Ile Pro Gly Leu Glu Asp Glu
Val Gln Pro 1155 1160 1165Pro Asn Leu Ile Pro Arg Arg Pro Ser Pro
Tyr Glu Lys Thr Arg Asn 1170 1175 1180Gln Arg Ile Gln Glu Leu Ile
Asp Glu Met Arg Lys Lys Glu Met Leu1185 1190 1195 1200Gly Lys
Phe650PRTHomo sapiens 6Asp Asp Gly Trp Ser Pro Trp Ser Glu Trp Thr
Ser Cys Ser Thr Ser1 5 10 15Cys Gly Asn Gly Ile Gln Gln Arg Gly Arg
Ser Cys Asp Ser Leu Asn 20 25 30Asn Arg Cys Glu Gly Ser Ser Val Gln
Thr Arg Thr Cys His Ile Gln 35 40 45Glu Cys 50757PRTHomo sapiens
7Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val Thr1 5
10 15Cys Gly Asp Gly Val Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro
Ser 20 25 30Pro Gln Met Asn Gly Lys Pro Cys Glu Gly Glu Ala Arg Glu
Thr Lys 35 40 45Ala Cys Lys Lys Asp Ala Cys Pro Ile 50 55857PRTHomo
sapiens 8Asn Gly Gly Trp Gly Pro Trp Ser Pro Trp Asp Ile Cys Ser
Val Thr1 5 10 15Cys Gly Gly Gly Val Gln Lys Arg Ser Arg Leu Cys Asn
Asn Pro Thr 20 25 30Pro Gln Phe Gly Gly Lys Asp Cys Val Gly Asp Val
Thr Glu Asn Gln 35 40 45Ile Cys Asn Lys Gln Asp Cys Pro Ile 50
55950PRTHomo sapiens 9Glu Glu Gly Trp Ser Pro Trp Ala Glu Trp Thr
Gln Cys Ser Val Thr1 5 10 15Cys Gly Ser Gly Thr Gln Gln Arg Gly Arg
Ser Cys Asp Val Thr Ser 20 25 30Asn Thr Cys Leu Gly Pro Ser Ile Gln
Thr Arg Ala Cys Ser Leu Ser 35 40 45Lys Cys 501057PRTHomo sapiens
10Asp Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys Ser Val Thr1
5 10 15Cys Gly Val Gly Asn Ile Thr Arg Ile Arg Leu Cys Asn Ser Pro
Val 20 25 30Pro Gln Met Gly Gly Lys Asn Cys Lys Gly Ser Gly Arg Glu
Thr Lys 35 40 45Ala Cys Gln Gly Ala Pro Cys Pro Ile 50
551156PRTHomo sapiens 11Asp Gly Arg Trp Ser Pro Trp Ser Pro Trp Ser
Ala Cys Thr Val Thr1 5 10 15Cys Ala Gly Gly Ile Arg Glu Arg Thr Arg
Val Cys Asn Ser Pro Glu 20 25 30Pro Gln Tyr Gly Gly Lys Ala Cys Val
Gly Asp Val Gln Glu Arg Gln 35 40 45Met Cys Asn Lys Arg Ser Cys Pro
50 55123974DNAHomo sapiens 12ggtacctaag tgagtagggc gtccgatcga
cggacgcctt ttttttgaat tcgtaatcat 60ggtcatagct gtttcctgtg tgaaattgtt
atccgctcac aattccacac aacatacgag 120ccggaagcat aaagtgtaaa
gcctggggtg cctaatgagt gagctaactc acattaattg 180cgttgcgctc
actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
240tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
tcctcgctca 300ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
atcagctcac tcaaaggcgg 360taatacggtt atccacagaa tcaggggata
acgcaggaaa gaacatgtga gcaaaaggcc 420agcaaaaggc caggaaccgt
aaaaaggccg cgttgctggc gtttttccat aggctccgcc 480cccctgacga
gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
540tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
gttccgaccc 600tgccgcttac cggatacctg tccgcctttc tcccttcggg
aagcgtggcg ctttctcata 660gctcacgctg taggtatctc agttcggtgt
aggtcgttcg ctccaagctg ggctgtgtgc 720acgaaccccc cgttcagccc
gaccgctgcg ccttatccgg taactatcgt cttgagtcca 780acccggtaag
acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
840cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
ggctacacta 900gaagaacagt atttggtatc tgcgctctgc tgaagccagt
taccttcgga aaaagagttg 960gtagctcttg atccggcaaa caaaccaccg
ctggtagcgg tggttttttt gtttgcaagc 1020agcagattac gcgcagaaaa
aaaggatctc aagaagatcc tttgatcttt tctacggggt 1080ctgacgctca
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcgtcga
1140caattcgcgc gcgaaggcga agcggcatgc atttacgttg acaccatcga
atggtgcaaa 1200acctttcgcg gtatggcatg atagcgcccg gaagagagtc
aattcagggt ggtgaatgtg 1260aaaccagtaa cgttatacga tgtcgcagag
tatgccggtg tctcttatca gaccgtttcc 1320cgcgtggtga accaggccag
ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg 1380atggcggagc
tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
1440ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
aattgtcgcg 1500gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
tggtgtcgat ggtagaacga 1560agcggcgtcg aagcctgtaa agcggcggtg
cacaatcttc tcgcgcaacg cgtcagtggg 1620ctgatcatta actatccgct
ggatgaccag gatgccattg ctgtggaagc tgcctgcact 1680aatgttccgg
cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
1740tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg
tcaccagcaa 1800atcgcgctgt tagcgggccc attaagttct gtctcggcgc
gtctgcgtct ggctggctgg 1860cataaatatc tcactcgcaa tcaaattcag
ccgatagcgg aacgggaagg cgactggagt 1920gccatgtccg gttttcaaca
aaccatgcaa atgctgaatg agggcatcgt tcccactgcg 1980atgctggttg
ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
2040ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
cagctcatgt 2100tatatcccgc cgttaaccac catcaaacag gattttcgcc
tgctggggca aaccagcgtg 2160gaccgcttgc tgcaactctc tcagggccag
gcggtgaagg gcaatcagct gttgcccgtc 2220tcactggtga aaagaaaaac
caccctggcg cccaatacgc aaaccgcctc tccccgcgcg 2280ttggccgatt
cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
2340gcgcaacgca attaatgtaa gttagcgcga attgtcgacc aaagcggcca
tcgtgcctcc 2400ccactcctgc agttcggggg catggatgcg cggatagccg
ctgctggttt cctggatgcc 2460gacggatttg cactgccggt agaactccgc
gaggtcgtcc agcctcaggc agcagctgaa 2520ccaactcgcg aggggatcga
gcccggggtg ggcgaagaac tccagcatga gatccccgcg 2580ctggaggatc
atccagccgg cgtcccggaa aacgattccg aagcccaacc tttcatagaa
2640ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt
cggtcatttc 2700gaaccccaga gtcccgctca gaagaactcg tcaagaaggc
gatagaaggc gatgcgctgc 2760gaatcgggag cggcgatacc gtaaagcacg
aggaagcggt cagcccattc gccgccaagc 2820tcttcagcaa tatcacgggt
agccaacgct atgtcctgat agcggtccgc cacacccagc 2880cggccacagt
cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag
2940gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt
gagcctggcg 3000aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca
gatcatcctg atcgacaaga 3060ccggcttcca tccgagtacg tgctcgctcg
atgcgatgtt tcgcttggtg gtcgaatggg 3120caggtagccg gatcaagcgt
atgcagccgc cgcattgcat cagccatgat ggatactttc 3180tcggcaggag
caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc
3240cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
gcccgtcgtg 3300gccagccacg atagccgcgc tgcctcgtcc tgcagttcat
tcagggcacc ggacaggtcg 3360gtcttgacaa aaagaaccgg gcgcccctgc
gctgacagcc ggaacacggc ggcatcagag 3420cagccgattg tctgttgtgc
ccagtcatag ccgaatagcc tctccaccca agcggccgga 3480gaacctgcgt
gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga
3540tcagatcttg atcccctgcg ccatcagatc cttggcggca agaaagccat
ccagtttact 3600ttgcagggct tcccaacctt accagagggc gccccagctg
gcaattccgg ttcgcttgct 3660gtccataaaa ccgcccagtc tagctatcgc
catgtaagcc cactgcaagc tacctgcttt 3720ctctttgcgc ttgcgttttc
ccttgtccag atagcccagt agctgacatt catccggggt 3780cagcaccgtt
tctgcggact ggctttctac gtgttccgct tcctttagca gcccttgcgc
3840cctgagtgct tgcggcagcg tgaagcttaa aaaactgcaa aaaatagttt
gacttgtgag 3900cggataacaa ttaagatgta cccaattgtg agcggataac
aatttcacac attaaagagg 3960agaaattaca tatg 397413112DNAHomo sapiens
13aagcttaaaa aactgcaaaa aatagtttga cttgtgagcg gataacaatt aagatgtacc
60caattgtgag cggataacaa tttcacacat taaagaggag aaattacata tg
11214542DNAMus musculusMisc_feature(3)N is any nucleic acid
14gtncgaattt cggcacgaga nnttagacgc cttttcatgg aagctgggga atgtgggggc
60cttggggaga ctgttcgaga acgtgcggtg gaggagtcca gtacacgatg agggaatgtg
120acaacccagt cccaaagaat ggagggaagt actgtgaagg caaacgagtg
cgctacagat 180cctgtaacct tgaggactgt ccagacaata atggaaaaac
ctttagagag gaacaatgtg 240aagcacacaa cgagttttca aaagcttcct
ttgggagtgg gcctgcggtg gaatggattc 300ccaagtacgc tggcgtctca
ccaaaggaca ggtgcaagtt catgttgcca agccaaaggc 360nttggctant
tctttcgttt tgcagcccaa ggttgttagg tgggtantcc atgttaggcc
420cagattncac ctttgtctgt gtgcaaggac agtgtgttaa aagttggttg
tgatccgcnt 480cntagattcc aaaaggagtt ttgttaatgt ggtgttttcn
gggggaatgg tctantttta 540aa 54215320DNAUnknownDescription of
Unknown OrganismSequence from Applicant's cDNA clone HPLBM11R
15cagagaacat tcgccccact cttcaatgac ccatgctgaa aaagtgggga tagcattgaa
60agattccttc ttcttcttta cgaagtaggt gtatttaatt ttaggtcgaa gggcattgcc
120cacagtaaga acctggatgg tcaagggctc tttgagaggg ctaaagctgc
gaattctttc 180caatgccgca gaggagccgc tgtacctcaa gacaacacct
ttgtacataa tgtcttgctc 240taaggtggac aaagtgtagt caccattaag
aatatatgtg ccatcagcag ctttgatggc 300aagaaagctg cccttgttcc
32016316DNAEimeria tenella 16aatgccgaga cattaatgga cagcctgctt
ccgagtgtgc aaaggaagtg aagccagcca 60gcaccagacc ttgtgcagac catccctgcc
cccagtggca gctgggggag tggtcatcat 120gttctaagac ctgtgggaag
ggttacaaaa aaagaagctt gaagtgtctg tcccatgatg 180gaggggtgtt
atctcatgag agctgtgatc ctttaaagaa acctaaacat ttcatagact
240tttgcacaat ggcagaatgc agttaagtgg tttaagtggt gttagctttg
agggcaaggc 300aaagtgagga
agggct 31617383DNACaenorhabditis elegansMisc_feature(160)N is any
nucleic acid 17gtcgacccac gcgtccggat ggtactccat gtagcccaga
ttccacctct gtctgtgtgc 60aaggacagtg tgtaaaagct ggttgtgatc gcatcataga
ctccaaaaag aagtttgata 120aatgtggtgt ttgcggggga aatggatcta
cttgtaaaan aatatcagga tcagttacta 180gtgcaaaacc tgggatatca
tgatatcatc acaattccaa ctgggagcca ccaacatcga 240agtgaaacag
cggaaccaga ggggatccag ggaacaatgg gcagctttct tgccatcaaa
300gctgctggat ggcacatata ttcttnaatg gtgactacac tttgtccacc
ttagaganag 360acattntgtg acaaagngnt tgt 38318404DNACrotalus
atroxMisc_feature(21)N is any nucleic acid 18cccacgcgtc cgcccacggt
nccgggactt gtgtgggtcc cagacatgtg atactcttgg 60gatggctgat gttggaactg
tgtgtgatcc gagcagaagc tgctccgtca tagaagatga 120tggtttacaa
gctgccttca ccacagccca tgaattaggc cacgtgttta acatgccaca
180tgatggatgc aaagcagtgt gccagcctta aatggtgtga accagggatt
cccacatgat 240ggcgtcaatg ctttccaacc tgggaccaca gccagccttg
ggtcctcctt gcagtggcct 300nacatggatt gacatcattt ctgggatgaa
tggtncatgg gggaatgttt tgattggaca 360agccttcaga atnccctnac
annttcccag gggttctccc tggg 40419152DNAHomo
sapiensMisc_feature(105)N is any nucleic acid 19atcgtagaag
atgaaaaatg gggcccagag gtgtccgaca atggggggct tacactgcgt 60aacttctgca
actggcagcg gcgtttcaac cagcccagcg accgncaccc agagcactac
120gncacggcca tcctnctcac cagacagaac tt
152204180DNAUnknownDescription of Unknown OrganismGenBank Clone
Accession Number D67076 20gcagctccga gctaggtgct atcgcaaggc
cagagcgcac agcccggcgg agagagcaga 60tccttgctca gatcgagtca aatcgggcca
aggcggagga cgaagagtcc aggctcctat 120tctggacttg ttccccagct
ccgggggcgc ttctaggtcc tgcagcagcc agcagtgcgg 180agccaccaac
tcggtgctgg aatgaaaaaa ttcccgcgcg ccagtgcaga atctttctaa
240gtgacccgga gcttcgggtg ctagctctgc acgaactttc ccatcaaagt
gatcgtgaat 300tttaagcatc aggagcaggc cagcgaagct ctacgcgtct
aaacgtctat ccagaccaag 360agttctctgc ggtgcagggt gcggtgccat
gcagccaaaa gtcccttttg gggtcacgca 420agcagaagcc ctgctccgac
atgggggacg tccagcgggc agcgagatct cggggctctc 480tgtccgcaca
catgctgttg ctgctcctcg cttccataac aatgctgcta tgtgcgcggg
540gcgcacacgg gcgccccacg gaggaagatg aggagctggt cctgccctcg
ctggagcgcg 600ccccgggcca cgattccacc accacacgcc ttcgtctgga
cgcctttggc cagcagctac 660atctgaagtt gcagccggac agcggtttct
tggcgcctgg cttcaccctg cagactgtgg 720ggcgcagtcc cgggtccgag
gcacaacatc tggaccccac cggggacctg gctcactgct 780tctactctgg
cacggtgaac ggtgatcccg gctctgccgc agccctcagc ctctgtgaag
840gtgtgcgtgg tgccttctac ctacaaggag aggagttctt cattcagcca
gcgcctggag 900tggccaccga gcgcctggcc cctgccgtgc ccgaggagga
gtcatccgca cggccgcagt 960tccacatcct gaggcgaagg cggcggggca
gtggcggcgc caagtgcggc gtcatggacg 1020acgagaccct gccaaccagc
gactcgcgac ccgagagcca gaacacccgg aaccagtggc 1080ctgtgcggga
ccccacgcct caggacgcgg gaaagccatc aggaccagga agcataagga
1140agaagcgatt tgtgtccagc ccccgttatg tggaaaccat gctcgtagct
gaccagtcca 1200tggccgactt ccacggcagc ggtctaaagc attaccttct
aaccctgttc tcggtggcag 1260ccaggtttta caagcatccc agcattagga
attcaattag cctggtggtg gtgaagatct 1320tggtcatata cgaggagcag
aagggaccag aagttacctc caatgcagct ctcacccttc 1380ggaatttctg
cagctggcag aaacaacaca acagccccag tgaccgggat ccagagcact
1440atgacactgc aattctgttc accagacagg atttatgtgg ctcccacacg
tgtgacactc 1500tcggaatggc agatgttgga accgtatgtg accccagcag
gagctgctca gtcatagaag 1560atgatggttt gcaagccgcc ttcaccacag
cccatgaatt gggccatgtg tttaacatgc 1620cgcacgatga tgctaagcac
tgtgccagct tgaatggtgt gagtggcgat tctcatctga 1680tggcctcgat
gctctccagc ttagaccata gccagccctg gtcaccttgc agtgcctaca
1740tggtcacgtc cttcctagat aatggacacg gggaatgttt gatggacaag
ccccagaatc 1800caatcaagct cccttctgat cttcccggta ccttgtacga
tgccaaccgc cagtgtcagt 1860ttacattcgg agaggaatcc aagcactgcc
ctgatgcagc cagcacatgt actaccctgt 1920ggtgcactgg cacctccggt
ggcttactgg tgtgccaaac aaaacacttc ccttgggcag 1980atggcaccag
ctgtggagaa gggaagtggt gtgtcagtgg caagtgcgtg aacaagacag
2040acatgaagca ttttgctact cctgttcatg gaagctgggg accatgggga
ccgtggggag 2100actgctcaag aacctgtggt ggtggagttc aatacacaat
gagagaatgt gacaacccag 2160tcccaaagaa cggagggaag tactgtgaag
gcaaacgagt ccgctacagg tcctgtaaca 2220tcgaggactg tccagacaat
aacggaaaaa cgttcagaga ggagcagtgc gaggcgcaca 2280atgagttttc
caaagcttcc tttgggaatg agcccactgt agagtggaca cccaagtacg
2340ccggcgtctc gccaaaggac aggtgcaagc tcacctgtga agccaaaggc
attggctact 2400ttttcgtctt acagcccaag gttgtagatg gcactccctg
tagtccagac tctacctctg 2460tctgtgtgca agggcagtgt gtgaaagctg
gctgtgatcg catcatagac tccaaaaaga 2520agtttgataa gtgtggcgtt
tgtggaggaa acggttccac atgcaagaag atgtcaggaa 2580tagtcactag
tacaagacct gggtatcatg acattgtcac aattcctgct ggagccacca
2640acattgaagt gaaacatcgg aatcaaaggg ggtccagaaa caatggcagc
tttctggcta 2700ttagagccgc tgatggtacc tatattctga atggaaactt
cactctgtcc acactagagc 2760aagacctcac ctacaaaggt actgtcttaa
ggtacagtgg ttcctcggct gcgctggaaa 2820gaatccgcag ctttagtcca
ctcaaagaac ccttaaccat ccaggttctt atggtaggcc 2880atgctctccg
acccaaaatt aaattcacct actttatgaa gaagaagaca gagtcattca
2940acgccattcc cacattttct gagtgggtga ttgaagagtg gggggagtgc
tccaagacat 3000gcggctcagg ttggcagaga agagtagtgc agtgcagaga
cattaacgga caccctgctt 3060ccgaatgtgc aaaggaagtg aagccagcca
gtaccagacc ttgtgcagac cttccttgcc 3120cacactggca ggtgggggat
tggtcaccat gttccaaaac ttgcgggaag ggttacaaga 3180agagaacctt
gaaatgtgtg tcccacgatg ggggcgtgtt atcaaatgag agctgtgatc
3240ctttgaagaa gccaaagcat tacattgact tttgcacact gacacagtgc
agttaagagg 3300cgttagagga caaggtagcg tggggagggg ctgatacact
gagtgcaaga gtactggagg 3360gatccagtga gtcaaaccag taagcagtga
ggtgtggcaa ggaggtgtgt gtaggggata 3420catagcaaag gaggtagatc
aggacactac cctgccagtt acattctgat aaggtagtta 3480atgaggcaca
gtagcatctg aaagaccata cagagcacta aggagcccca aagcactatt
3540agtatctctt ttcttatatc tatcgcccaa ataattttca gagtctggca
gaagccctgt 3600tgcactgtac taactagata cttcttatca caaagattgg
gaaaggcaaa gcagaaagat 3660ggtaagactg ggtttcaaac aaggcttggt
ttcaatcact ggaggcaagg aggaggggac 3720aaacaagatc attattcgaa
gtcgctggtt gctgtggttt tacggaaggt tgatgcatca 3780ttcctatcaa
cagtgaaaag ttcagcttgt tcaacgtgac agaaaggctc atctccgtga
3840aagagctcct gatttcttct tacaccatct cagttcttaa ctatagttca
tgttgaggta 3900gaaacaattc atctatttat aaaatgtaca ttggaaaaaa
aaagtgaagt ttatgaggta 3960cacataaaaa ctgaaggaaa caatgagcaa
catgcctcct gctttgcttc ctcctgaggt 4020aaacctgcct ggggattgag
gttgtttaag attatccatg gctcacaaga ggcagtaaaa 4080taatacatgt
tgtgccagag ttagaatggg gtatagagat cagggtccca tgagatgggg
4140aacatggtga tcactcatct cacatgggag gctgctgcag
4180219248DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number AB001735 21gcagctccga gctaggtgct atcgcaaggc
cagagcgcac agcccggcgg agagagcaga 60tccttgctca gatcgagtca aatcggggcc
aaggcggagg acgaagagtc caggctccta 120ttctggactt gttccccagc
tccgggggcg cttctaggtc ctgcagcagc caggagtgcg 180gagccaccaa
ctcggtgctg gaatgaaaaa attcccgcgc gccagtgcag aatctttcta
240agtgacccgg agcttcgggt gctagctctg cacgaacttt cccatcaaag
tgatcgtgaa 300ttttaagcat caggagcagg ccagcgaagc tctacgcgtc
taaacgtcta tccagaccaa 360gagttctctg cggtgcaggg tgcggtgcca
tgcagccaaa agtccctttg gggtcacgca 420agcagaagcc ctgctccgac
atgggggacg tccagcgggc agcgagatct cggggctctc 480tgtccgcaca
catgctgttg ctgctcctcg cttccataac aatgctgcta tgtgcgcggg
540gcgcacacgg gcgccccacg gaggaagatg aggagctggt cctgccctcg
ctggagcgcg 600ccccgggcca cgattccacc accacacgcc ttcgtctgga
cgcctttggc cagcagctac 660atctgaagtt gcagccggac agcggtttct
tggcgcctgg cttcaccctg cagactgtgg 720ggcgcagtcc cgggtccgag
gcacaacatc tggaccccac cggggacctg gctcactgct 780tctactctgg
cacggtgaac ggtgatcccg gctctgccgc agccctcagc ctctgtgaag
840gtgtgcgtgg tgccttctac ctacaaggag aggagttctt cattcagcca
gcgcctggag 900tggccaccga gcgcctggcc cctgccgtgc ccgaggagga
gtcatccgca cggccgcagt 960tccacatcct gaggcgaagg cggcggggca
gtggcggcgc caagtgcggc gtcatggacg 1020acgagaccct gccaaccagc
gactcgcgac ccgagagcca gaacacccgg aaccagtggc 1080ctgtgcggga
ccccacgcct caggacgcgg gaaagccatc aggtataaga gtgaccccca
1140tctctcagtc tttacgaggc gtgacttggg gtcacactcc agatcgcctc
taaatgcgaa 1200tgactcagac ttgcagtgaa ttgaagttct gggtcgtgac
cttcccgctc cccccccccc 1260aaaaaaagtg tgaccatact ctgctagaac
acttatttgc ccgaatagtt aataatttga 1320gaaagagaga aagaatcgga
ggtcctgtag ataagggcta agcgtttcct ccgcgaagcc 1380aataacccga
ctccttacac tggagaatct ctctccatcc ctttaatgcc tttagtgaat
1440gtatgagttc actttaacta ggttgtagtt tcgcgctgag ttttgtaacg
tcagtccgtg 1500tgagcacgta gcgctcaaag gagggcggag tagaggagcc
atggtgacct ggatgtgcgt 1560tcaggagcct gggcaacggc agtggtgatc
tcatttctgt ggccttccgt ctgtcccctt 1620cccccatttg aaaagctgac
cccgatggct ggtggctccg ttgggcccct ctgcagaacc 1680tgcttgggag
gtctttgctt ggttcgcccc gcctccacgc gcctcctacc tcggcctcgt
1740tgctcgcact ccctctcccg gcagaggttg gactccccag cgctgtggaa
tgttagcctg 1800gactgatcct ccctgctaca cattcgcctg actctgccgt
gttcagtctc taccagccag 1860ttagttcttt ttaatcattc aaatttcttt
ttgccctttt ctagatttct ccctcttttc 1920cgacttgtcc ctaggagctg
gtattcatat cctactttac gatttctctg accgctgagt 1980ctcagcagcc
cgaaaaaggc cattttccaa attggcaacc ctggtttgag aaaggaactt
2040attccccccg gggcactggg agtgagagga ggcaggaaaa cactgctggg
cagagtgggt 2100ggtcctagtg cccggaactg gatcaagcag agaaccccct
gggacccctt gaatgagaga 2160gctgagcctt acagactgag actcctcaag
ccccacccct tggctgagct ccccgccctg 2220ccccatgcct tccacgtgga
gctggatgat ctcattcggg atttcagccc tggcttcaat 2280agtgaaaggg
tgactcaggg cgtccgcctg cttctcttgc caagttttta ctacagctgg
2340gtagaaatga tagccatact gcctcactca ggctgtggag tcttcaaaga
ccacaaaaga 2400aatctgcgga cacatatata gacagtttga tcactctgtt
gcttgctttg ttttgttttg 2460ttttgtctta tttaaagcaa aagaaaaaag
acttaaaaat aactcacagt ttttagaaga 2520tgcaaatatt tgttttattt
ttgttccagg tgtatttcag ttttatttac tttgactagg 2580ttgactttcc
taatataccc cgagaaggtc actattagga gaaggactgc ccatgagcaa
2640acttcctttt ctttttacag gaccaggaag cataaggaag aagcgatttg
tgtccagccc 2700ccgttatgtg gaaaccatgc tcgtggctga ccagtccatg
gccgacttcc acggcagcgg 2760tctaaagcat taccttctaa ccctgttctc
ggtggcagcc aggttttaca agcatcccag 2820cattaggaat tcaattagcc
tggtggtggt gaagatcttg gtcatatatg aggagcagaa 2880gggaccagaa
gttacctcca atgcagctct cacccttcgg aatttctgca actggcagaa
2940acaacacaac agccccagtg accgggatcc agagcactat gacactgcaa
ttctgttcac 3000cagacaggta agacaggagc ttatcaacca tttcatcaac
tcaactcgga ggtcagcctt 3060gtgttggatg ggatgagagg gtgggggtgt
ggcggagagg aaacccagaa ggggatgaca 3120tttgaaatgt aaacaaaata
accaattaaa aaaaaaaggc atctcatctg tattgcctca 3180tttcctttcg
gttataggct agctcaatct gtcttgctta tttctatttt aaacttccac
3240atctcaagtt ctacagttct attttaaaag cattacaggg aatcttgctt
agagtcagtc 3300cttcaagccc agcaataatg aatggacagg cttcaaagtg
catgtgaaga cacgcccaac 3360tgaagagcta agtatcactc tctcctactt
aaaagggatt tcccttgcct ctttgtagga 3420tttatgtggc tcccacacgt
gtgacactct cgggatggca gatgttggaa ctgtatgtga 3480ccccagcagg
agctgctcag tcatagaaga tgatggtttg caagccgcct tcaccacagc
3540ccacgaattg ggtaagtcgg cttcagagta caagttaagc ccaaatgcat
ggatacaacc 3600caataagtca atctgatgtg acgagagaga aaacatctca
gactatgttg ctacctcagc 3660caccagcaat tttagaaggg gtagggtata
ttttccacga tttcaagtat ggtcttacta 3720ggacaggaga aagtggtaca
aacatttgaa cgttgacatt tttatacttg ccctgatcaa 3780agtgagtatg
agccccaata caggttgtct aataagagag ccattgagcc tcactcaata
3840atacagctga atgtccttct tgtctgcttc ccaggccatg tgtttaacat
gccgcacgat 3900gatgctaagc actgtgccag cttgaatggt gtgactggcg
attctcatct gatggcctcg 3960atgctctcca gcttagacca tagccagccc
tggtcacctt gcagtgccta catggtcacg 4020tccttcctag ataatggaca
cggtaagatg acagctcctc tttccagatg gtgttcaacc 4080ttccttgtgt
agggctctct ctggctaagt gagctccatg gctcttgctc atttcccctc
4140cttcagagtt ttctctggca ggatcataag tagtagatct ttacctccat
tgcatcctgc 4200tcccaaagtc cattcattca taaacaataa cttctcgcca
ttgtaaaatc agaagtcccc 4260tattgaggat aacgtctcga taaaaatcta
aagttcccta gcattgattt tcccaaaaat 4320gcatgatttc accaaacatg
tattaataat tgcctctttt ttcttttcct tttttttttt 4380tattatttta
ggggaatgtt tgatggacaa gccccagaat ccaatcaagc tcccttctga
4440tcttcccggt accttgtacg atgccaaccg ccagtgtcag tttacattcg
gagaggaatc 4500caagcactgc cctgatgcag ccagcacatg tactaccctg
tggtgcactg gcacctccgg 4560tggcttactg gtgtgccaaa caaaacactt
cccttgggca gatggcacca gctgtggaga 4620agggaagtgg tgtgtcagtg
gcaagtgcgt gaacaagaca gacatgaagc attttgctgt 4680gagttttccc
aatgaaacat atccgtttgc aactcagggt tgagaagggc aaagtgatgg
4740tttagttcct ttcctagaca aactcctcta cctgtgtcct gtagtgggac
tatgagatgg 4800tagcgtattt tgagaattga ttgtctgttt tacatttttc
tctgattccc taaaatgtct 4860ttatagttct aacactgata tctgtatctc
catttagact cctgttcatg gaagctgggg 4920accatgggga ccgtggggag
actgctcaag aacctgtggt ggtggagttc aatacacaat 4980gagagaatgt
gacaacccag tcccaaagaa cggagggaag tactgtgaag gcaaacgagt
5040ccgctacagg tcctgtaaca tcgaggactg tccagacaat aacggtgagt
catactggac 5100ttcagctctc agaaaccggg caaaggcggc gtgccacaac
atgtggttgg aagttggaaa 5160ctgggaacat catcgccgtc gttctctttt
caggaaaaac gttcagagag gagcagtgcg 5220aggcgcacaa tgagttttcc
aaagcttcct ttgggaatga gcccactgta gagtggacac 5280ccaagtacgc
cggcgtctcg ccaaaggaca ggtgcaagct cacctgtgaa gccaaaggca
5340ttggctactt tttcgtctta cagcccaagg taggtgcttt tacacttgaa
tctttgcaaa 5400ggagcctcag ctgggcttgc tgccatgcca tacaaatgtt
tgggctgtct ttacctattg 5460atctgtgttc cgttttgaat ttggaatact
tctaaatgca ggaacaactc cttgctttgg 5520gatttgttgt tgccttctgt
tgggaaggaa gcttaaatct agctagcact taaaagagtc 5580ttgcatgtgt
ttaatattgc ttctctatcc ccaaagaatg gccctttgaa aactcaagag
5640ccctctctgt ataactaggt ttcacataca aaaattcatg gttagataaa
ttatatatta 5700acatggcacc caggagtttt agaaagtagt ccaaagtact
tgttactggg tacctagcag 5760ccgcacatac gagcacacta actaaggtaa
gagtttgaga attaaaaatt catcgttgga 5820acatgtactt tgaccaaaga
gactcgccat ttcttttggt gttttgcaga aaggataaat 5880cctgctttga
agaagaaaat tgaatgaaat ttgcttaagc ttgtcatgta ttcttagcat
5940tataagatag caaactatat ccaagttgtg gatgaagtat ttagcaagtg
atttataaag 6000taccttcaac tacagcatat tattctaggt actgaccatg
gaacaataat cagtgtgaca 6060gtgaaccctg cttccattga cctaggccag
caaatatata aaatcaagac atttataagc 6120cttacagata gctatatgaa
ctgttgaaaa agccaaaatg aaagtgaaca tgtggcacgt 6180gacaaggaga
ctacttgtag cctgggagga gagcattccc agttgccatc acatcagatg
6240tttaaccacc atggtgcatg ttgtctccac aggttgtaga tggcactccc
tgtagtccag 6300actctacctc tgtctgtgtg caagggcagt gtgtgaaagc
tggctgtgat cgcatcatag 6360actccaaaaa gaagtttgat aagtgtggcg
tttgtggagg aaacggttcc acatgcaaga 6420agatgtcagg aatagtcact
agtacaaggt gagtttcaga acgctcactt ctgcagtaga 6480cacgctgtgt
tgctcagttg gtccctagca tctacaagac cttgggttca atccgcatgc
6540atgtacctgt agtcccagtg tatgggagac agagacaagt gtgacaagac
ggtcagatgt 6600tcaggtcatc tttgctacat agtgactttc agttcacctt
ggggaacatg aaaaacctga 6660ctggaaacac aaacacacac aaaacaatta
acccaggtac ttcatgtaat cccagtgttc 6720agtaggctga cttgggagga
tggttgctat aaggcctagg ttagcttggt ctacataatg 6780agttccagta
taacctggcc cacaagtgaa ccctaaagtt aattaatcga cacatgaaac
6840aaaacacatg ctttggagac cctgtaattt tgatatacga ttttgtagga
ctaaggaaaa 6900gtcacattta aaagaattgc ctatttttaa agcaatgtga
ttgattaact cattgaaaga 6960catatacctg ttttctttgt ccacagacct
gggtatcatg acattgtcac aattcctgct 7020ggagccacca acattgaagt
gaaacatcgg aatcaaaggg ggtccagaaa caatggcagc 7080tttctggcta
ttagagccgc tgatggtacc tatattctga atggaaactt cactctgtcc
7140acactagagc aagacctcac ctacaaaggt actgtcttaa ggtacagtgg
ttcctcggct 7200gcgctggaga gaatccgcag ctttagtcca ctcaaagaac
ccttaaccat ccaggttctt 7260atggtaggcc atgctctccg acccaaaatt
aaattcacct actttatgaa gaagaagaca 7320gagtcattca acgccattcc
cacattttct gagtgggtga ttgaagagtg gggggagtgc 7380tccaagacat
gcggctcagg ttggcagaga agagtagtgc agtgcagaga cattaatgga
7440caccctgctt ccgaatgtgc aaaggaagtg aagccagcca gtaccagacc
ttgtgcagac 7500cttccttgcc cacactggca ggtgggggat tggtcaccat
gttccaaaac ttgcgggaag 7560ggttacaaga agagaacctt gaaatgtgtg
tcccacgatg ggggcgtgtt atcaaatgag 7620agctgtgatc ctttgaagaa
gccaaagcat tacattgact tttgcacact gacacagtgc 7680agttaagagg
cgttagagga caaggtagcg tggggagggg ctgatacact gagtgctgga
7740gggatccagt gagtcaaacc agtaagcagt gaggtgtggc aaggaggtgt
gtgtagggga 7800tacatagcaa aggaggtaga tcaggacact accctgccag
ttacattctg ataaggtagt 7860taatgaggca cagtagcatc tgaaagacca
tacagagcac taaggagccc caaagcacta 7920ttagtatctc ttttcttata
tctatcgccc aaataatttt cagagtctgg cagaagccct 7980gttgcactgt
actgactaga tacttcttat cacaaagatt gggaaaggca aagcagaaag
8040atggtaagac tgggtttcaa acaaggcttg gtttctatca ctggaggcaa
ggaggagggg 8100acaaacaaga tcattattcg aagtcgctgg ttgctgtggt
tttacggaag gttgatgcat 8160cattcctatc aacagtgaaa agttcagctt
gttcaacgtg acagaaaggc tcatctccgt 8220gaaagagctc ctgatttctt
cttacaccat ctcagttctt aactataatt catgttgagg 8280tagaaacaat
tcatctattt ataaaatgta cattggaaaa aaaaaagtga agtttatgag
8340gtacacataa aaactgaagg aaacaatgag caacatgcct cctgctttgc
ttcctcctga 8400ggtaaacctg cctggggatt gaggttgttt aagattatcc
atggctcaca agaggcagta 8460aaataataca tgttgtgcca gagttagaat
ggggtataga gatcagggtc ccatgagatg 8520gggaacatgg tgatcactca
tctcacatgg gaggctgctg cagggtagca ggtccactcc 8580tggcagctgg
tccaacagtc gtatcctggt gaatgtctgt tcagctcttc tactgagaga
8640gaatatgact gtttccatat gtatatgtat atagtaaaat atgttactat
gaattgcatg 8700tactttataa gtattggtgt gtctgttcct tctaagaagg
actatagttt ataataaatg 8760cctataataa catatttatt tttatacatt
tatttctaat gataaaacct ttaagttata 8820tcgcttttgt aaaagtgcat
ataaaaatag agtatttata caatatatgt taactagaaa 8880taataaaaga
acacttttga atgtgtatgc ctattttctg gagtgggatt aacttctggg
8940caagaaatct gatgagacac aaacattgga cttcaagaca gttttaaaat
ttgggtaaat 9000gaactgtatt tcctgtttat agacgtacta ataaaaaaga
agttgatgat gtctttagtg 9060gtaagattgt tactaatgtg gttggcaaat
tgctgtaaag agccagatag taagcattta 9120tggcattgta ggctatcttt
cctgccacaa ccatgtgaca gtgagtgctt tgtaggactg 9180agagcagcca
taaatgacat gtaaatgata aactgtggct gtgctttaat aaaactttat 9240ttacaaaa
9248225722DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number X14787 22ggacgcacag gcattccccg cgcccctcca
gccctcgccg
ccctcgccac cgctcccggc 60cgccgcgctc cggtacacac aggatccctg ctgggcacca
acagctccac catggggctg 120gcctggggac taggcgtcct gttcctgatg
catgtgtgtg gcaccaaccg cattccagag 180tctggcggag acaacagcgt
gtttgacatc tttgaactca ccggggccgc ccgcaagggg 240tctgggcgcc
gactggtgaa gggccccgac ccttccagcc cagctttccg catcgaggat
300gccaacctga tcccccctgt gcctgatgac aagttccaag acctggtgga
tgctgtgcgg 360gcagaaaagg gtttcctcct tctggcatcc ctgaggcaga
tgaagaagac ccggggcacg 420ctgctggccc tggagcggaa agaccactct
ggccaggtct tcagcgtggt gtccaatggc 480aaggcgggca ccctggacct
cagcctgacc gtccaaggaa agcagcacgt ggtgtctgtg 540gaagaagctc
tcctggcaac cggccagtgg aagagcatca ccctgtttgt gcaggaagac
600agggcccagc tgtacatcga ctgtgaaaag atggagaatg ctgagttgga
cgtccccatc 660caaagcgtct tcaccagaga cctggccagc atcgccagac
tccgcatcgc aaaggggggc 720gtcaatgaca atttccaggg ggtgctgcag
aatgtgaggt ttgtctttgg aaccacacca 780gaagacatcc tcaggaacaa
aggctgctcc agctctacca gtgtcctcct cacccttgac 840aacaacgtgg
tgaatggttc cagccctgcc atccgcacta actacattgg ccacaagaca
900aaggacttgc aagccatctg cggcatctcc tgtgatgagc tgtccagcat
ggtcctggaa 960ctcaggggcc tgcgcaccat tgtgaccacg ctgcaggaca
gcatccgcaa agtgactgaa 1020gagaacaaag agttggccaa tgagctgagg
cggcctcccc tatgctatca caacggagtt 1080cagtacagaa ataacgagga
atggactgtt gatagctgca ctgagtgtca ctgtcagaac 1140tcagttacca
tctgcaaaaa ggtgtcctgc cccatcatgc cctgctccaa tgccacagtt
1200cctgatggag aatgctgtcc tcgctgttgg cccagcgact ctgcggacga
tggctggtct 1260ccatggtccg agtggacctc ctgttctacg agctgtggca
atggaattca gcagcgcggc 1320cgctcctgcg atagcctcaa caaccgatgt
gagggctcct cggtccagac acggacctgc 1380cacattcagg agtgtgacaa
aagatttaaa caggatggtg gctggagcca ctggtccccg 1440tggtcatctt
gttctgtgac atgtggtgat ggtgtgatca caaggatccg gctctgcaac
1500tctcccagcc cccagatgaa tgggaaaccc tgtgaaggcg aagcgcggga
gaccaaagcc 1560tgcaagaaag acgcctgccc catcaatgga ggctggggtc
cttggtcacc atgggacatc 1620tgttctgtca cctgtggagg aggggtacag
aaacgtagtc gtctctgcaa caaccccgca 1680ccccagtttg gaggcaagga
ctgcgttggt gatgtaacag aaaaccagat ctgcaacaag 1740caggactgtc
caattgatgg atgcctgtcc aatccctgct ttgccggcgt gaagtgtact
1800agctaccctg atggcagctg gaaatgtggt gcttgtcccc ctggttacag
tggaaatggc 1860atccagtgca cagatgttga tgagtgcaaa gaagtgcctg
atgcctgctt caaccacaat 1920ggagagcacc ggtgtgagaa cacggacccc
ggctacaact gcctgccctg ccccccacgc 1980ttcaccggct cacagccctt
cggccagggt gtcgaacatg ccacggccaa caaacaggtg 2040tgcaagcccc
gtaacccctg cacggatggg acccacgact gcaacaagaa cgccaagtgc
2100aactacctgg gccactatag cgaccccatg taccgctgcg agtgcaagcc
tggctacgct 2160ggcaatggca tcatctgcgg ggaggacaca gacctggatg
gctggcccaa tgagaacctg 2220gtgtgcgtgg ccaatgcgac ttaccactgc
aaaaaggata attgccccaa ccttcccaac 2280tcagggcagg aagactatga
caaggatgga attggtgatg cctgtgatga tgacgatgac 2340aatgataaaa
ttccagatga cagggacaac tgtccattcc attacaaccc agctcagtat
2400gactatgaca gagatgatgt gggagaccgc tgtgacaact gtccctacaa
ccacaaccca 2460gatcaggcag acacagacaa caatggggaa ggagacgcct
gtgctgcaga cattgatgga 2520gacggtatcc tcaatgaacg ggacaactgc
cagtacgtct acaatgtgga ccagagagac 2580actgatatgg atggggttgg
agatcagtgt gacaattgcc ccttggaaca caatccggat 2640cagctggact
ctgactcaga ccgcattgga gatacctgtg acaacaatca ggatattgat
2700gaagatggcc accagaacaa tctggacaac tgtccctatg tgcccaatgc
caaccaggct 2760gaccatgaca aagatggcaa gggagatgcc tgtgaccacg
atgatgacaa cgatggcatt 2820cctgatgaca aggacaactg cagactcgtg
cccaatcccg accagaagga ctctgacggc 2880gatggtcgag gtgatgcctg
caaagatgat tttgaccatg acagtgtgcc agacatcgat 2940gacatctgtc
ctgagaatgt tgacatcagt gagaccgatt tccgccgatt ccagatgatt
3000cctctggacc ccaaagggac atcccaaaat gaccctaact gggttgtacg
ccatcagggt 3060aaagaactcg tccagactgt caactgtgat cctggactcg
ctgtaggtta tgatgagttt 3120aatgctgtgg acttcagtgg caccttcttc
atcaacaccg aaagggacga tgactatgct 3180ggatttgtct ttggctacca
gtccagcagc cgcttttatg ttgtgatgtg gaagcaagtc 3240acccagtcct
actgggacac caaccccacg agggctcagg gatactcggg cctttctgtg
3300aaagttgtaa actccaccac agggcctggc gagcacctgc ggaacgccct
gtggcacaca 3360ggaaacaccc ctggccaggt gcgcaccctg tggcatgacc
ctcgtcacat aggctggaaa 3420gatttcaccg cctacagatg gcgtctcagc
cacaggccaa agacgggttt cattagagtg 3480gtgatgtatg aagggaagaa
aatcatggct gactcaggac ccatctatga taaaacctat 3540gctggtggta
gactagggtt gtttgtcttc tctcaagaaa tggtgttctt ctctgacctg
3600aaatacgaat gtagagatcc ctaatcatca aattgttgat tgaaagactg
atcataaacc 3660aatgctggta ttgcaccttc tggaactatg ggcttgagaa
aacccccagg atcacttctc 3720cttggcttcc ttcttttctg tgcttgcatc
agtgtggact cctagaacgt gcgacctgcc 3780tcaagaaaat gcagttttca
aaaacagact catcagcatt cagcctccaa tgaataagac 3840atcttccaag
catataaaca attgctttgg tttccttttg aaaaagcatc tacttgcttc
3900agttgggaag gtgcccattc cactctgcct ttgtcacaga gcagggtgct
attgtgaggc 3960catctctgag cagtggactc aaaagcattt tcaggcatgt
cagagaaggg aggactcact 4020agaattagca aacaaaacca ccctgacatc
ctccttcagg aacacgggga gcagaggcca 4080aagcactaag gggagggcgc
atacccgaga cgattgtatg aagaaaatat ggaggaactg 4140ttacatgttc
ggtactaagt cattttcagg ggattgaaag actattgctg gatttcatga
4200tgctgactgg cgttagctga ttaacccatg taaataggca cttaaataga
agcaggaaag 4260ggagacaaag actggcttct ggacttcctc cctgatcccc
acccttactc atcaccttgc 4320agtggccaga attagggaat cagaatcaaa
ccagtgtaag gcagtgctgg ctgccattgc 4380ctggtcacat tgaaattggt
ggcttcattc tagatgtagc ttgtgcagat gtagcaggaa 4440aataggaaaa
cctaccatct cagtgagcac cagctgcctc ccaaaggagg ggcagccgtg
4500cttatatttt tatggttaca atggcacaaa attattatca acctaactaa
aacattcctt 4560ttctcttttt tccgtaatta ctaggtagtt ttctaattct
ctcttttgga agtatgattt 4620ttttaaagtc tttacgatgt aaaatattta
ttttttactt attctggaag atctggctga 4680aggattattc atggaacagg
aagaagcgta aagactatcc atgtcatctt tgttgagagt 4740cttcgtgact
gtaagattgt aaatacagat tatttattaa ctctgttctg cctggaaatt
4800taggcttcat acggaaagtg tttgagagca agtagttgac atttatcagc
aaatctcttg 4860caagaacagc acaaggaaaa tcagtctaat aagctgctct
gccccttgtg ctcagagtgg 4920atgttatggg attccttttt tctctgtttt
atcttttcaa gtggaattag ttggttatcc 4980atttgcaaat gttttaaatt
gcaaagaaag ccatgaggtc ttcaatactg ttttacccca 5040tcccttgtgc
atatttccag ggagaaggaa agcatataca cttttttctt tcatttttcc
5100aaaagagaaa aaaatgacaa aaggtgaaac ttacatacaa atattacctc
atttgttgtg 5160tgactgagta aagaattttt ggatcaagcg gaaagagttt
aagtgtctaa caaacttaaa 5220gctactgtag tacctaaaaa gtcagtgttg
tacatagcat aaaaactctg cagagaagta 5280ttcccaataa ggaaatagca
ttgaaatgtt aaatacaatt tctgaaagtt atgttttttt 5340tctatcatct
ggtataccat tgctttattt ttataaatta ttttctcatt gccattggaa
5400tagaatattc agattgtgta gatatgctat ttaaataatt tatcaggaaa
tactgcctgt 5460agagttagta tttctatttt tatataatgt ttgcacactg
aattgaagaa ttgttggttt 5520tttctttttt ttgttttttt tttttttttt
tttttttttg cttttgacct cccattttta 5580ctatttgcca ataccttttt
ctaggaatgt gctttttttt gtacacattt ttatccattt 5640tacattctaa
agcagtgtaa gttgtatatt actgtttctt atgtacaagg aacaacaata
5700aatcatatgg aaatttatat tt 57222342521DNAUnknownDescription of
Unknown Organism GenBank Clone Accession Number U64857 23gatcgttttc
cagacatttt tgttctctgt tcatttcctt atcgtattca aaaagttatc 60acaaatgacc
ttctctatct gtctgcgtct cttttaactc tcaccgtttg ggacctttca
120aatagttttt cgctatcaaa tctaaacatt agttgcgttg actcgacatt
tgacccctca 180ctatcatctc cagttctctt ttttgttaca ctttagcagt
ggcagcagag agcaagtagg 240tggagccaaa gtgtgcgcca ttcatcggga
aaaattgtgt tcttcatcaa attttgggca 300atttactcgg gatttgcgct
aatttggaaa caaaaattca aattcctgcc aattgttttg 360tttgcttttt
ttcttttttt tttgctcctc ccatctctca tcaaattgct cttttttcga
420ttctaacata tcagccatct tcagagtgtg tcactaaccc ccatttttat
tcaaggttag 480tgatatagta tcctaactac agacgtcaca ccatgaggtt
gctgctcttc tcggcagccc 540ttcttctgtg ctccgtccca acgtgggcct
tctctctgtc atcattcttc ggaagcgatg 600ttgcacaagt aagcaagctc
tcctatacct agaatcttgt aaattgaaaa ctctaatttc 660cagaagccat
accttcatcc aaactcccca ccggagcgtg acccggcgag ttccagaatg
720aagagacagg catatcaagt gtacgttgat ggagatgttt ccgttactgt
tgacaagtct 780ggacaaaagg aaaccggcaa ctggggacca tgggtgcccg
agaacgagtg ctcacgttcg 840tgtggtggag gagttcaact cgagaagaga
cagtgcaggt tcgtggactt ttcatttttt 900agggaatttc ctagacgttc
taaaagctta ttttcaaaaa ttttggtttc ctgatcttca 960tgcctttatg
aacgtggtga aagatcaacc taggctagcc tgtgacatac attttttgaa
1020gcagatccaa ctttatcaag agccatcgaa ttctcgtttt aaagtgtttt
ttttttctga 1080taactttttt ctaatagctt tacccatttt tatgtcaaga
ctgaaagcaa tgaatcacaa 1140gaggctatct acgtttgttt ttgaagctct
gtaggaatca tcttaaaaaa ttaagtaaag 1200taatggagat gaaattctaa
ttttttaaaa tcataatcat tactttctgt attatcttca 1260agttcaaact
tttcaaacgg ttattctcaa gaaactcaca tagaatttta acaatttcct
1320ctatctattt cttgcaagca acccaccgaa ctcaaatctt atccaaacta
aacttttagt 1380ggtgactgca ctggagcttc agtccgctac atctcgtgta
acttgaacgc atgcgagtct 1440ggtactgatt tccgtgctga gcaatgctcc
aaattcaacg atgaggctct tgatggaaac 1500taccacaagt ggactccata
caagggaaag aacaagtaag ttaactttct tcaagatgtt 1560tttctaattt
tcgagttttc aggtgcgagc tcgtctgtaa gccagaatct ggaaacttct
1620actacaagtg ggctgataag gttgttgatg gaaccaagtg cgactccaag
agcaacgata 1680tctgtgttga tggggaatgt cttccagttg gatgtgacgg
aaagcttgga tcttgtaagt 1740ttaaaattta attcaaaatc ttcatttcat
gccgaatatt tcagctctca aattcgacaa 1800gtgcggaaag tgcgatggag
atggttctac ctgcaagact attgaaggac gtttcgatga 1860gcgcaatctc
tctccaggat accatgatat tatcaaactt ccagaaggag ccaccaacat
1920taagattcag gaagccagaa agagcaccaa caacttggct ctgaagaacg
gttccgatca 1980cttttatttg aatggaaatg gattgatcca agttgagaag
gaggttgaag tcggaggaac 2040tatcttcgtt tacgatgacg ctgaaccaga
aactctcagt gctcaaggac cactctccga 2100ggagctcacc gttgctcttc
tcttcagaaa gggaagccgt gatactgcta tcaagtacga 2160gttctctatt
ccacttgagg aggaagttga ctacatgtac aagtttgaca actggactcc
2220gtgctctgta tcatgcggaa agggtgttca aacccgtaat ctctactgta
ttgatggaaa 2280gaacaaggga cgcgttgagg atgatctctg cgaggagaac
aatgccacaa agccagagtt 2340cgaaaagagc tgtgaaactg ttgactgtga
agccgaatgg ttcactggag actgggaatc 2400ttgctcatcc acctgcggag
atcaaggaca gcaataccgt gtcgtctact gccatcaagt 2460attcgctaac
ggacgtcgtg ttaccgttga ggatggaaac tgcaccgttg agagaccacc
2520agtaaagcag acttgcaatc ggtaagttga ttttataaat gcataaacaa
ctctgtgaat 2580ctatttgttt atgcgatgct atccatatat attaccagat
ggtgttggtg cccaaaactt 2640ataaacaatt attttctctt tgcagttttg
cctgcccaga gtggcaagct ggtccgtggt 2700cggcttgctc agagaagtgt
ggagacgcct tccaatacag atcggtgacc tgccgcagtg 2760agaaggaagg
agaagaggga aaactcttgg ccgctgatgc ttgcccagct gatgagcaag
2820agaagttcga cacagagaga acttgcaatt tgggaccatg cgagggactt
acatttgtca 2880ctggagaatg gaacttggtt agattttgca aaatatgggg
acctggggaa aagcatacta 2940aataagatca actttatgaa acaaataatt
tttagtgcac ccgctgcaac gatactgagg 3000agactcgtga agtcacctgc
aaggactccc aaggaagagc ctatccactc gagaagtgtt 3060tggttgataa
ctccaccgag attccaactg atactaggtg agtcattcca gatatgacat
3120tgaacttgga ttaatttttt tcttccagat catgcgccac ccaaccacca
tgtgagtacg 3180agtggaccgt cagtgagtgg agcaagtgta ccaccgaatg
cggacacgga cacaagactc 3240gtcgtgttat ctgtgccatc caccaaaacg
gaggactcga ggttgttgat gaaggacact 3300gtcaagctga gaagccagaa
ggaaagacta actgcaccaa tgaggagaag tgtactggaa 3360catggtacac
atcttcatgg tccgagtgta ccgctgaatg tggtggtgga tcccaagatc
3420gtgtcgctgt ttgcttgaac tacgataaga agccagttcc agaatggtgc
gacgaagccg 3480tcaagccatc tgagaaacaa gattgtaacg ttgatgactg
cccaacttgc gttgactctg 3540agttcggatg ctgcccagat aactctactt
ttgctaccgg agaattcaac ttcggatgct 3600ctaactgctc ggaaacagaa
ttcggatgct gtgctgacaa tgttaccgtt gccactggac 3660ctaactccaa
gggatgcgaa gaattcgttg agtctccact taaccttgaa gctgatgttg
3720ccaatgctga cgctgaagct tcaggagatg ctccagaact ctgcagcgtc
acaaacgaga 3780acggagaagc tgttgatgtt gagtgtgcca ccattgctcc
aatcactgct cttcttggag 3840atggggaact tatcggaaat gatactgatg
cttccaatga gaccatacac tgctcgaaga 3900ccgaattcgg atgctgtcca
gattggtaca ccgccgcctc tggaaagggt aacgaaggat 3960gcccatcgtt
cactcttgga ggatgtaacg agactcaatt cggatgttgt cacgatgatg
4020tcactcttgc tcgtggagcc aaccttgaag gatgcggaga gccatcttgc
gctgcttccc 4080tctatggatg ctgtaaagat cgtaagacaa ttgccttcgg
accacactat tctggatgtg 4140agcgatcatc cttcccatgt gagcttagcg
acttcggatg ctgcccagat ggtgagactg 4200ctgctcttgg aaagaatgga
accggatgcg gagagaactg cttgaccacc aagttcggat 4260gctgccctga
tggaaagacc accgccaagg ggtcccacaa cgagggatgc ggatgcgagt
4320tcgcccaata cggatgctgc ccagacggaa aatcagttgc caagggagcc
ggattttacg 4380gatgcccaga aagctgcgcc cagagccagt tcggatgctg
cccagacgga aagactcgtg 4440ctcgcggaga gaacaaggaa ggatgtccat
gccagtacac ccgttacgga tgctgcccag 4500atggggagac tactgctctt
ggaccacgca atgatggatg tgataactgc cgctacgcca 4560agcacggatg
ttgcccagat ggagagacca aggctcttgg accagatgga gccggatgcc
4620caccaactac cacgccacca ttcctcatgg gaggaactgt tgccccacat
aaaatcgccg 4680cctgtaatca gacacaagaa agtggaaccg tctgcggagc
cggatacaag cttgtaagta 4740attaacctca tgaaaaagaa ttggagcaac
acatttcatg tataaatatt tcaatttcag 4800gcatggcatt atgataccac
tgagggacgt tgcaaccagt tctggtacgg aggatgcggt 4860ggaaatgaca
acaactttgc tagccaggat atgtgcgaga ctatctgcgt cgaaccacca
4920ggcaagggaa gatgttacct gccacgtgtt gatggaccac tccggtgtga
ccaacttcag 4980ccaagatact attatgatca ttccaagaag cactgtgtgg
ccttctggtg gagaggatgt 5040ctcggaaatg ccaacaactt caactctttc
gaagaatgct ccatgttctg taaggacgtt 5100ggaccgtacg atgctccaac
caccgctgct ccaccaccac caccacagca aaatgctcag 5160caataccttc
caactccaga agttcaacag attgagattc aatctgctga gcaacctcaa
5220ccacaacagc cacaacaaca gcaacagcaa caacagcaac aaccacagca
accacgtcaa 5280tcaatggaag acatctgcag atcccgccaa gacgccggac
catgcgagac ttactccgat 5340caatggttct acaacgcttt cagccaagaa
tgcgaaacct tcacttatgg aggatgtgga 5400ggaaatctca atcgtttccg
cagcaaggat gaatgcgagc agcgttgttt cttcgttcac 5460ggagctcagc
catccgctgc ccggcaggaa caagctcagc cagcagctca accagctcaa
5520ccagctcagc caagtaacat cgtctctcca ccacaacagt cagctagtcc
agttgtggtt 5580ccatgtaagt tctttagaat gcatttattt cttactataa
gtttctataa gttcgcatgt 5640gaagcatccc catttcagcg aacagcaaac
aacgcgatgc ttgccacctc aacgttgacc 5700aaggacgttg taagggggct
tttgactcct ggtactacga agttgccacc ggatcctgcg 5760tcacattcaa
gtacaccgga tgcggaggaa acgccaacag atttgctagc aaggatcagt
5820gcgagtcact ctgtgtgaag ccagcttctg aagctgcttc agccggaatt
ggtatgcttt 5880gagttataga gaatgttcac tatttttgtt aaatgtttga
gtaaatgaga aactggctca 5940gtttgaaaat gtttgcacca tgtttcaaaa
tagtttttga gttgaatagt tgaggccatg 6000aaaatcttaa ttacactcca
gaagtacatt ttaaaacatt tttgagaatt aggtcttcaa 6060aaaaaggttt
aatattgagg tttcaaatta gaaatattaa tatacgggga tttgggttta
6120aaactgattt ttaaaatctt atttttgaag tttcgctttg atattcgtgc
aaaaaaaaaa 6180ccaacttttt cagacggtgc agctggaatc aactcagttt
gtgacgaagc caaggacacc 6240ggaccgtgca ccaactttgt cacgaaatgg
tactacaaca aagccgacgg aacctgcaac 6300cgattccatt acggtggatg
ccaaggaacc aacaatcgat tcgacaacga gcaacagtgc 6360aaggctgctt
gtcaaaatca taaggatgct tgtcaacttc caaaggttca aggaccatgc
6420tctggaaagc attcctatta ttactacaac actgccagtc atcaatgcga
gacgttcact 6480tatggtggct gcctcggaaa tactaacaga ttcgctacca
ttgaggagtg tcaagcgaga 6540tgcccgagta agttctaagt taatagtgat
atatgctttg tttccccttt attctttgac 6600aattttcaaa tactttttgc
ataattacct tatttctatt cccttctgtt tcccattttc 6660ctccacccgc
tacaaattgt ttcccgtact ctctcctttc tcactttccc gtccgaaggg
6720acacggcaat gctgcctaaa tgaactgcct aataatattt atgaattttc
caattttcta 6780aaaaaaaaca attctctcaa aaaattccct gccgttccgc
cactgctttc ttcacccatt 6840gttgcgctat tttttttaaa taaatgaata
aagctgaaat agttaacagt ttctgaaatt 6900gcatgtaagt ttgtagtgta
tcagtgtgtt tgtcgtgaaa gttttttttt acctgcatga 6960tttcctgaac
tgcatgaaac tgttcttatt acgttttaga tttgctgaag tgtgctagaa
7020gtgtgatttt gtttcagaag acgaccagac tacaacaaca tcacaaccag
aagagctccc 7080aagtttgcca cttgttcaag aagatcctca gccacgaccg
gcattttcat tgaagtaagc 7140acgtgtagtc caagtgccta cttctcgtat
gaccaaaaaa tttaatataa ggtttccaag 7200tattaaggaa tcagtagcat
gtaaattgtg tggattgttc tcctgggttg atgggttttt 7260ttctcactca
caatcagata tggagtagct tatatgggaa tttatttgag aaatagaata
7320tgtcataaca tccaaattta attattaaaa agttgtgaag tttctcatta
tgtatataaa 7380attcgccttt caaataagaa caaaaattaa ctgtatgaaa
gagctgaatt caatttgaaa 7440ttgagaaaat aactggttca aaaagaagaa
aaacgttgga aaatctagac gtaaatctat 7500ggattttctt ttcaggtcgg
ggaaatttcg acgattttta tattttcaaa aatcattcac 7560aaatatacac
caaaaattat ttttaccata ataaaatacg gaatttcact ggattactgt
7620agtattcatg taaggttact gtattgttac tctagggata ctacaagaat
atttttgcaa 7680agttgtaaga agtatagaga ttactgtaga ttgaaaatct
agacaaaaat cattttccgt 7740aataatctgt ggggatagaa tgttgaaggc
acaaggctta taaagcacca tgggaaaaaa 7800ttttaacagt gattttttta
agcatatcct ctttcccagg aaatccactt ttcaaatata 7860ttcccactaa
actctttaag acaatccttc gcccatagtc gtcgccgtga tgctccattt
7920gcacgttccg tatccgcccg tcaccatact cctgattccg aagaggaacg
agttgactgt 7980tatgctgttc cagatccagg atcttgcggg taataaatct
cacctatcca ttacaaccat 8040taccgtctta atgattcaga gactaccgtc
ttgtttggca ctactctgcc acgagtaact 8100catgccgtca attctactat
ggtggatgtg ctgggaatac gaatcgcttc gagacccggg 8160ataaatgtga
aacatcgtgt gttgctaaga ttgaagaacg cgtggaaagt gtgtcagaag
8220cttcaaaatc tctggaagag gttagactaa cggatccaag gatggattct
cactttggat 8280atcatgatcc agaagttgat caaatcgaag aagaagctga
atatgtcatt gttgataccg 8340gagctctacc tgaattatgc atgcttccag
aacaaagagg gtcttgttat gataacattt 8400tgagatggag gtaagtcaaa
tcaagaatag aaaattcgaa aatccgaaaa actttataat 8460tatactaaaa
gcaaaatctt aaaatctttc agattcgact ctgaaaagtc tcaatgtgta
8520accttcatgt attctggatg taatccaaat gcaaatcact tcactagtca
ggttagtttc 8580attattttgt gtcctttcgt ggaactggcc ccttggtttc
taacttgatc ttctccttcc 8640gaatacccaa tttgagcacc gctggctcac
tttttcgacg gtgacgttcc tcaattctag 8700cggcctctgt attttctgag
cactcttgag caacagtttc ctcactggaa atgtttgttt 8760ttcaagaggg
agtgagagag agaaataaac gtacaatttt tgaagccgca catgatttgt
8820tagaagtcga tgccgttctg cagtatcctt catgtttcgt agttgtttct
gtagtaattt 8880ttatggatta ggaactaaga aatcatcact cactgcggta
gttgcatttt tgtgcatgca 8940tcttcccata aaagcaacaa atgcaacaac
tgatagagcc gccacacaaa ttgcaataat 9000tcgaagtcga tttctaattc
ctttctttac tttttgtcta tgctcagctg ctttttcgat 9060gtgcttcttc
ttgctggggt cgagctcgca atgaggaaat ggttcgatga gtggaccgtg
9120ttttttgcat tgttcacaac ggcgtccagt gtatttgtct gggcagtcac
acgaaagagt 9180gcggtttttg aaatctgaaa attttaaatt taagaacagg
atctatagca gttttgccca 9240tcacagtcct
atgtctatat taaaaaaaat tatcggacat taaaaaaaat gttttctcat
9300tttttcagta tttctataaa aactgcattc gcatttaatc ataactttta
atcgttaaaa 9360acttagtctt taagtacctg gggatccgta aacacagaca
atttcatcac aataatcgcc 9420ttcaaatccc acatcacaga tgcatcttcc
atttctcaaa aacccctcga cacatttact 9480tgtatattgg cattcacttc
caaaatatga gcccacacat tcacatcggt cccccttcca 9540ttctcctttg
tttccgcact gaaataattc aatagatttt ggaagtttag ggcctcaaaa
9600atataccttt tccgctggcc gatagtcaca catttcacct ttccatccga
cttcgcaaat 9660gcacggctca ctgaatagaa ggctttccgg gtcgaagcgg
aatccgaccg agagtccgtg 9720aactgtaaat tgaaaatttg taattccaaa
aaaaaaacag cttttgcaaa aatcgtccaa 9780aagaatttta gagttagaca
ttatttttct caaaaagttc aaagttgtat cagttttaaa 9840ataaaatatt
taataggatt gtagagcttg ttagaaaaaa taaaagctac ttgaaaaaag
9900aaaggtatcc aaaaaggtat tgagatagtt tcaagcaact ctatttgtaa
actgtcgagt 9960ttttaagttc tacaaatctc ttataacatc gctacatcta
ctatcaaact ttgaaaaaaa 10020accataccac attcaaaatg ttcacattta
tctccagtct gtcccttgat acaatgacaa 10080atccctccag catagattcc
tccattacga cattcggctc tcggatcatc cagagcaaca 10140ttgtctagaa
tacttctctt ttgaagaata cgatgcacgt cgctcaatat attttcatct
10200agatctagtg agtcatctcg tgattgtgct tttgttgttg ataaaaatag
gaagagtaaa 10260gtggaaaatt gtaaacagta catagcgtta gatactgaca
agtctactat caattgattt 10320atttattgcg tcttgaaagg ggtatcaatg
agagaaatag ggagatgggt aaaatgcatt 10380tataagagaa tacaaaagat
gacgtaattg attaatcaga gatcagttga aaatactttt 10440aagtatcaat
tattatctgt gaagacagtc acgtgactct gactcgaact caatttgcat
10500gttgatagtt ccaatgttaa agaaagtctt tgggttttct ccagatgaaa
caaatgattt 10560tggaatatta aacgtgactc ttctctgaca aggtttgagt
ccgtcatcac aatcgtgata 10620gatattaagt tttggatcaa tagtcatcac
ttcggaagtg tgtccggtaa gaaggaattg 10680accaagagag tctgtagttc
cttcggcaag aagatcgtca agatccggtc ctgaaaaaaa 10740cttttatttt
gaaaaatttc aatgagttgc ttcatgttag aatttggaat ttttaaagat
10800gttagcaatt ggtatttaaa tgttcaagct aacgtaatta gagttattca
aacaagcttt 10860atataaaaac tttgtgtaag attcggtcta attagaacat
caatttttaa cgcagctgat 10920aaaaaacttt aatttcaagc ttcacataat
tctacttacc ggtatcatca tcgtagagct 10980tcaccttcgt gttagccagt
ggtttgtctc cacacatcag aacaccctta actccagctg 11040attgggtgaa
tacagcttcg gagccaattg cacaaagtat gaaaagtgat gaaatgcacg
11100cgagtcgtga cattattttt gtctgaaaat acaaacactg actgatctga
ccttcatcgg 11160agaaactctc ttatagcaca gttggttaga aaaagatacg
gagaggagaa gtgggaaatc 11220gaattgacca aacaaaagaa ctggttttca
cttgaaatag aagacgatga aagatataca 11280acagagaaga tcggaagtga
ttcatctgga gaagaaaatt gagaggagca acttcttgta 11340ttttccactt
atttatatac ccaatagaat tcacctgatt ctttccgatt tgtgtacatt
11400tcgctgacta acgtgtgctt cttcggtttt gtcatttctt attgttcatt
gaaaataaac 11460agaacaaagc aatcataagg tcgaaaatcc catttagaga
tcaagaggtg tacctttaat 11520tgtgcggcat ggcatagttt tatcttgctg
aactctcacc aattgatgag tatgtcagta 11580gaatggattc catccgatcg
ttgctccacg gtgatctctt ccgccgcctt ttcatccacc 11640atacccgttg
tgtatggctg gcaactgtga acagcgcctc agtggaatgt ttagtttgat
11700atacagttta aaataatttt ctaaactaaa gaaatcagtt tttgaaacca
gtcttgtagg 11760catgtcgggc gcaggcacgc taacgtgaaa aatagaattt
cgagtggtta actattttat 11820tttcaattaa aatacaatca actacacaat
gaatgacccg gataaatgaa atacaaatac 11880aagaatttaa aaaaaacatg
gaaatttaaa cttttccatc atctcccttt gctggaatat 11940tatatttcat
tcgataagct tccaattcgg cttttctctg atcggatcgt acactgtgtc
12000tctcatccat ctcttgttga gctgtcattc tcttctcatt ccatttctga
gcttttgctt 12060ttttgtggat tctgttgtat tctttgcact tgcagcagca
ataacagaag caggcaatga 12120gaattacagc aatgattcca gcgcagatgg
caataacgat tgcggcggct gatgtgctca 12180cccagcagac attgtatttg
acatgcttga tattacagtc tggatagtac cagtcgaatg 12240gcatacatct
tttcgttttt ccaccacacc agaagcaatt ctgaaaaaat gtgtttttga
12300aattttcaat atgtttgctt ataaaattga atttaatttt tcaaacagtg
tttcagaaac 12360tcaacttctg aaattaggaa agtattctca attgagagct
gtttttgtat taaaagtttc 12420agtttagaac tacaggtgtg aaaaaatctg
agcaagtgaa caccaacgta ttgcatcaca 12480gtttacgcgt caatttattc
gagtgttcat tgtagagaaa gttaggtcac cttccagaaa 12540attaagaaac
ttgtttcaga catttttgct cttttagagg aatttttttt tagaggaaac
12600acgcaagttt ctttgaaaac aaaaacaaaa tatatttttt atccacttac
cgagcccttg 12660ccaacacatg tttcacaagt gttcaaatcg ttcgatccaa
ttctacagta ttcttgtttc 12720tctgaccatg tcatgttatc cgcacatact
gatactagaa caattgagaa aaagagtagt 12780aatcggtgaa tcatcgttct
gaaaaatcaa taaatagtaa caacttgagc aagtctcgta 12840actgagcgac
aaaaccaaag tagtaatgaa atagaaagat agaaaggtaa actcaaaggg
12900ctcgcgtgtg tttgtctatc gagtgccaat gagttttagg agtagcgaca
gaaataagtt 12960ggcagaagaa gaacatacga actatgtcgg gctacaagat
tcttgtgttt actttttgaa 13020aaagaaaatg catttgagaa aatgcaaatg
ttcggcagaa atcgaatgga gtttagagca 13080gaatggtaaa aataaaggtg
gatcagcaaa aatagttgaa caaatatttt gtagatttca 13140tgaaagataa
caaaaaaaaa taaatacaga aaacaatata tgacgtattt ttcaatcatt
13200gtttttgtat agtgcaaatt cagtagttgt acctgttata agtacagcga
agttatacat 13260tttagagtgg gtcttgtcac gatccatatt ttttgaacgc
aatatttgaa atccaaaaaa 13320aaataaagaa actaggcgcc aagaagctat
agtagctata cgcataaatt gtgaatacct 13380tgaattacat taaattccaa
caaaatagga aaatcatata aaaacgaagt tagttgtcaa 13440ttcaaaaacg
tttttaaaat tgttcataag cgccgagctg tccccctcag ttttcgttta
13500ttcagctttt ctctctctct ctattctcta tcgtcaccta tatttcatag
tccccttatc 13560caaaagtgga agtgaatgag gatggaaata tgataccgca
tgcttcaaaa aaatttgctt 13620atgagaaacc aacatttgaa aatttccagg
aaacttgtga acgagcctgt ggtaaatgga 13680gaaatgtggc agtgtgcgag
ttgccggccg aacacggaga ttgccaactt gcgattccca 13740ggtatgtact
gttgacacat tttacaaatg ggatgggaag tggtcggtga tcaggtggaa
13800atgttgatgg caaggtttta aatagatgta gtaactgaaa acaaaatgac
agatgtacat 13860acataaatta ggattaaaac aaaaatacta tgcggagtca
ggtgactaat ttttctggaa 13920attccagaat ttgaaaatgt ttttctctgt
ttgaaagtag aacgggacct tttacaaaat 13980aggctgaggt aggtaggctg
tagaaagtgc ctttggtgtc tttgtaattt ttgttttcaa 14040aaaatcactt
gtaagcacat gaaaatcaca tgaataatga tgtaaaattt agaaaattag
14100tataaagaag atttacattt taataataat aattccagat ggtaccatga
cccaaaaaca 14160tcccaatgtc aaatgatgat gtggactgga tgcggaggaa
atggaaacgc gttctcttca 14220aaagcagact gtgaatctct ttgccgagtt
gagacattat ggtccaacaa cactgacttc 14280tgtacattgg aacgatcggc
cggtccatgt acagattcta tttcaatgtg gtatttcgat 14340tcaactcatc
tcgattgtaa gccattcact tatggaggtt gccgtggaaa tcagaatcga
14400ttcgttagca aagagcaatg tcagcagagc tgccgtcctg gagacacaaa
atctgaggat 14460atctgcacac tccgcccaga gccgggaccg tgtcggctgg
gactcgagaa atacttttac 14520gacccggtga tccaatcctg tcatatgttc
cattatggag gttgtgaggg aaatgcaaac 14580cggttcgatt cagagttgga
ctgcttccga cgatgctcga gtgtcaaggt tgaagcaagt 14640gaaagcgaga
gagtgggaca gctgacgtct gcatccacgc cagttattta tattgttaac
14700aaaacagcga tttttgttgg aaatactgta agttattaat tttaattcga
agatttctta 14760atatttaaac tggtcccatg agagtttggt tcattttccg
acaatagact gcaaaattga 14820taacttttca tgaacacttt agccgatttt
agctagtttt gtttattaaa atttggtaat 14880tcaaaataaa aaccttacgc
cactccactt ttgaatactt gtcaaataca ttttttcagt 14940tccgaatccg
atgcaacagt tacggagtgc ttccaataac atggtacaag aacggaggtc
15000tcctccagtt cggctcgcga atcactgaag agaatgatga cactttggaa
attgtggatg 15060ctttaactgc tgacgccggt gtctacactt gcattgccgg
ccaggatagt acaatgagcg 15120agggagtcga ggttgtgatc aagagacttc
ctggtcacag aactacatct cgtccaatgc 15180tgacaccatc caagaacttc
tccttgggaa ccccaccgac accatctcca tctacagttt 15240ctacaacacc
cttccgaatc tatacgcctg gatctgctcc atctgatgct cgtgtaagcc
15300gcccgacaag caattcctgt atggatgtgg gtaacgcgag cacgtgcgat
ttgatcgtga 15360agaacggttt gtgcgggaag aagcgatatg gaacattctg
ctgtcacact tgcacccggg 15420ttcataattt taaattttaa gtttggattt
tttgatttca aattttcatt aatcttttaa 15480tgttttctcc ttcataatat
ctccattgcg agatctcttt ttcccttctc ttcctatact 15540ttcccctcag
acaattggct aattactcgt tcgttccagt aaataaatat gaatttattt
15600cttcttccta tactttggta tacataatca tggcatgaaa tacaagacaa
aaaaaacaag 15660aaaaaacaat ccacttgaaa tccattcagg tgtgaactaa
catcttactc tattaacttc 15720gtgccattac ttccacttat tttgcctatt
cactaatgaa gtctctgaga attattttct 15780gtctaactct gctgattgca
agcttcccag ctcagcggag ccgccgaaaa cagaaatttg 15840tacgccttcc
tagtgggttc acgtttcctg cggatgcggc gagtaatttt caaagagatg
15900cgtatattcc agcgacggta aattttcgct ttttgttaaa tgaatttcag
gcttcaaatt 15960attttctagg acaaaaattt aaagtaggct tgcgcatact
catttccctg ccttacctgc 16020caacaggcta gcttttggag agaaatcaaa
agtttggtgt ctgtaaatct aagctttccg 16080aagcgtccga aagtttttgg
gaatccgcta tacactttaa gattgataaa tatttgaatc 16140aggtttattt
tgcactatta aggcgtgtag gcactaggcc ggcaaagctc gcctacgggg
16200agccttacaa tcaagtatta ttcatgaagg tcttgatttg gttacagaat
tccatctaaa 16260attacttata caaaaacatg aaaaatttca gtttgccccg
ccatctgaga agattcttca 16320agctccacca cgctatttaa ctggagaaca
caatccagct tatggtaggc ccaatttttt 16380atctgatttt ctaaatttaa
cttcaagctc acaataccga tgtgcaagga atgaactacg 16440ctgagtacaa
gcaagcgatg gccccacaac cacatccagt cgatgcttat tctccaccac
16500ctcctgcacc aatggtccca ccggttactg tagttgaacc acctgcaatg
ccgtatgaaa 16560tgactacgat tgcatctgtt ggaccactta ctactcccgc
atcagtcggc ttgaagaagg 16620gaaagtttgt gattttagtt aattgatctt
tcaagtaatt ggatacaatt tccagcatcg 16680gaggaattgc tcaaaacttg
aacgacaggt acaccagctt aacaccagaa gctcaacgtg 16740ctcagaaagg
tcatacctat acggctctgg gcggtggaca attctatcaa agtttacttg
16800gaggggtaag atgcaaggtt agaacttaca aactcaattc attttacaga
aaggaggccc 16860cggaggattc tccccactct cgttctttct aaacggcggt
ctaggaggta ctggtggtgg 16920tggtaacaat ggattcttcg ttccggtgcc
tgtagtcatt ccgcctccac cgccaccgcc 16980accaggacca aactgtttca
cgaacccgtc gggattcctt tgctgtaacg tgacacttga 17040gaaaactatg
gaagacgcgt acctggccgc aaaagcagat ggtgcatcac tgtgcaatgt
17100acagaaaatg gcaactgcag tgcaagcggt ggggtttatg gatttcattt
tataatgtaa 17160tgtgctcttc cctagaattg aataagctta caacttgaat
tacgacttga attacaactt 17220gaataagctt aaaatatcca ccaaatttca
gcaagccgaa aaaaaattcg gaacaacttt 17280cgaatcagtc gctgctcatt
cggacttcgt cgcaaaaatt aattttgccg gtgacctgaa 17340ctgtaaaata
gaaatcgatg ggaaattcat actagcgtac gcaactccaa tcgccgagca
17400agaggtgaac attgtcgatg ctagctcatt cttctcggga gctgctgata
aggatttgga 17460tggtgtcaat ggtaccaagc ccacctacat tgtctacggt
cccattaaat aatggagggt 17520ctagctttaa agatttctgt atattaaagc
tgaaatgtga attaattgtt tatttgccaa 17580tcacaataaa gttggaaata
tcatttgaat agttcgaaag ttttcaatcg gaatgggaga 17640aaattcgaaa
atttaggtgg aggtgaaaag ttgatgaagt aacacaatta actgtgctcg
17700aatcctgaat agaaggagaa aagagcctat aaacagattt tcaatttaca
catattacac 17760aacaattcag gaagaagaca gtagttgcaa aagaaaatac
gtagaaaaaa gagtgaagga 17820ctggcgggat gtcagtttgg atgtacaaat
agaactcctg aagcataaga aacagaagaa 17880tcgaccgatg atcgaacctg
aaatggattt attgttgatt gaaaaatatt aagcaattct 17940gaatctctac
cttgtttgat tgtgtgtaat gcaagaatct aaactcgtga gtgtgattgt
18000tactgatccg gaaatgttcg gctgcttgca gcattatcaa tatcggatta
cgcccacaaa 18060tcgtgttctg ggtctttttg aggtagtcat taaaagctgc
cggattaagc gtctcaattg 18120cgctcattcc ctgcttatcc atattggtta
tctgctcata aatcggaata gaactatgac 18180gatcgtacgg agaaaagctg
aagcgttctc cccaatggca aaagtccgaa gagatcacaa 18240acaagtttct
tggatcctcc atgtaatgag caaaaatatt tccatacgtt tgctgcctag
18300atcctggtaa agatccaaca agtaccggaa caatggtgta acgttttgaa
cccataacct 18360ttgcaataaa tgggagttgc atttcaatac tatgctctga
ttcttcatct cggcgatcca 18420tcaaatcgaa atgacgagtg gcacgaagct
cctcgttaac tgcaaagggc aatgttgtaa 18480aagatgtact aagagtgcaa
tagattactt ttgtgatcaa cgatcaagtc gccgagtgga 18540gttctgtact
tgctgcatgt ggttatagca catccattta gagcaacaac gtgagatggg
18600ccaagaatga agactctttc actgaaagtt attgagtaag ccctgttgcc
aagtacaaat 18660ttcaacaact cacactgctg atgaaacaac ttgtttgaaa
gcatatgcag ctgtttctcc 18720acaatacgaa tatcccgcat gtctgaaagt
tatcagaaaa taaatattaa atgcatttag 18780agtattacgg tgaaatcaac
gctcgagccg ttccaatccg tggaccggcg ttgtcaagcc 18840attttgtgag
ttgccgatca agatctcgct ggttggcgtt gtaccatgat ccggcatgtg
18900aggcagatct cgtgtgctcg ccgaatccgt ttagtgacat tttaaattca
gatggtctga 18960atattaaagt tttgataaat tgttgtatac gacttgatta
atatgtttag tagggttttc 19020aactactgtg tgtttcccaa atagtcaaca
ttgaaaaatg gaaaagtttg aatttaaata 19080ttcaaataat tttaattaat
taatattaaa attcacaata cagtgtaaca tcacacttaa 19140ttcaagatgt
tctaaaaata tgagccatcg ggctagctct acttcacgaa ttcgaatcaa
19200gtccggggaa ctggctcgaa agaaaataaa tttttaattt ggtttatgtc
cgaaatagaa 19260atgggaatct ggtttttcat tctgaataat ttccgagaaa
cacttacaaa ataaaattca 19320gatatcttgc aaaaggaagg ccaaatgtcc
tgagaaatag agcacgagag ttttgaaata 19380cctgcaacaa caggatttgc
ttctattttg ttttttgaac tgaattttaa actattatct 19440attctgaaaa
cattttttgt ccaaaaaaaa tcaagaacaa tttagagcaa aatgtggcaa
19500tccgaaaatg ttgatgcaac aaaaaagtgt tttttttttc attgaatttc
agttttgaaa 19560actgatttct ttccaaaaaa aaaacgaagg aaaattttga
gaaaaaagtg aaaatccaaa 19620aatgctgatt ttggtttttt tttcaaaaaa
aaagcatttt gcaaagtgtg tgcttttttt 19680cgaaagtttc agaaccttga
gacaaaaaac caaaattgtg ttcccgagtg aagcccgcca 19740cgtggacatg
gtcagacgaa tcttgttcgt gttcgcagcc aattttcatt tttgctgaac
19800gcataattgt tcaaagaaga ttcggtctaa aaagacgaaa ttgaaataga
ttgtggaatc 19860ctttgaaatt ttcttttgac aaaaggtcac cgttattcaa
aaattgagat ggtctcgtga 19920ctaaaattaa acaatcaaga taatcatgat
tgtgggcctg ttttaaaata cacttttcaa 19980aaacgaaatg taggctccaa
tccaaactgc gcatcaagac caagaatata aaatttttaa 20040actcgggaga
cgtagagaaa ctttgaatat taaacatcgc cgtcaagttt ccgtcagagc
20100gcgcctgaaa ttttttagag gcttctttca aaaagctacc catacaaata
atcataagaa 20160aaacgtttta aaactttgca ttccacccaa aaatgtctga
aattacccgt aaaaagaatg 20220tgtgaaggga gtgatttgag ggttctgtca
aacagtttga ctgtttcgcg ttcgacgtgt 20280ctcgacgtgg atggtattga
agaggaccgc gctgatcttg tgctggtcgt cgtcgtcttg 20340tcggaccgcc
gcgagtagtc ttcagtctac caattacctg aaaatttgac actttttgtg
20400atgtgaaact ggctgcctga agcaatgcca tataataatc ataataataa
taatgaagag 20460ggatgaggat gcatgccaaa agaatgaaag gaaagacgct
cttctacaac accagccgat 20520agtatttaga agaaaaagaa gactaaaaag
agagtattgg gtgatgggag aaagaacaca 20580ataggggagg cagtgaaata
gaacgagaac aatggaatcg gcagacattt gacactagag 20640gggccactgt
ttcagtcttt ttcgcacttg aatattggaa gagggccaag aaggggagtt
20700ccaagaatgg aaaaagtggt aggtttgtag aaaatctgcc tttttttttt
taaaatttcg 20760tgttcactac tttatttcgt gttcactcgt ttatgtcttc
cattataggc aggcaaagtt 20820tcatgcctac atacctgcct catgcctatt
tgactttcaa tataaaactt gatttttggc 20880attcttcatt ttataacaat
tgtaactaat aataagcttt gcaaagtttt ctgaaagaaa 20940ttgtctaaat
tttcctggta cactgaacat ttttcggtat aaaatctatg cgtatcaagc
21000ctatttctaa gagccgtaag tattttcagc tgaaaatgta aaccacggag
tcaatattta 21060cttcgtatca tccatcttcc attccgtctt gtttacacct
acggcaggta tttagacacg 21120aatgattgtt tttctcgttg cctaatactt
tttcccccga aatattccca tattccagtt 21180ctgaacaatg cacttttcag
cggtcatcgg gtccatccag ccctcattca gccctttcat 21240ttatcttcgt
ttctactttt agacgaaaat gcaaaaaaaa gagaaaaaga cactctcttt
21300tgacgctcac attcgctcac attgctgtgg tagaaaaaca ctcactcggt
ggctgctggg 21360aagggaaaac gagaaaatgt ttggtcacgc aatacgccta
tatctttgat ttgactttga 21420atctttatac atttttcacg gggttcaaaa
acaattatga agaaaattgt ttgattaaat 21480tagaatgtag attctttata
ttttcaatca aaaattaatt ttggaaaaat aactatccaa 21540aaaacgaaaa
aagtaataaa tgagtacttg aaagtgaaat ggggcaatta aacaagataa
21600aaaagactaa aacgtgagac atctcacaac gggtcacggg caagaagtac
acgagaaatc 21660gaacgtgagt ggggaggcag agacactcag ctgactgcct
ggcctgacgc tcgctcacaa 21720aacgctctca ctctcttcct cgctttgccc
gctctccgcc ccgggtcgtc agttcggtcg 21780atccatgttt gttcattttt
ataggtgaaa atttatgtaa gggaacggaa aatgtaaagt 21840gatcgtggga
aaatagaaaa acaattacat tgtaactttt ctggaccaag ttgtacccag
21900atgcaatatg tatatttttc tcagaaaata ctgtgttggg tttcgacagg
atcgatttat 21960caaaagcaaa cgagtgtgcg tctcaacgag cactaaagtt
cccaactaga gcatccttgt 22020tgtggtagaa ctacatagaa atttttaatt
ttgatttcaa tagcttttct cttgttttct 22080caaaatttat tgaaaaactt
atttactata aaacgaccaa cgacggatct ggaaactaca 22140gtactcctta
atgcaaaagg caacgaaaaa tcagccagtg acttattttt tgttctggat
22200aaaaatcggg aatatttgca ttttgaattc gcactgtatc gataaacaaa
acaccgaaga 22260tcacgccaaa atgactattg taactaacag gtacgagaaa
gggacgcttg ttctacaaaa 22320ataattcaac aaattttccc caaaaaaatg
tgaagtccgc aattctcgta gttttacgta 22380aatcaaaccg agcatgacac
tctgacacca cgtgcgcctg aagatgtgcc tgcctaccat 22440ggatgcttta
catttgctag ttccatgaca ccccatcctt tcagcttcca agatgaagga
22500gttcggagaa aattcgaaaa aatattgaga aaaataaccc aaaacattct
gaaacattgc 22560ggaaaaaagt tagaaattat gtcgaatata tctgaaccaa
tcaacaattt caaataaaat 22620acaaaaaaaa attggaagac cttaaatagt
ctccgcccat attttggctt caaatgaccg 22680tacttcggaa tatggccgat
ggccgtggca agacctccaa tcgtagtttt gagcggtcag 22740taagtgaaga
ttaaaatagg aacagtaccg taagatcagc ccaggtgcgg atgtgggata
22800gaggaactga aaataatcga agaagcatga taactaagcc acgtggccac
gttcgttttt 22860gcgatgttaa tagatcgcca cttcgtccat tgtcgttttg
tttgtactaa gtctccttag 22920caattctctc gaaggcgggc cattgctatt
agtaaaataa gctaccaatt ttacctttca 22980atacattcat tcactgatgg
ttttcctatc aggtgatcat ttttttgttc ttctcaatta 23040cactatctaa
aaatgatgaa gtttttgctt cgcggctatt tggttgaagt gatgatatat
23100ccattgattg tcgtctccac ttgtgctctt tttacgtctt acaacttctt
tttaagtgtt 23160ttgcgtattc actgtttcat ttattttttg cagaaaatga
gcctgttcag caaatttttc 23220ggaggcatga tgcaagaagc tccgattact
ccacaagaat ctattcaaaa acttcgggaa 23280acagaagata ttcttgagaa
gaaacaagaa ttcttggaga aaaaaattga cgacgtaagt 23340tggaagatca
gttttggtcg aattaatcac attaaaaagt gctgaaatcg aaatttttaa
23400actctcgagt ctcaagtgac tgtgacgtaa ttaaaacatt gctcagcatt
tacattgttt 23460actgacgtct tttcgaagtt tagtcgagca aatccaaaaa
agagcaataa aaatttctgc 23520tacgatacgt ttgggaaatt ggaatcatag
ttttttaaac tccatttttc aaaaaataca 23580ttattagaaa atcagtaagt
ttcggaaatt atttgagaaa cgtttcagga aagcaaaatg 23640ccgtgaagta
tggaacaaaa aacaagcgga tggctctcca gtgtttgagt aggaagaaag
23700ctttcgagaa gcagttgatc catattgacg gagttttggc tactctcgaa
catcaggttg 23760gtatataaaa atattagaga aataaattga ataacacggt
ttttcttcca gagagaaacc 23820ctcgaaaatg cttcaacgaa tgctgaagtt
ctcacggtta tgaaacttgc tagcgatgcg 23880ttgaaagcgg ttcataataa
catggatagc gaccaagttc gtgatatgat ggataacata 23940gatgaacaac
gagaagtggc gaaggaaatc gcggatgcta tttcaaaccc tggctttaac
24000aacgcaattg acgaggccga tttgctgcgc gagttggtgg atcttgaaca
ggttcgtcta 24060taccaccaac atcgtgtaat tattagaaaa tataccagga
agcacttgac aaagatttgc 24120ttgatgcgag agctccccca gtcacgcttc
cggatactcc caatattgca cttccagcct 24180ccagaccgag agctaaagaa
gctgacaagg atctagaaga cctcgaaagt tgggcaaact 24240aacttctcta
agtcactttc atatttaatt ttcggctatt tttgtttcat ttgcatcccc
24300ttcatcaatc
ctaccattct ccggagattc tcctaaatca actttctaat tacgacaaat
24360tcaaatagtt gaatgatttc tgtttagcca tttcattcga aacaaatttc
cccaaggcta 24420cgatcaacac tcatcaaaat tgtaacatat tatcgagctt
tttggaaatt tgtcatttta 24480tacatcttgg tccctttctc caaaatcttc
caagcatgca ttaaagttcc aacttttatt 24540aaaaattcat tctggcaaac
atgttatttg taccggttga aaacgaaaac caagcgagaa 24600atagttacat
ctcagatctc cctaacgatg gctcaacccc tttgacgctc atttactaat
24660gtttatactt ttgctcattt actaatgaat ggctcattta ctaacttgct
gagatttttt 24720aatttactac tgctaattgt aagatatata tcatttatca
tttactatat ataaagcgct 24780tattccgttt gtccatagtt tgtagtctat
gtagtctttg tagtctgtga cgttttggct 24840tctggaagga tagtgagttg
ggcttagtgt agggatatag ggggtactgt agtggtacaa 24900tagtggtacg
gtaggagtac tgtatgatta cggtagtttc agaaaaatta gttttcagct
24960ccagaagtcg ggggccgcgc cggaggtgcg gtccacggct ggttttacat
aaggtagttc 25020caaaaaatgt cctacttcca attactcata actcagttag
cgcgctatag ctatagcgtt 25080tgagtttaaa aaaattgtgg ccaactgaaa
tgctgtttgt cagagatgcg agctctaaaa 25140gatgatcgaa atattctatt
tctgcggatc tagaatattt cgatcatctt ttggagctga 25200catctccgca
atcgctaaag ataactaaaa ggtaccaatt aacaaaatgt gttttacaat
25260attgccaaca acattttagg tttctttcgc tgattgtttc cttttggttt
tggtgatggt 25320cccggagtgg tttttttcgc tggttctact attttttgga
tcggcaggct ctgaacaatt 25380ggttgtacaa tcttcttcaa cttcatcaaa
ttatccagag ttatgttgtc gcttctgctg 25440tccaacatat tcatgcattt
gacggaactc ttcaactttc tgcattgtca ctggattctt 25500ctttttcgat
tttattttat gaaaacttta ctatcataaa caatagtatt tatcatgtta
25560caaatcagtt tggaatgatc tccttcattc aaaattctta atgatcagtc
gattcactct 25620tagagccacg aaaaatgtgg gacaattgtt tgagaagtga
aaaatagtta ttaatgttgc 25680aattagttgt acatataagt aatacatgaa
aatacatctt aaaaatacag ttactactag 25740gtattattgc ttaaaattgt
gttccaatct gccagtacta tgagcgtaat tcgttgatcc 25800aatcttcgaa
tagccgtgag cacaggcttc gccggcactg cacacaaact tcacgattgc
25860acgatttgca gaggtagagg acgaacgact ttcctgtaat tggcgaaata
ttgttttaag 25920ataaagttag taggaacgat cgtactgttt ttagaacgag
actgtctagc tggtggccgc 25980atcgagcatt gatggcatcc aagaccttga
acttcttcgc tgaatgatat acgatgcttg 26040aatatggatc cactgaaaat
tgaggttata gtagattatt gggagctatt atgatttcac 26100ccatgaagaa
ctgcgtcagt aactcgtttc agattctcgc tatccttttc accgcttttt
26160cgttgtaatt ctatgagaaa acggtagaat ttggtgacat ttgtcgagtt
aaacaattcc 26220acgaggcaga caaacatctg aaatttgcgt tttttccaca
aatgcataaa ctttcaataa 26280aacaaaccgc ttctagggca acatcagcta
aactgtgatc atgctcgtat tcggcgttta 26340gcgagaagca taaatggtag
aataaatgaa agatatcggt aggttcgcgg gaatccggat 26400tgtagtcttt
gagataatca acgcaatttt gtttcagatt cgtcatcagg tatttgtcgc
26460atagcctgag aactgtgcac acgttttgtt ctgaaaataa atttggcatt
cattgaaact 26520acatcgatca tgaactacca tcaataacat ccggatataa
accaagagaa ttgggagaaa 26580tgacagtgat caacttgaga atatcttccg
gtgactcatc aaggatattc agctgcttta 26640tggcgccttc aaggaaaaac
ttgttctcca tcaagatgcg gaaataatcc gaatttcttg 26700caaagattgc
tggatccaca aagtactttt gattaccaac aataataggc cagtttcgaa
26760gcttgcttgt cgactcaaaa tcgacctgaa agaaaaatcg aaaaattcca
atttaaaaaa 26820cgtttgttta cgtaatcgga tccttctagg aaggtttcat
gacttgttgt cggctgcatt 26880agaatgacgt ttacggggaa atcattattt
attccgaaac gtgcttgggc ttgttctgtt 26940tgctgaaatt ttgaaaggtt
ctccgaatat taagcgaaaa aaacttacat taataatata 27000aggtctcata
gcgccgagta gctaaacaat taatatttga ttacaagttt ggaaagatct
27060ttctgagctc gatcaggaag aaaaacttct tgaaacttta gaagatgaaa
tgtgtgctac 27120cgtataaact ttaaaggtgc atgaataaat ttctcctttt
ggtcctgcga cgattaaact 27180ttttaatcaa ttctctgggc tagtttttat
tcaataacta gaaatgttgt ttatttttgt 27240tccctactta aatcatatgt
tattttcttt ttcctttgtg tcttacaggc ttttttagct 27300gaagaaatag
caattttccg ataaaatttg ttgctctatg ttaaaggcgc atgcatttat
27360ttgagagacg ggtctcgcaa cgtgctcact cctcggcccg atttgttctt
cgtttgcgcg 27420gttttcaggc ctttaaaaga tagttccgtc gtttttttct
caatttctgc tgaaataagg 27480tttaattaaa tttattttca aaatcttggt
aaacatttaa actcatatat tcagaatttt 27540cattcctctt tcacccagaa
aaccgaattt caatattaag attaagaaca catctagaac 27600atgcaaaaaa
cacaattgct atctctctac tttcatttta aggctgattt tttgaagaaa
27660aatcatgaaa tacgtccatt attgttgtat cccttgtttg catccaaagt
tgactcgatt 27720gatctcttaa atgtggtatt ccgttcgaaa ttcgattgat
ttttagaagt taacacattc 27780ggaatgatga taattcgtat caaaccaaaa
ttgtcttctt ttcgcctttt ttgtgcagtg 27840tcagcattaa acaaaacgag
aatattgaaa gttacgtggc gtttgcatct ctcaccacga 27900tgacatcacg
aaatgcagac gacaaagacc ggtgaaaaat agtgcgctga atggtgaaaa
27960cttgcgaaga taacgtgtta cgggttgaga gagaaaacat tccgcgagac
aatgcttttg 28020gtgagaggcg cagatggttc agagaacact agagaaaacc
gcgcctctgt ccgctcacag 28080ccagccccat caagcctctt cgggcatcga
cgcatagaca cacatcattt tgccccaatt 28140tcctttcatt ccgtcaagta
tttcgcaact aatcgttatt gctcattaca atacacattt 28200tacagaagtt
cctcttcttc tacttggtcc gaccgcatca gataactggg agatccagtt
28260gtgcatgttc ttgtgcccac acaaactcgc gcccatttac aattttatga
tcgacaaccc 28320tcaagaaggt aagcatttaa acgtgttggc cgtgcgtctc
aaaaaattgt taaaaaacct 28380ggcgacacgc gtttttccac aatttcattc
cctagggcat tttgtatttg aagtaattct 28440attacgcgta cgcaatcgga
cgaatcctgc aggtttgttg gtagtcaatt ttatcaagtc 28500gactgcctct
tatgctttct gaaaaaagag aatgacagtt ttcgctaagt agtactaaag
28560cgatctttta tctttggcaa aaccttgata taagcattat cacagcatat
catgcagatt 28620gatttagagt taagcatgaa atgtgcaagg ctaaaataaa
ttacaaaata agtccatagt 28680ccattttagt aacagtatac atcagctgat
agaatcacat gcgtaatgac aggtctaaaa 28740cattatcaaa caaaagacat
tacaaaaaca agaaaaatac aatataatag aacgactatt 28800tgaaatgagc
gtagttaaat tcggaacttc aatagattat catacgcgct tttaaaaaaa
28860tgtgtgttcc cttttctccg cgtttgcccg ctacaaaccg gtgagtcgga
aggcataatc 28920gggttgaaaa aaaagtatca aacactgatg gtgtcttttt
tagggaggtt gtccagaaag 28980agaaagaaac tgaagatttg cgaatcgata
gcgtcgtcat ctctcgacgc cagtgaagtc 29040aagatcggtt acaatagtgt
atgcgattcc caaaatccac atatcaaccg gactcgtgat 29100atttatcatt
tgtaagtact aacaagagat gtgaacgtat ttacactcaa cattagcaaa
29160ttccagaaga agatctaaac aaaaactatc gaaatggctc tcaacgtgaa
ccgcgctgtc 29220gctgatccat tctaccgcta caagatgccc aagctgtcag
caaaagtcga aggcaaagga 29280aacggaatca aaacggtcat ttccaacatg
tctgagatcg cgaaagctct cgagcgtccg 29340ccgatgtgta tgtttatcgc
cagttggctc gccattggac acaaaaataa ccattgtttt 29400tcagacccca
cgaagtactt tggctgtgag ctcggggctc aaacgaactt cgatgccaag
29460aacgagcgtt acattgtcaa cggcgagcat gatgccaaca agctccagga
tattttagat 29520ggtttcatta aaaagtttgt gctttgcaaa tcatgtgaaa
acccggaaac tcagttggta 29580cgagatcatt gaattaataa tctgtctaat
tttattattt cagtttgtcc gtaaaaataa 29640catcaagagc aagtgcaagg
catgtggatg ttcgttcgac attgatctca aacataagct 29700gtctacattc
atcatgaaga atcctccaaa gattgatgtc gatttttgta agtatcgttt
29760actaacattt ttcgattgaa cttatgcaaa attctgccaa aaattctatt
tgcattttaa 29820atcctttcaa ttcgattttc cgtgtgcttc cagtgcatac
aaacatgcta atttttggtt 29880tccagccaaa gccgaacaaa agaatggaaa
gaagacatcg ggtgctgacg ccgccgccgc 29940cgtggctgcc gacataatcc
acaacagcga caaaggcagt tcgaatgatg acgacgacga 30000cgattgggaa
cctgaaccag tcgagccgaa tggcatgctg tcggcgggaa tgggcaagct
30060cgtgctggac aaggatcttg agaagagcga agaacagcgt ctcgacatgc
ttcacacatt 30120cttcttgaaa gccaaggaag aaggtaagaa ttctgagcat
tgataaaaag tattctcgtt 30180atttcagata gaatttctga tgccaaggga
caaactgctc tacgtgacga agctgagaga 30240cttgagctga agcaaaaagc
atctctcctt ctcgcgaacg tttttcttga tgagaaagta 30300atcactgaca
aacaaatcag caaacaccgc aatcttctgc ttcgcttcac gttgaatgac
30360aagaaagctc aaagatacct gttgggagga gttgagcaag taattcacaa
acatgaagcg 30420gaacttctgt ctaaatcagc tcacatcatt aagtcattgt
atgatgaaga tgtctgcgaa 30480gaggattcgc ttatttcatg gggagagaag
gttagtacca aatggagctt tgtttcgaat 30540taaagtttat atttacagcc
gtcgagtaag tatgtctcca aatcttttgc caagaagatt 30600attgagaact
ctcaaccagt gctcaactgg ctgaaagaag cggaagaaga aaccgaagaa
30660gagtccgacg atgagattgc ggtaagaaat atcagatttg tttttttttt
ttcaatggtt 30720ggttttcagt tcggaggaga cgtcaaggag agtgaattcc
ttcgtcaaca gaaggagaag 30780gctgctagag aagctcagca aaaatcagcc
aaggctacaa acggcaatgc tgctgctgca 30840tccggagcaa atgatgaaga
ggacttggat attgatgaca tttaattgta cagatgcttt 30900tttaaaattt
acctgggcta cttatgtttt ttgtgtattt cttcccatat tcgaaccaat
30960tcaactaatt tcgaagaagc ctcagttttt ttttgctttc tccccctttc
aatagtaagc 31020atcatttcat ttctgtcttc tgtcttttct gttcctacgc
tgttttccct tcaccaaatc 31080caattcattt attcgtaaag tcattactat
ttgttgttaa tcgtaaacat ttgggaatat 31140tcttgttcaa ttcagtctta
tattacaaaa acacaatgtt caaaaaaaaa gaatcacttc 31200agatgggaac
ccgtcgaatt cggcggtccg atggagaata cacattgttt tttcggaaag
31260ttagcccatt ttcaaatcat cacccagctg atttcatttg cgacgaagcg
ataaattgta 31320aagagccgaa aaccttttgc tgctcggaac agtactatat
gtacaataag gcttcactat 31380tgatggattc aaaactgatg gcagcgattc
tagaagcaac ttgtccgaaa acaatgaaga 31440caatgtgttc taaatggtcg
ttgaaaggat ggaaggattc ggtgtaagtt ttaaatcagt 31500ttgataataa
aatatgtttt tcttttacag atgggatgag aacaaagaag aagtgatgag
31560aataggatgc ttggcaaaat tccgtgcttc tcgccatctt cgttatgctc
tttttctcac 31620aactggtagc aaactagtcg aatgtagtcc gttcgataaa
atatggggaa tcggttagtt 31680tccaacggat cgtcttattc ttccatcgcc
catcacaatg caatcagaat cttcaaactg 31740gaaatgtttt gaaatcattg
aaatcatctt tgagctgata tggtgacgga agaaaaggac 31800gtctgaaaat
ggctgaatta ttataggaaa agatatgcaa gccgcacaat gggctccatt
31860gagctctggc aagaatctgc tgggaaagat tttggatgga atccgagagg
aattgtggga 31920tgattcaaat tacaagttag ctctggaatc agaaaattat
tattatataa aattactatt 31980tcagagatga acgagaagaa gtggagaaac
gaatggaaac tgaaagagat tatctattca 32040ctgctataga gcacatggac
ttgatgtaca aagaaagagc aacaaaaaga gtattgtaag 32100aatcagaaaa
tctgcgtaat tgtcgacaga aataacgtat tccagattgt tcgaggaaga
32160attgttaact gatgatagat cctacatcac accagatatt cagaggctcc
ttcccgactg 32220ggcttggccg ccgatcctcg tgaaaaacga gcctattcaa
ccatcgctgc ctgtaataat 32280cgatttccct aggtacttgc cttgatcttt
aatttatcag aattaacttt caaattccag 32340atcatctcca cttcgagcag
ctgaaatatc acgtaggaag agcacatctc attcgacaag 32400cttgagtaaa
aggcggtacc tcaggagcag atcgagaagt ctgtccaaaa gcccggctcg
32460aagacgctcc agacatcttt cccgaagtgg atcccgtaca ccagctcaac
ggcattccag 32520aagatccgaa agtacatctc gaagacgttc cggacggcac
tctagaagtc gatctagaag 32580cccaccacga aaacgtccgg tacgccgatc
aagaagcaga tccaggagca ggacaccaaa 32640ccgaaattgg acaagagcac
ggagcagaac aagaagtcag gctaaaagta gcagcacttt 32700aacctggcca
ctgagcccat cgagaagcag aagtaacagt aatgaaagga atttgaaaga
32760gaagaaagac cggaaaaaga aaaaatctga gaagaaacgg aagcatcatt
ctaaatccag 32820aaaacaccgt tctaaaagat ccgaatccag agaagaacgt
cacagaagac ggaaggagaa 32880gaaaagagag aaaaagaaga aacgacgtcg
gagaagttcc actacttcag attaaacttt 32940atttttgaaa actagtcata
actttaaaag tcataacttt tttaaaagtc ataacactgg 33000tttaatatca
aatgtctttt caaatattct ctatttattt attcttcgta attaaactga
33060gattaagtac tgggtatatc attaataaaa ttacgatact ttgccgaata
aatcagttat 33120aattacaatc tgtctgctgg tgaaaattgt acatgctatt
ttcttgttcc tcattctttt 33180ttcattctct gtaaggtttt gttcgttttt
tggaaaattc tgagagtagc cggaaaaaaa 33240aaaaaaaaaa actaaatacc
tacagtaatg ccagaggcat atgctcaata attatcaaaa 33300attagttttc
cgcggcgaga cccatcccca caaaagtatg actcccttga aagtcgtaaa
33360tgacaatttc ttgaaacaag aacatttgta tattaacgaa acacaaaatt
ccgagaatgc 33420gtattgagca gcatatttgc cgagccaaat atctcgtagc
gaaaactaca ttaattctta 33480aaaacactac tgtagcgctt gtgtcgattt
acgggctctt tgaattatca ttgatttatc 33540gatagaatat ttaaaaaata
aattcatttc gaaattagag cccataaatc gacacaaaca 33600ctacagtagc
catttaaaga attactgtag ttttcgctat gagatatttt gcgcatcaaa
33660tatgttgcgc aatacgcatt ctcagaattg tgtcttccgt aataatagac
agtggcttcg 33720ctaaaaacta agaacaaagt aaattaaagt ttttttctgt
tcacttcaaa ttttacacga 33780tcttgaagca aagttcaaaa gagcatgaat
caattggaaa gtgttcaatg caccctacag 33840atatgatttc ggggcagtgt
aaactacagg gcacagacat aaaaatttaa attgttgaag 33900actaaaatat
aaacatatga attcaagggt cataataaat gtattttttt aaataatatt
33960tattaaatgt atgcatacaa ttaaatacaa cataattatc aaatacaaat
attataattg 34020caacctgtcg gacaacaact ttgctgaggt gtcgtgtgac
agtcagaatc cttgtcacac 34080cagctgaccg gctcagagac gatacatcgg
aagttgagat gagtgactgg tggacattgc 34140cgacgcgttg gagcacaaca
ctcacgatat cgagtcatgt cgatgcagcg ctgaaactca 34200ggaaactatg
tggaatttag gtggatcacc caaccagctg cccttcaccg cactgataat
34260ttggagtgca gtacatgtaa tgggcagagc attgctgcat ttgcatcaca
atcaatgaat 34320ttgcaaaggg cctggagatt ggcttggctg aaagagttga
tattatttct attgatataa 34380taccctaaat ttacgaaaat tatgctaaat
taggatttta gttataatcc tcgtcacatc 34440tgatctctga aaacttaaaa
atatcctttt tggtagtgtg gcaccaaatt cgtgctgtaa 34500cagagaccaa
aaacactact ttttcgacat ttcctctcct tgcagcgaaa aataaaattt
34560tttgaaaatc tgtgttttct catacccgga aaaaaccaac aaaaacggcc
ttgttccaaa 34620ggcggtgagt atttctattt tatgaaagtg gccgagattt
ctctttttct acgccaagta 34680gttaattctt cgcggcaaga cccatcaatt
ttctaacctc taatctcttt ttcaacatga 34740atatccacgt catcatagaa
tttgcactcg ggcttataga tttggagcct ttgaaagtat 34800atgcaccagt
ctatatgggt gttgggaaac gaataggcag tagttttttg gaccaattgt
34860agaatagaca gtagtaatag ggaagaatat aagaatttca taattcagat
ttcaataaaa 34920aataaattta attgagaaaa aaaacggttg atattctttt
gtttaagcag acaagtatgc 34980ggaagtgaat cttgagcacc tcgtaaatca
cgggaggcgt acttgtacag aagagagata 35040agggattaag aggcgcaagc
tttgccactt tgaagttaaa aaataaagaa agagacatgc 35100aaattggtgg
acaaatagcg gaaggttagc gggaggtggg aggggggaca ggtgcatgta
35160acacaatgga ttttacaata ggaatattga aaatacgcat atgggaaatc
ggaacagata 35220tgaaggtgtc aatatttgag gtcaactgtc tggtttttcc
ccgatttttg aattttttga 35280aaaaaagtgc ataattcaca gattgaaatt
ggaaattggt cgagaaaaga ataaggagtg 35340ttatgaattg atggtggcaa
caaaacacaa attctacatt tgtaccaaaa tgcccactaa 35400aatgggcata
ttcgcacaca ttccacacaa attgcataca tattccacaa tggggaatat
35460tttgaatatt tagattaata aagatgaaat aattgagttt tatttgtaat
taaaatattt 35520ttctgtttat cattaattga aaatgttgaa ttacttttta
atagacgaat catcaaagaa 35580cttgatccct gcattatcag gcaatcctac
ataacctttc aacgttgtcg ttttaccaat 35640tgcaacattt ctcgctactg
gaacacgcat actggaatac gatgacgatt ccaattggaa 35700gaatatattg
gtgcccggtt ggaagttaac aattgaattg ttgttaagcg ataaaggata
35760cacattgata acatccaaaa gttcagttat gtatatccat ccgtataaat
cttgcgatct 35820tccattcacc aaaagctggt cgccatcttg tataggaatg
aatggagtta aggatcccgt 35880aacagtacga gttgtgagcg tagttccact
gaaaattact aaatatttag ttcaaaggtt 35940ttctgttact actttttggt
tgcaacaact ctgagaaatt ttagttttca ccaaaatttt 36000tcgattttgt
acagaattgc acaatatatt ttggaatagc aagaaattgt tcagtgaatg
36060tcaaatctga caaaaaaaaa tttttttaaa aggtgcctat caatttttaa
aaatgttcta 36120atattttgtt ggaaagtttc aataatttca ctacatttac
tatttctttt ttaggcctat 36180tttgggtatt caaaatatta accacacgac
cttcaataca ggaaaactgt caaatttttt 36240ttaaattatg aacaattaac
tcactttaca ttttgtcctc cattccttgt agttaatata 36300agacttccca
acgcttcttg agaactattc gaaataatat aaatcttcga atttcttcct
36360actatatatc ctagtgtgtt gctcgttgca acgtctagag tatccaatat
aaacccacta 36420gaagctgata taaagaaaaa taatagaaat atatttttca
ttttttccaa atgactaaat 36480gaccaacttc aagacatttt atatgcttaa
aatcacgtca cagaactata atcatgttga 36540tttttgatag aaaatgataa
gaaatgcgac caaaatgtgt attttctccg tttgtcctct 36600gaatgagtca
aattcacgta aaacttggca tttgtcacag tgtgtcagac acaaggcaca
36660tatgtattta ccggactttt caagacttta ttattattga gatcaaacca
gattacagaa 36720gacgggagaa aggtaccaac aaatatcaga atattgcaaa
aaaaaattaa aaatttcaaa 36780acgcaaactt caaactagga gagctaattc
aaactttgaa atcatgttcc ataaccggta 36840gcatttgttc ggtgacttgt
ttgacagccc attgaaggaa gagaagtact cccgacaggc 36900tgaaacatat
gaaatagtcc aggccttcca ttagagaatg tgatgtttga aggaagaaca
36960atgggacgta gagtactccg aatagagcag taagtccatt gatgagctga
aacagtaaat 37020aatcgaaaag ttagtaaata tgttcaagga atggaagtaa
accggaatta tccgagtatg 37080ggcgttttat agttttttct ctttttttga
cttcgttttt catcctatta aaatatcatc 37140ggttttttcg agttccagaa
aaaatattta aaaaatcatc cgaaatccga acacaaaatc 37200cgaaggctac
tccaaggtaa gttaacccta ctcggcaaat ctctcgtcct ggagcgcgga
37260cggggcgcga ctagatcacg ggttcgcgct ccagtcaccc tttttttcgc
gcttcttacg 37320cgccacgtcc gcgcttcagg aggagcgatt tgcggagtac
cttttatgca ttcagactgg 37380tacttaaaaa ttaatcgatt tttttaaaaa
gtgtcataaa ctttttctac gtctttttct 37440gacacaatgt tgaaccgtac
tagattgttg taaacacggt cttcaaattt gattttcgcg 37500aaaaaatttg
aataattttt ttctaacttt tttcttttta aaatcttacc acacttagca
37560aataaccatg aagcacaact tcataagtgg atcctatttt tcgtttgaag
aggcaaaata 37620ctgaaaacaa aagagctgat atggagcaag acacgtggat
ccagaagagt atacgcacaa 37680tcacactatc cccttcgatt ttgacgcggt
acagaattct ggaatttttt tttgaacttt 37740aatggattgc gattcaaaag
aaaacgtagc ttaatctcca gttaaagctg attttcattg 37800caaaatgtat
ttagaaaaaa ctcacgctaa taaggcggag agtattgtct gtagaaccgc
37860catgattact gtagatgcat agagtgagaa tgagcacata taagcgctcg
gctgtttttg 37920aacgacaatc gaattggccg ccatcatctc attcttcgac
ctcccgtttt atttctgaaa 37980atatatgaca ctttttaaat gaattgacag
aaatctgatg ctaactacat tttaacttgt 38040aggagtggtt caaatgattc
ataaagggaa tacaatttct gaatgatcaa agaagaaaga 38100aaaaaaatat
tggtgaatgt ataatttttt aggggtaaag taaataaata aacacaaggc
38160cgaagattag caagagtttg gggataaccc ccgtgaagaa aaatatgaaa
aaaaatggtt 38220tgaaagaatt aaaaaaatcc tttcaaattt gagattcaaa
ttttgttcat ctgttctgtt 38280cgaacattga gcagaagaag cttttaccaa
taaatccaaa atttgttaag agaatatagt 38340ttaaggatat cacccagttc
aaaatagtag ttcaaaaact cgagtcttaa ttttttcagt 38400attcgaattt
ttacagtaca ttgatcgttt cgttatttga tcgctttttg ataaaacaaa
38460aaatagataa tgaagctgcc aagtttaaaa aaatcggggc taaggctaat
ggagcataca 38520cggtatatca ctacctggat attagtttta gacttcatca
gatatttagt cagaaaagta 38580cgtcaagaag tcggatacga aatgtataaa
tttcttaaaa cttaaaactt cgagatatcc 38640agactgtggc tctcaagctt
cagtgcttgg agaaatagtt taatagtcag aatatgtttt 38700aaatttctta
atttttctga agaagtcgta aaagtataaa tgttgctaga tcaaacactc
38760tagaaaacct tcaccacttg agaatactcc agtctcaaat tttccctcga
cgcggaagtg 38820tagaagggcg cgagattcag aagtaggtga aaattagacg
gaaaactctc tcaaaattga 38880aatcaatgaa taggacaact gagacaatgt
gcaggtgtat gtgtatgcac atggcaccca 38940cgtacacgca tacatcttat
gttagagaag tacgtgtgct ccgctcatca tgtcttctcc 39000ttctcctaca
tctacatttt ttgctccgtg agccacgccg ggaaaaacga cgacgacgac
39060ggcgacgggg gacgactact cgactctaat tggccctaaa cgcaagtaaa
tttttaggca 39120atgtatgttt gcgagagttg agagccccac cgccacgagg
agaagtgggg gaagattccg 39180aagagattcc ccctcctcct tctgatcacc
tcgtctttcc ttttttgttc catttccgtg 39240aaaaagctgt ggaagggagg
agaagaactt accggctaaa tggaaaaaaa ggaactctaa 39300cttattctga
ctctacggaa ataggaagcc tacttgtcaa ttagaccgcc ctcgcacaga
39360tttctttttt
tttgtagata caaatataaa aactaactgc gtgtgatgca gcagatatct
39420tgaattggaa agtgtcagtg ctcagaggga atagccaatc attgacagaa
atttgactac 39480ttcagaagga atcaactaga acatttgacg cctgaaacct
aacaagaaaa atctataatt 39540tggagatccc tagattgatg ccaactttat
taaaaactaa gtatacttat atatatacga 39600tttttttaaa aataaacctg
attgtctgaa tttctacaag attgcgacca aattttccgt 39660atttccaaaa
tctaatatta ggggtttcta ctaaaattca acgagaactc ttaacattat
39720ggttatttta acacatggtt caccgccggc tcaaacttca ttcttagtcc
tctgattttt 39780ggtaaatcga cgcctacgtc tcaacaatta gtttgtgcag
aaaataagta aaaagagttg 39840tgctccatct tgcacacata cacatcgcct
gtaatgaaga ggttcggagt cagatgacta 39900ggcgtagaaa tgtgcgaaat
tcacggataa cagagatttt tgatgtttca tcagacttac 39960acgttttgga
agtatgaatt gggtctagac aacggagtgg cagatgttcg gaaaattttg
40020cagaaaagag aacctaagag cgttgatggt ttggtgacta acgaacttaa
aagaaaattg 40080gtcattgaaa attttaaaat tttaaatttt gcttgcagtt
catctttctc tattaacaaa 40140aattattttg tagcttttct caatttcagg
caattaaaac atttcaattt attcttctat 40200tatggaagtt tatctctaat
tgaaactctc caattttgat caaagaacaa acgttctcgt 40260tgtttgaaaa
aaaaaacagt tcttttttga aactcgcgcg caaattatta accaatcatc
40320ctcgtttgcg cgcaaaattg tagaaaaaat catttaaatt tatcaaaaat
agtttaccat 40380tctgatgagt ttttcatata caaaaatgcc ctggcaattg
ttgttttctc tgaaatagca 40440cataataatt gaactctacc cacataaagt
tcgttctgaa aaacacctta caattattgt 40500gattgagagc caccccaaga
gggattagaa aaacggatgt aatctgtata ccttcgagat 40560tcgtttattt
ccttgtataa ccaatagcag gaaaattaca gctttttcta agtaagcggt
40620gaaactagag agattctata gaatatgggc gttaataatt gtatgttaaa
gttttagaat 40680aacacaagtc cagagtaagg gcaagaaaag taatgagcaa
cggaaaccag catgcaagac 40740acccgaattc cggttctctt ctgaaactaa
aagttgcgtg tactaaacct taaaccagca 40800gctggctagt ctcaagaaat
aatagaaaaa aggaaggaat gaagatatgg gaataataca 40860aattgaaaat
gttgtgtgag ctccgaataa ttttcaatat caaaaattta tgaattgtgt
40920ggacggctgt gtgtgcgtgt gcgtatgcgt cggcaagaaa aagaagcgac
cgaataagaa 40980aatggttgat tcagtgaaca aaaaaagaga gaaagatatc
caaacaaaat tattcaaaac 41040tattatcaat cggtaggtat tgctctagag
cacacctttc tggacactca gcagacatgc 41100gtagagaggg attatgtggt
acatatagtg gatggaggaa cagatattta taaatactta 41160tggaaaagag
gatgaagata ggatgaggta gatgaattga gaagatttta aaatgataat
41220ggatattgaa tttgaataag gagattctaa attatccgaa gaacacaaac
tatatcaaga 41280ctacaaaata atctagacga gtcccagttt tgcaaggtaa
ggattaatct taaaaggatc 41340ttttaaatat ttatttcaat gctcctataa
attttaaaaa gtaggtgcat tctaatatgt 41400acagtgatta ggagatatgt
gacgttacgt gaggtctcga taaagtacgg tattcgagct 41460aaatttcaaa
cattgtcaag gtagattcgg tacacagcca ccataaatgt tccactaaaa
41520atgtgttgtc cttctccttt ggaacacaaa tctagctgct gaactttttc
acttcactac 41580atgtcaatgg gattgatatg catctaggac atttttttgg
ttatcaatag tccgcatagc 41640ttgcgtaacc aatacaaccg attgtccaaa
aaaatttgaa cactacaaaa cgtatttatt 41700attcggatac ccgttgcatt
tcaatacaca agttgatact tgctgcccct cggggctctc 41760agacactcat
tgactgaaaa cagacgattg ctcgtcgtcg tagtctgaag gctcggagag
41820ctgaggaaga tatgaggaca taatgaattg atgtgtgaga atgagaaaat
gaaaaaggaa 41880aaatgagaaa aaaaagatga tgaagaatgt acaaatgaat
aatcaagtag caatgacgag 41940aaaagaacca ggtccttttg gcaggcaatt
ttcgaaattt tcagatcaaa tttgtcgcca 42000ttgcttctgg attaataatg
gatgacgctt tgacaatggt gctcaataca agtgcaaaca 42060gattggtttg
ggatggcgta tagaaataga gccggtgaga cgatgtgatg aagttctgag
42120agacgagatg tgatcgaggc gtttgtagtc gaggcaaacc gaggccgcat
atggggttcc 42180gataggcaat cggagaccag tgtccatctg aaagagataa
aagttattcg agttgtgaat 42240gttgcaagga aaattaaagg tacagtagag
acaatcgaga cttttttcgg gaggacgcca 42300tctaaaaact gtggaagcac
gtggctttgg tagcttgatg tcacagaagt tgattccata 42360agaattacat
tagaaagctt gcgacgctaa atggataaat ctggtaacgg cttcctaata
42420gcaagttaag ttttttcaca ataaattttt cagaattgaa tagatgcatt
ttataactta 42480cacatcgagt gggcacgttg gtggacaaga caagccccga t
42521244434DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number X04665 24gccgccctcg ccaccgctcc cggccgccgc
gctccggtac acacaggatc cctgctgggc 60accaacagct ccaccatggg gctggcctgg
ggactaggcg tcctgttcct gatgcatgtg 120tgtggcacca accgcattcc
agagtctggc ggagacaaca gcgtgtttga catctttgaa 180ctcaccgggg
ccgcccgcaa ggggtctggg cgccgactgg tgaagggccc cgacccttcc
240agcccagctt tccgcatcga ggatgccaac ctgatccccc ctgtgcctga
tgacaagttc 300caagacctgg tggatgctgt gcggacagaa aagggtttcc
tccttctggc atccctgagg 360cagatgaaga agacccgggg cacgctgctg
gccctggagc ggaaagacca ctctggccag 420gtcttcagcg tggtgtccaa
tggcaaggcg ggcaccctgg acctcagcct gaccgtccaa 480ggaaagcagc
acgtggtgtc tgtggaagaa gctctcctgg caaccggcca gtggaagagc
540atcaccctgt ttgtgcagga agacagggcc cagctgtaca tcgactgtga
aaagatggag 600aatgctgagt tggacgtccc catccaaagc gtcttcacca
gagacctggc cagcatcgcc 660agactccgca tcgcaaaggg gggcgtcaat
gacaatttcc agggggtgct gcagaatgtg 720aggtttgtct ttggaaccac
accagaagac atcctcagga acaaaggctg ctccagctct 780accagtgtcc
tcctcaccct tgacaacaac gtggtgaatg gttccagccc tgccatccgc
840actaactaca ttggccacaa gacaaaggac ttgcaagcca tctgcggcat
ctcctgtgat 900gagctgtcca gcatggtcct ggaactcagg ggcctgcgca
ccattgtgac cacgctgcag 960gacagcatcc gcaaagtgac tgaagagaac
aaagagttgg ccaatgagct gaggcggcct 1020cccctatgct atcacaacgg
agttcagtac agaaataacg aggaatggac tgttgatagc 1080tgcactgagt
gtcactgtca gaactcagtt accatctgca aaaaggtgtc ctgccccatc
1140atgccctgct ccaatgccac agttcctgat ggagaatgct gtcctcgctg
ttggcccagc 1200gactctgcgg acgatggctg gtctccatgg tccgagtgga
cctcctgttc tacgagctgt 1260ggcaatggaa ttcagcagcg cggccgctcc
tgcgatagcc tcaacaaccg atgtgagggc 1320tcctcggtcc agacacggac
ctgccacatt caggagtgtg acaagagatt taaacaggat 1380ggtggctgga
gccactggtc cccgtggtca tcttgttctg tgacatgtgg tgatggtgtg
1440atcacaagga tccggctctg caactctccc agcccccaga tgaacgggaa
accctgtgaa 1500ggcgaagcgc gggagaccaa agcctgcaag aaagacgcct
gccccatcaa tggaggctgg 1560ggtccttggt caccatggga catctgttct
gtcacctgtg gaggaggggt acagaaacgt 1620agtcgtctct gcaacaaccc
cacaccccag tttggaggca aggactgcgt tggtgatgta 1680acagaaaacc
agatctgcaa caagcaggac tgtccaattg atggatgcct gtccaatccc
1740tgctttgccg gcgtgaagtg tactagctac cctgatggca gctggaaatg
tggtgcttgt 1800ccccctggtt acagtggaaa tggcatccag tgcacagatg
ttgatgagtg caaagaagtg 1860cctgatgcct gcttcaacca caatggagag
caccggtgtg agaacacgga ccccggctac 1920aactgcctgc cctgcccccc
acgcttcacc ggctcacagc ccttcggcca gggtgtcgaa 1980catgccacgg
ccaacaaaca ggtgtgcaag ccccgtaacc cctgcacgga tgggacccac
2040gactgcaaca agaacgccaa gtgcaactac ctgggccact atagcgaccc
catgtaccgc 2100tgcgagtgca agcctggcta cgctggcaat ggcatcatct
gcggggagga cacagacctg 2160gatggctggc ccaatgagaa cctggtgtgc
gtggccaatg cgacttacca ctgcaaaaag 2220gataattgcc ccaaccttcc
caactcaggg caggaagact atgacaagga tggaattggt 2280gatgcctgtg
atgatgacga tgacaatgat aaaattccag atgacaggga caactgtcca
2340ttccattaca acccagctca gtatgactat gacagagatg atgtgggaga
ccgctgtgac 2400aactgtccct acaaccacaa cccagatcag gcagacacag
acaacaatgg ggaaggagac 2460gcctgtgctg cagacattga tggagacggt
atcctcaatg aacgggacaa ctgccagtac 2520gtctacaatg tggaccagag
agacactgat atggatgggg ttggagatca gtgtgacaat 2580tgccccttgg
aacacaatcc ggatcagctg gactctgact cagaccgcat tggagatacc
2640tgtgacaaca atcaggatat tgatgaagat ggccaccaga acaatctgga
caactgtccc 2700tatgtgccca atgccaacca ggctgaccat gacaaagatg
gcaagggaga tgcctgtgac 2760cacgatgatg acaacgatgg cattcctgat
gacaaggaca actgcagact cgtgcccaat 2820cccgaccaga aggactctga
cggcgatggt cgaggtgatg cctgcaaaga tgattttgac 2880catgacagtg
tgccagacat cgatgacatc tgtcctgaga atgttgacat cagtgagacc
2940gatttccgcc gattccagat gattcctctg gaccccaaag ggacatccca
aaatgaccct 3000aactgggttg tacgccatca gggtaaagaa ctcgtccaga
ctgtcaactg tgatcctgga 3060ctcgctgtag gttatgatga gtttaatgct
gtggacttca gtggcacctt cttcatcaac 3120accgaaaggg acgatgacta
tgctggattt gtctttggct accagtccag cagccgcttt 3180tatgttgtga
tgtggaagca agtcacccag tcctactggg acaccaaccc cacgagggct
3240cagggatact cgggcctttc tgtgaaagtt gtaaactcca ccacagggcc
tggcgagcac 3300ctgcggaacg ccctgtggca cacaggaaac acccctggcc
aggtgcgcac cctgtggcat 3360gaccctcgtc acataggctg gaaagatttc
accgcctaca gatggcgtct cagccacagg 3420ccaaagacgg gtttcattag
agtggtgatg tatgaaggga agaaaatcat ggctgactca 3480ggacccatct
atgataaaac ctatgctggt ggtagactag ggttgtttgt cttctctcaa
3540gaaatggtgt tcttctctga cctgaaatac gaatgtagag atccctaatc
atcaaattgt 3600tgattgaaag actgatcata aaccaatgct ggtattgcac
cttctggaac tatgggcttg 3660agaaaacccc caggatcact tctccttggc
ttccttcttt tctgtgcttg catcagtgtg 3720gactcctaga acgtgcgacc
tgcctcaaga aaatgcagtt ttcaaaaaca gactcagcat 3780tcagcctcca
atgaataaga catcttccaa gcatataaac aattgctttg gtttcctttt
3840gaaaaagcat ctacttgctt cagttgggaa ggtgcccatt ccactctgcc
tttgtcacag 3900agcagggtgc tattgtgagg ccatctctga gcagtggact
caaaagcatt ttcaggcatg 3960tcagagaagg gaggactcac tagaattagc
aaacaaaacc accctgacat cctccttcag 4020gaacacgggg agcagaggcc
aaagcactaa ggggagggcg catacccgag acgattgtat 4080gaagaaaata
tggaggaact gttacatgtt cggtactaag tcattttcag gggattgaaa
4140gactattgct ggatttcatg atgctgactg gcgttagctg attaacccat
gtaaataggc 4200acttaaatag aagcaggaaa gggagacaaa gactggcttc
tggacttcct ccctgatccc 4260cacccttact catcacctgc agtggccaga
attagggaat cagaatcgaa accagtgtaa 4320ggcagtgctg gctgccattg
cctggtcaca ttgaaattgg tggcttcatt ctagatgtag 4380cttgtgcaga
tgtagcagga aaataggaaa acctaccatc tcagtgagca ccag
4434252837DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number M64866 25agagagccag tccgatgtct gcagcctccc
tggccaggcc tctcctctcc tgccgcagct 60agtccccctc aggacagaca gagtactggc
gtcggtcacc attcacttgc aaacacacca 120ggtcacgtga agaaacttcc
tggtgacact caggctgtag ctgtgcactc ttcaaccacg 180aggttggttt
tctcctaagt gtcacaggtg gagacaagat gctctgggca ctggccctgc
240tggctctggg catagggcca agagcttctg ctggtgacca cgtcaaggac
acttcatttg 300accttttcag catcagcaac attaaccgga agaccatcgg
tgccaagcag ttccgagggc 360ctgaccccgg ggtgcccgcc taccgttttg
tacggtttga ctacatcccc ccagtgaaca 420cagatgatct caacaggatt
gtcaagcttg caaggagaaa ggagggcttc ttcctcacag 480cccaactgaa
gcaggaccgc aagtctcggg gaacgctcct ggtgttggaa ggccccggca
540cctcccagag gcagtttgag attgtgtcca atggcccagg ggacactttg
gacctcaact 600actgggtaga aggcaatcag cataccaact tcctggagga
tgtgggcctg gctgactccc 660agtggaagaa tgtgactgtg caggtggcca
gtgacaccta tagcctgtat gtgggctgcg 720atcttatcga cagtgtcacc
ctggaagaac cattctatga gcagctagaa gtagacagga 780gcaggatgta
cgtggccaaa ggtgcatctc gagagagtca cttcaggggc ttgctgcaga
840atgtccatct cgtgtttgca gattctgtgg aagatatctt aagcaagaaa
agctgtcaac 900acagccaggg agctgaagtc aacaccatca gtgaacatac
agagactctc catctgagcc 960ctcacatcac cacagatctc gtggtccagg
gtgtggagaa ggcacaggag gtgtgtacgc 1020actcctgcga ggagttgagc
aacatgatga atgagctctc tggactgcac gtcatggtga 1080accagctgag
caagaacctg gagagagtgt ctagtgataa ccagttcctt ttggagctca
1140ttgggggccc tctgaagaca agaaacatgt cagcctgtgt gcaggagggc
cgaatctttg 1200cagaaaatga aacctgggtt gtggatagtt gtaccacatg
cacctgcaag aaatttaaaa 1260cagtctgcca tcagatcacc tgctcacctg
caacttgtgc caacccatct tttgtggaag 1320gcgagtgctg tccatcctgt
tcacactctg cagacagtga tgagggctgg tctccgtggg 1380cagagtggac
cgagtgttct gtcacctgtg gctctgggac ccagcagaga ggccggtctt
1440gtgatgtcac cagcaacacc tgcctgggcc cctccattca gacaaggaca
tgcagcctgg 1500gcaaatgtga tacgagaatc cgtcagaatg gaggctggag
tcactggtca ccctggtctt 1560catgctccgt gacttgtgga gttggcaatg
tcacccgcat acgtctctgc aactcaccag 1620tgccccagat gggtggcaag
aactgcaagg gcagcggccg ggaaaccaaa ccctgtcagc 1680gtgatccgtg
cccaattgat ggccgctgga gcccctggtc cccttggtca gcctgcacag
1740ttacctgtgc tggagggatc cgtgagcgct cacgtgtttg caacagccct
gagccccagt 1800atggagggaa ggactgtgtc ggggatgtga cagaacacca
aatgtgcaac aagagaagct 1860gccctattga tgggtgctta tccaacccgt
gttttcctgg agccaagtgc aacagcttcc 1920ctgatgggtc ctggtcctgt
ggctcctgcc cagtgggctt tctgggcaat ggtacccact 1980gtgaggacct
ggatgagtgt gctgtggtca cagatatttg cttctcaact aacaaagctc
2040cccgctgtgt caacaccaac ccgggcttcc actgcctgcc ttgtccacca
cgctacaagg 2100ggaaccaacc cttcggtgtt ggcctggagg atgctaggac
agaaaaacaa gtgtgtgagc 2160cagagaatcc atgtaaggac aagactcaca
gctgccacaa gaatgcagag tgcatctacc 2220tgggccactt tagtgacccc
atgtacaagt gtgagtgcca gattggctac gcaggtgatg 2280ggctcatctg
cggggaggac tcagacctgg atggctggcc caacaacaac ctggtgtgtg
2340ctactaatgc cacctaccac tgcatcaagg acaactgccc caaactgcca
aattccgggc 2400aggaggattt tgataaggat ggaatcggag atgcttgtga
cgaggacgat gacaatgacg 2460gtgtgagcga tgagaaggac aattgccagc
ttctcttcaa tccccgtcaa ttagactatg 2520acaaggatga ggttggagac
cgctgtgaca actgccccta tgtgcacaac ccagcacaga 2580tcgacacaga
caacaatggc gagggggatg cctgctctgt ggacattgac ggagacgatg
2640ttttcaatga gcgagacaat tgtccatatg tctacaacac tgaccagaga
gatacggatg 2700gtgatggcgt gggtgaccac tgtgacaatt gtcctctgat
gcacaaccca gatcagatcg 2760atcaggacaa tgatctcgtt ggagaccagt
gtgacaacaa tgaggacata gatgatgacg 2820gccaccagaa caaccaa
2837264108DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number L07803 26agagagccag tccgatgtct gcagcctccc
tggccaggcc tctcctctcc tgccgcagct 60agtccccctc aggacagaca gagtactggc
gtcggtcacc attcacttgc aaacacacca 120ggtcacgtga agaaacttcc
tggtgacact caggctgtag ctgtgcactc ttcaaccacg 180aggttggttt
tctcctaagt gtcacaggtg gagacaagat gctctgggca ctggccctgc
240tggctctggg catagggcca agagcttctg ctggtgacca cgtcaaggac
acttcatttg 300accttttcag catcagcaac attaaccgga agaccatcgg
tgccaagcag ttccgagggc 360ctgaccccgg ggtgcccgcc taccgttttg
tacggtttga ctacatcccc ccagtgaaca 420cagatgatct caacaggatt
gtcaagcttg caaggagaaa ggagggcttc ttcctcacag 480cccaactgaa
gcaggaccgc aagtctcggg gaacgctcct ggtgttggaa ggccccggca
540cctcccagag gcagtttgag attgtgtcca atggcccagg ggacactttg
gacctcaact 600actgggtaga aggcaatcag cataccaact tcctggagga
tgtgggcctg gctgactccc 660agtggaagaa tgtgactgtg caggtggcca
gtgacaccta tagcctgtat gtgggctgcg 720atcttatcga cagtgtcacc
ctggaagaac cattctatga gcagctagaa gtagacagga 780gcaggatgta
cgtggccaaa ggtgcatctc gagagagtca cttcaggggc ttgctgcaga
840atgtccatct cgtgtttgca gattctgtgg aagatatctt aagcaagaaa
agctgtcaac 900acagccaggg agctgaagtc aacaccatca gtgaacatac
agagactctc catctgagcc 960ctcacatcac cacagatctc gtggtccagg
gtgtggagaa ggcacaggag gtgtgtacgc 1020actcctgcga ggagttgagc
aacatgatga atgagctctc tggactgcac gtcatggtga 1080accagctgag
caagaacctg gagagagtgt ctagtgataa ccagttcctt ttggagctca
1140ttgggggccc tctgaagaca agaaacatgt cagcctgtgt gcaggagggc
cgaatctttg 1200cagaaaatga aacctgggtt gtggatagtt gtaccacatg
cacctgcaag aaatttaaaa 1260cagtctgcca tcagatcacc tgctcacctg
caacttgtgc caacccatct tttgtggaag 1320gcgagtgctg tccatcctgt
tcacactctg cagacagtga tgagggctgg tctccgtggg 1380cagagtggac
cgagtgttct gtcacctgtg gctctgggac ccagcagaga ggccggtctt
1440gtgatgtcac cagcaacacc tgcctgggcc cctccattca gacaaggaca
tgcagcctgg 1500gcaaatgtga tacgagaatc cgtcagaatg gaggctggag
tcactggtca ccctggtctt 1560catgctccgt gacttgtgga gttggcaatg
tcacccgcat acgtctctgc aactcaccag 1620tgccccagat gggtggcaag
aactgcaagg gcagcggccg ggaaaccaaa ccctgtcagc 1680gtgatccgtg
cccaattgat ggccgctgga gcccctggtc cccttggtca gcctgcacag
1740ttacctgtgc tggagggatc cgtgagcgct cacgtgtttg caacagccct
gagccccagt 1800atggagggaa ggactgtgtc ggggatgtga cagaacacca
aatgtgcaac aagagaagct 1860gccctattga tgggtgctta tccaacccgt
gttttcctgg agccaagtgc aacagcttcc 1920ctgatgggtc ctggtcctgt
ggctcctgcc cagtgggctt tctgggcaat ggtacccact 1980gtgaggacct
ggatgagtgt gctgtggtca cagatatttg cttctcaact aacaaagctc
2040cccgctgtgt caacaccaac ccgggcttcc actgcctgcc ttgtccacca
cgctacaagg 2100ggaaccaacc cttcggtgtt ggcctggagg atgctaggac
agaaaaacaa gtgtgtgagc 2160cagagaatcc atgtaaggac aagactcaca
gctgccacaa gaatgcagag tgcatctacc 2220tgggccactt tagtgacccc
atgtacaagt gtgagtgcca gattggctac gcaggtgatg 2280ggctcatctg
cggggaggac tcagacctgg atggctggcc caacaacaac ctggtgtgtg
2340ctactaatgc cacctaccac tgcatcaagg acaactgccc caaactgcca
aattccgggc 2400aggaggattt tgataaggat ggaatcggag atgcttgtga
cgaggacgat gacaatgacg 2460gtgtgagcga tgagaaggac aattgccagc
ttctcttcaa tccccgtcaa ttagactatg 2520acaaggatga ggttggagac
cgctgtgaca actgccccta tgtgcacaac ccagcacaga 2580tcgacacaga
caacaatggc gagggggatg cctgctctgt ggacattgac ggagacgatg
2640ttttcaatga gcgagacaat tgtccatatg tctacaacac tgaccagaga
gatacggatg 2700gtgatggcgt gggtgaccac tgtgacaatt gtcctctgat
gcacaaccca gatcagatcg 2760atcaggacaa tgatctcgtt ggagaccagt
gtgacaacaa tgaggacata gatgatgacg 2820gccaccagaa caaccaagac
aactgcccat acatctccaa ctccaaccag gctgaccatg 2880acaacgacgg
caagggcgat gcctgcgact ctgatgatga caatgatggt gttccagatg
2940acagggacaa ctgtcggctt gtgttcaacc cagaccagga agactcggac
ggtgacggcc 3000gaggtgacat ttgtaaagat gactttgaca atgataatgt
cccagatatt gatgatgtgt 3060gccctgagaa caatgccatc actgagacag
acttcagaaa cttccagatg gtccctctgg 3120atcccaaggg gaccacacaa
attgatccca actgggtaat tcgtcaccaa ggcaaagagc 3180tggtgcagac
agcaaactca gaccctggca tcgctgtagg tttcgacgag tttgggtctg
3240tggacttcag tggcactttc tatgtcaaca ctgaccggga tgatgactac
gctggctttg 3300tctttggcta tcagtcaagc agccgcttct atgtggtgat
gtggaagcag gtgacccaga 3360cctactggga agacaagccc agtcgggctt
acggctactc tggtgtgtca ctcaaagtgg 3420taaactccac gactggtact
ggcgagcacc tgaggaatgc cctgtggcac acgggaaaca 3480cagaaggcca
ggtccggact ctatggcatg accccaaaaa cattggctgg aaagactaca
3540ctgcctacag gtggcacctg attcacaggc ctaagacagg ctacatgaga
gtcttagtgc 3600atgaaggaaa gcaagtcatg gctgactcag gaccaattta
tgaccaaacc tacgctggtg 3660gacggctggg cctgtttgtc ttctcccaag
agatggtcta tttctcggac ctcaagtatg 3720agtgcagaga tgcctagaga
gcagggctcc agctccagca atgtgctgca aacacccctt 3780cttagacaca
tcagtccatc ttggcacttg tggcttttct gtcatttggc atttcctgtt
3840tcttgacctt aactgagtgg atctacacct ccttcatcag caccaagtcc
aagtgtcttc 3900aaaggagaaa catcaattgc actccaagag cttccagcct
gctgctggaa aacatttgga 3960tgagatatga ggctcaccgt ggagcgaaga
ccgagcattc cgctgtgttg ccttttcttg 4020tttgtttaaa aagaatgacg
tttacatgta aatgtaatta cttgcagtat ttatgtgtat 4080atggagtcga
agggagcttt agagcaca 410827820DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number U08006 27tcgaccagag
gaggggaggc cagttcctct cccaagggtg ccacacaccc ctccctgttc 60atcaccagac
aggcccttcc
ttcttagcca tatgctaacc ttctcctccc tgggaaattt 120cctctgcagg
agccaaagca gatgggagct ggagttgctg gagctcctgg tctgtatgca
180gagcaggcat ccaggaaagg agaagagagt gtgacaatcc agcacctcag
aatggagggg 240cctcgtgttc agggcggaaa gtacagacgc aggcttgctg
agggcctctg gacacaggct 300ggaccagatg ctgtggatgt cgacccctgc
actgactatt ggataaagac ttctttcaac 360taagagaaga tgcaaatcag
cacacttttt tctttgttct gccagcttcc aggcctaaga 420ctaggttttg
ctgtctacag ccaactattc tattagttac aaaactcaat cattttattc
480agcaactgga tgttgactgt taactagaag ctctgtccta cttacagcac
tttggatcat 540caaaaaaata aagtaaaata gaaaactgag aaaactcaat
ccatgaccag ggagaactta 600caggatgtta gagacaaaac aagcagacac
ctgaaacaat caacgcccaa taaaacaaag 660taggatgaaa attctcttag
ttctttgata acaatttgtt cactcataga aacattatta 720attggtaggg
taagcagaca ctctgaaaca atgagaaaaa tactaaaaat tgacttgagt
780tatttcaaat tgcctcattg acctgttata tcataactct
820282397DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number M16974 28tttttttttt catcctactt tgttttattg
ggcgttgatt gttacaggtc ccagcctgta 60gacatctttt actccaattt cctgaataga
tagctttatt ccttcaaggt aatatagtgc 120ggtggcttct ggctgagatg
tttgctgttg ttttcttcat cttgtctttg atgacttgtc 180agcctggggt
aactgcacag gagaaggtga accagagagt aagacgggca gctacacccg
240cagcagttac ctgccagctg agcaactggt cagagtggac agattgcttt
ccgtgccagg 300acaaaaagta ccgacaccgg agcctcttgc agccaaacaa
gtttggggga accatctgca 360gtggtgacat ctgggatcaa gccagctgct
ccagttctac aacttgtgta aggcaagcac 420agtgtggaca ggatttccag
tgtaaggaga caggtcgctg cctgaaacgc caccttgtgt 480gtaatggaga
ccaggactgc cttgatggct ctgatgagga cgactgtgaa gatgtcaggg
540ccattgacga agactgcagc cagtatgaac caattccagg atcacagaag
gcagccttgg 600ggtacaatat cctgacccag gaagatgctc agagtgtgta
cgatgccagt tattatgggg 660gccagtgtga gacggtatac aatggggaat
ggagggagct tcgatatgac tccacctgtg 720aacgtctcta ctatggagat
gatgagaaat actttcggaa accctacaac tttctgaagt 780accactttga
agccctggca gatactggaa tctcctcaga gttttatgat aatgcaaatg
840accttctttc caaagttaaa aaagacaagt ctgactcatt tggagtgacc
atcggcatag 900gcccagccgg cagcccttta ttggtgggtg taggtgtatc
ccactcacaa gacacttcat 960tcttgaacga attaaacaag tataatgaga
agaaattcat tttcacaaga atcttcacaa 1020aggtgcagac tgcacatttt
aagatgagga aggatgacat tatgctggat gaaggaatgc 1080tgcagtcatt
aatggagctt ccagatcagt acaattatgg catgtatgcc aagttcatca
1140atgactatgg cacccattac atcacatctg gatccatggg tggcatttat
gaatatatcc 1200tggtgattga caaagcaaaa atggaatccc ttggtattac
cagcagagat atcacgacat 1260gttttggagg ctccttgggc attcaatatg
aagacaaaat aaatgttggt ggaggtttat 1320caggagacca ttgtaaaaaa
tttggaggtg gcaaaactga aagggccagg aaggccatgg 1380ctgtggaaga
cattatttct cgggtgcgag gtggcagttc tggctggagc ggtggcttgg
1440cacagaacag gagcaccatt acataccgtt cctgggggag gtcattaaag
tataatcctg 1500ttgttatcga ttttgagatg cagcctatcc acgaggtgct
gcggcacaca agcctggggc 1560ctctggaggc caagcgccag aacctgcgcc
gcgccttgga ccagtatctg atggaattca 1620atgcctgccg atgtgggcct
tgcttcaaca atggggtgcc catcctcgag ggcaccagct 1680gcaggtgcca
gtgccgcctg ggtagcttgg gtgctgcctg tgagcaaaca cagacagaag
1740gagccaaagc agatgggagc tggagttgct ggagctcctg gtctgtatgc
agagcaggca 1800tccaggaaag gagaagagag tgtgacaatc cagcacctca
gaatggaggg gcctcgtgtc 1860cagggcggaa agtacagacg caggcttgct
gagggcctct ggacacaggc tggaccagat 1920gctgtggatg tcgacccctg
cactgactat tggataaaga cttctttcaa ctaagagaag 1980atgcaaatca
gcacactttt ttctttgttc tgccagcttc caggcctaag actaggtttt
2040gctgtctaca gccaactatt ctattagtta caaaactcaa tcattttatt
cagcaactgg 2100atgttgactg ttaactagaa gctctgtcct acttacagca
ctttggatca tcaaaaaaat 2160aaagtaaaat agaaaactga gaaaactcaa
tccatgacca gggagaactt acaggatgtt 2220agagacaaaa caagcagaca
cctgaaacaa tcaacgccca ataaaacaaa gtaggatgaa 2280aattctctta
gttctttgat aacaatttgt tcactcatag aaacattatt aattggtagg
2340gtaagcagac actctgaaac aatgagaaaa atactaaaaa ttgacttgag ttatttc
2397294100DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number L13855 29ggatcccccc gctccgctac catcttcatc
gacctcaccc aggacgacga ctgagctccc 60tcttcctcgc cgcggactgg ggcgaccctg
ttgctgctgc ggccgccgcc gctcctgccc 120ccacttcggc tcccgctcct
gctcctgctc ccggccccac tcctgttcct gttcctgttc 180ctgttcctgt
tcccggtcct gctccggctc ccggccccgc acccacctcc gctcctgctg
240cgggtctcca ggcccagaca aaataaaaaa agatatattt tttcagtccg
tctctcccgc 300ccggtgtctt ctatggctga gggagtctgg ctctcggggc
tctcgggtcg gctgggcggc 360tcggctggtt ggctggctgg cgagatggac
cgctccggcg cgcagcgtcc gcggctgctg 420tgatgggtgg gcggagcgcg
gaccggggat tatatacacg atgtgcatcc ataattgatg 480ttgtttgaga
aaaacaaagt cataaagtgg cactcagaca gcactttggc ctggcgcccg
540gccaccatct gagtgcccaa ccgggcccgg cggttacatc acccccacat
ggaccatcac 600ggcccattag caccaattgg ccagagtgtc gggagccacc
gctaattgca gtaacgcgcg 660gctgccagac tgcaatttac cgcgcgatac
tgcagtttac tgcagccgcg gtaaactgca 720gtacgcggcg gccgcaggaa
atctactgta gtatttggcg gcggcgcgcg gtactgcaac 780tgtagtaaac
tgtagctgca gtagagttac tgcagcgcca tcgggccggt gtggccgcca
840gggtaactgc acccgcagta aatttactgc agccggactt tgtgcgctgt
ggagaccgcg 900ccgaactggg acccccccga ctcccccccg actccccccc
gactcccccc cgactccccc 960ccgactcccc cccgactccc ccccgactcc
cccccgactc ccccgggacg cgtccgcgcc 1020tcgatgcgcc ccatcgcgcc
ccgttccgct tcgccacgct ccagttgccc cgcccccggc 1080acgtggcacg
tatttccccc ccgtaaatca agagggatta tgcggatgtc tagtttatgt
1140ctcaatttcc tctttccgga gataaaagcc gggacccccg cgccgaaaaa
ggatacacca 1200gccgcgatgt cgccgctcgt ggcggtgctg gtgttttttt
cggcggcgct gggggttcct 1260ggccccggcg tcgcgggaaa cccccgtggg
ctcgatgcca tcttcgaggc cccggtcacg 1320cccgcgcccc ccactcgcca
tcctcggcgc gaggagctgg agtgggacga tgaggatcac 1380ccgctgctgg
acctcgagcc gcccgtggga tcacgctgcc atccctacat cgcgtactcg
1440ctgccgccgg acatgaacgc cgtcacgagc gtggtcgtga agccctactg
ctcgccgccg 1500gaggtcatcc tgtgggcgtc tggcaccgcc tacctggtca
acccctttgt cgccatccag 1560gccctggccg tcggagagcc cttaaatgag
gcggccctca aggagctcgg agaggtggcc 1620gtgcacaagg actccctgcc
gccgctgcgc tataatggag ggccccccgc cgagtaagag 1680accctgcggc
ctgccgcccg gggtgcgcct cgtcgtgcct gccgccgccg ccgcttctgc
1740ctctaacgcc gccaccgccg ctgcagcagc agcccccgcc ggggccgggg
ccggggcctc 1800gaagccggcc cgaccccccg ccgccgcccg gcccgcgaag
ggcacgcccg cggcgtcggc 1860ggcaacaaca gccacggggg ccgacgcctc
cgccccggcc cccgaccccg gggcgcccac 1920gtgggacgcc ttcgccgccg
agttcgacgt ggccccctcg tggcgcgcgc tgctggagcc 1980cgagatcgcc
aagccgtacg cgcgcctgct gctggccgag taccgcggcc gctgcctgac
2040cgaggaggtg ctgcccgcgc gcgaggacgt gttcgcctgg acgcgcctca
cggcgcccga 2100ggacgtcaag gtggtcatca tcggccagga cccgtaccac
gggccgggcc aggcccacgg 2160gctggccttc agcgtccggc gcggggtgcc
gatccccccg agcctggcca acatcttcgc 2220ggcggtccgg gcgacgtacc
cgacgctgcc cgcgcccgcc cacggctgcc tggaggcctg 2280ggcgcgccgc
ggggtgctgc tgctgaacac gacgctgacc gtgcggcgcg gggtccccgg
2340ctcccacgcc ccgctcggct gggcgcggct cgtgcgcgcc gtcgtccagc
ggctctgcga 2400gacccgcccc aagctggtgt tcatgctctg gggcgcccac
gctcaaaagg cctgcgcgcc 2460ggacccgcgc cgccacaagg tgctcacctt
cagccatccg tcgccgctgg cccgcacgcc 2520cttcaggacc tgcccgcact
ttggagaggc gaacgcgtac ctcgtccaga cgggccgggc 2580ccccgtcgac
tggagcgtgg actgagtcgg gcgtgcgcgc acaccgccgg cggaggacga
2640ggagggggga ggggggtggg atggacggag gagagcggat gatggagccc
gcgctcgccg 2700gcgccccggc cagcgcgctg ccggtcctgg cggtgctgcg
cgagtgggga tgggccgtgg 2760aggaggtcga gccctccggg ccgtgcccgg
aggacgcgga cgcgccccgg gagagcgcac 2820cccctccccg ggagggggtg
cgcgggagcg aagacggaga ggggggcgtg gaagacggcg 2880aggaggggaa
ggcgacggag aaggaggaga cggaagacga ggaagacggg ggggacgaag
2940ggacgacgac ggcggcggcg ggcccgcgcc gggcgcagca cgtggagttt
gacacgctgt 3000ttatggtcgc gtccgtggac gagctcgggc gccggcggct
gacggacacg atccgccggg 3060acctggccgc ggccctggcc ggcctccccg
tcgcctgcac caagacgtcc gcgtttgcgc 3120gcggcgcgcg cggcccgcgc
ggcgcccccg ggcgcggcca taaaagcctg cagatgttta 3180tcctgtgccg
cagagcccac gcggcgcgcg tacgcgatca gctccggtcc gcggtgcgcg
3240cccgacgccc acgcgagccc cgcgcgcgcc cgacgagcgg acgggcgcgg
ccggccgcgc 3300cggtgttcat ccacgagttc atcacccccg agccggtgcg
gctgcaccgg gacaacgtgt 3360ttgcggcgcc atgagcacct tcggacgcgc
gtccgtggcc acggtcgatg actaccaccg 3420gttcctgcag gccaacgaga
cggccgcccg gcgcctggcc gcggcctccc gccgcgtctc 3480caccggcggg
ggcgagacgc gggccccgcg gtcctcgcgc ggcccccacg acgatgaggc
3540gcccctgcgc gccggcggcc tgggcaccgc ccgcgggcgc tcgcgccagc
gcggcgcgac 3600cgagccggac cccgtctacg ccaccgtcgt ccagcctacc
caccaccacc accagcagca 3660ccaccaccgc tctcagcatc cgcagcagca
gcaacaacag cagcgggccc cacgccgccg 3720cggcagcgtg cacgcctcgg
cgacggccgc ggacggaccc gagtcgtgcg cggccgcacc 3780cccgcgccgc
cgcggcagcg tgcacgcctc ggcgacggcc gccccggcgg tccagctgcc
3840ccggccccgg caacggagca tcaacgcctc gacgacggcc gccccgacgc
cccagctgcc 3900gagaccccgc cagcgcagcg tcaacgcctc ggcccgcgcc
gccgtcccct cgacggccac 3960cctcccgcgc ccccggaccc cgtcccgggg
ccggcgcgcg ccccccgcct catgctgtta 4020tcgcgatcaa taaagggcga
gcgcccacgg accagacaaa agacacaacc ggttcggtct 4080ctctgtccgc
gcacgcgcgg 41003038734DNAUnknownDescription of Unknown Organism
GenBank Clone Accession Number AL021529 30gatcctcgtg accgggtaca
ccgacgcctc ctggacgccg ctgttcgcca tcgcgggcgg 60ggtcgtcacc gacatcgggt
cgatgctctc gcacagttcc atcgtggccc gcgagttcca 120cgtcccgtcg
gtggtgaaca ccaaggacgc cacccagcgc atcaacaccg gcgacctgat
180cgtggtggac ggcgacgcgg gcacggtcga ggtcgtcgag agcgcggaca
ccgacccgca 240gggcccggcc ggggccgccg ggaccccggc cggagccacc
accgactgaa gccggccacc 300gccgcaacac cggaccacga ccgcccccgc
gaggggcgga ccacacccca gacgggagac 360gacccgatga tccccaacca
gtggtatccc atcgtcgagg cgcaggaggt gggcaacgac 420aaaccgctcg
gtgtgcgccg catgggccag gacctcgtgc tctggcgcga catcgacggc
480aacctcgtct gccagggcgc ccgctgcccg cacaagggcg ccaacctcgg
cgacggccgc 540atgaagggca acaccatcga atgcccgtac cacggcttcc
gctacggagc cgacggtgcc 600tgccgggtga tcccggcgat gggctccgag
gcccgcatcc ccggctcgct gcgggtaccc 660acctacccgg tccgggagca
gttcggcctg gtgtggatgt ggtggggcga cgagcgcccg 720acggccgacc
tgccgccggt ggcggccccg gccgaggtga cggacaaccg gaagctgtac
780gccaccaagc gctggacccg cccggtgcac tacacccgtt acatcgagag
cctgctcgag 840ttctaccacg tgacctacgt gcaccgggac cactggttca
actacatcga ctacctgctc 900ctgtacggca ccccgagcaa gttcggcctc
gacggccgcg agcggtacct ggccgccacc 960cggatcacca accaccgggt
ggagacggag gcggaggggc agaccatccg ctactccttc 1020gaccactgcc
aggaggacga ccccaccaac accacccact acgtcatcac gttcaccttc
1080ccgtgcatgg tgcacgtgca gaccgagcag ttcgagacca cctcctggct
ggtgcccatc 1140gacgaccaga acaccgagca catcctgcgc tggtacgagt
acgaacaggt caagcccgtc 1200ctgaggttcg aaccgctgcg ccgtctgctg
ccctgggcgt ccctctacat ggagaagtgg 1260gtgcaggacc cccaggacgt
ccgcatcatg gaacaccagg aacccaagat cagcgccggc 1320ggcgtgaaca
agttcatccc cgtcgacgag atgaacgcca agtacatctc gatgcgcgcc
1380aagctgatcg cggacgcctc ggccgcgccc tcgtcaccgg cgcgggcggc
ggagcccgag 1440ccggaagcgg cggggcgggg cggatcagcg gcccgtgcca
cgggcaacgg caggggagcg 1500gccggcggac gacgcggcac caagcccaag
gaggacgccg ccgcgcgccc gtagacccga 1560agacggggga cggacaagag
agagcgagag tgagagatgt acggcggata cgacgcgtcg 1620accggcccca
aggccctggt gacggccttc aacaccgtcg ccgtggccgg cgccgtgtgg
1680ttcctgttcg gcggcgcgga caccgtggcc gactggttcg gcaccgactt
cgacgaggcg 1740gtgaccctgc gccgggtcct gctggcgacc ctgtcggtgc
tctacctgct gcgcttcatc 1800gccacgaact tcgtgatgct ccagcgcaag
atggagtggt cggagtcggc caccatcggg 1860atctgggtcc tggtgatcca
cggcacgatg gcgtacttcg gcggcaccaa cgacgccggc 1920gtgagcgcgt
tcacctggct gggcgtcgtg ctgtacctcc tcgggtccta cctgaacacg
1980gggtcggagt accagcgcaa actctggaag aagcgcccgg agaacaaggg
caagctctac 2040accgaaggcc tgttcaagca ctcgatgcac atcaactact
tcggtgacgc cgtgctcttc 2100tccgggttcg cgctggtcac gggcaccccg
tgggccttcg ccatccccct gatcatggtc 2160tgcatgttcg tcttcctgaa
catccccatg ctcgacaagt acctcgccga gcgatacggc 2220gaggccttcg
acgagtacgc gtcccggacg gcgaagttcg tcccctacgt gtactgaccc
2280cgcccgtcac gcgcgtacgg cggcctcccc gggcgagggg ggccgccgta
ccgggtggca 2340accacagatc ccacagatcc ccacagatcc ccacagagcc
cctccacaga ccccctccag 2400agatccacag atcccctcca cagatccgag
acgaggcacg tatgaccgga gacattccct 2460tcggagaggc cgaggcgtcc
ctgaccgccg aggtgctgcg cgaggtcctg gccggcggcg 2520ccgaggcgtt
cgcccggctg acctccgacg agggcgccgt cgacgacttc ggcttcgacc
2580cggagctgac cgacgactac ctgctccccg ccctgcgcct gctgtacgag
aagtacttcc 2640gggtcgacct ggagggactg gagaacgtgc cggccgaggg
gggcgcactc ctggtcgcca 2700accactccgg caccctgccg ctcgacgccc
tgatgctcca ggtggcgctg cacgaccatc 2760acagcacgca ccgcaggctc
cggctgctcg ccgccgacct tgccttcgac ctccccgtcg 2820tccgtgacct
cgcccgcaag gccggccacg tacgcgcctg ccccgagaac gcgctgcggt
2880tgctcggctc cggcgaactg gtcggcgtga tgccggaggg ctacaagggg
ctcggcaagc 2940ccttcgagga gcgctaccgg ctgcagcgct tcggccgggg
aggcttcgcg gcggtggcac 3000tgcggtcgcg gcgccccatg gtgccgtgct
cgatcgtcgg cgccgaggag atctacccga 3060tgatcggctc ggcccccacc
ctggcccgga tgctgaagct gccgtacttc ccgatcaccc 3120cgaccttccc
gctgctgggc gcgctgggcc tgatcccgat gccgaccaag tggaccatcc
3180gcttcggtgc cccgatccac acggacggct tccccgagga cgccgcggag
gacccgctgg 3240tggtcgagaa gctcgccggc gaggtgaagg acaccatcca
gcacacgctc aacgagatgc 3300tggagggccg cggctccccg ttcgtctgag
ggccgcggct cccggttcgc ccgagggcgg 3360cggctcccgg ttcgcccgag
gaccgtccct ctcgtccggg gccccgcctc agccccccgc 3420cgacgatccc
cggcggcaga tgctgcgaac gctggcgaag gccagaacgg cgaggccgac
3480gagcgtgacg ccgccgccga ccagctccgc ggacagatgc atgggatctc
cctcaggggg 3540acgacggacg gtgatggtca tatagccatg cgaaccccgc
cgtccgcccg atccgcagcc 3600gcaccgcccc gcgaattcac ccgtagagca
gaccggtgcg gccgaggagg ggtggcgatt 3660gggtggtcgc gcgttcgaac
gcttacgatc ctctgttgtg tccaaactga ccgacgtgcc 3720caagcggatc
ctcatcgggc gcgcactgcg cagcgaccgg ctgggtgaaa cgctcctgcc
3780gaagcgcatc gcgcttcccg tgttcgcgtc cgacccgctg tcctccgtgg
cgtacgcgcc 3840cggcgaggtg ctgctcgtcc tgtccatcgc gggcgtgtcg
gcctaccact tcagcccgtg 3900gatcgcggtc gcggtcgtgg tcctgatgtt
caccgtggtc gcctcctacc ggcagaacgt 3960gcacgcctac ccgagcggcg
gcggcgacta cgaggtggcc accaccaacc tcgggcccaa 4020ggccggtctg
accgtcgcca gcgccctgct ggtcgactac gtcctgaccg tcgcggtctc
4080catctcctcc ggcatcgaga acctgggctc cgcgatcccc ttcgtcgtcg
agcacaaggt 4140cctgtgcgcg gtcgccgtga tcctgctgct cacgctgatg
aacctgcgcg gggtcaggga 4200gtcgggcacc ctgttcgcga ttccgacgta
cgtcttcgtc gcgggcgtct tcatcatgat 4260cgtgtggggg gcgttccgcg
gactggtcct ggacgacacc atgcgtgccc cgaccgcgga 4320ctacgagatc
aagccggagc acggcggcct ggccggcttc gcgctgatct tcctcctcct
4380gcgcgccttc tcctccggct gtgccgcgct caccggtgtc gaggcgatct
ccaacggcgt 4440cccggccttc cgcaagccca agtccaagaa cgcggggaac
accctcgcga tgatgggtct 4500gctggccgtc accatgttct gcggcatcat
cgcgctggcc gccgcgaccg acgtgcggat 4560gtcggagaac ccggccaccg
acctcttcca caacggcgtc gcggtcggcg cggactacgt 4620ccagcacccg
gtgatctcgc aggtcgccga ggcggtcttc ggcgagggca gcttcctgtt
4680catcgtgctg gccgcagcca ccgcgctggt cctcttcctc gccgccaaca
ccgcgtacaa 4740cggcttcccg ctgctcggct cgatcctcgc ccaggaccgc
tacctgccgc gccagctgca 4800cacccgcggc gaccgcctgg ccttctccaa
cggcatcgtg ctcctcgccg gagccgccat 4860gctcctggtc gtcgtctacg
gcgccgactc gacccggctg atccagctct acatcgtcgg 4920cgtcttcgtg
tccttcacgc tcagccagat cggcatggtc cgccactgga accgcaacct
4980ggccggcgag cgggaccagt ccaagcgacg ccacatgatg cgctcccgcg
cgatcaacgc 5040cttcggcgcc ttcttcaccg gcctcgtcct ggtggtggtc
ctggcgacca agttcacgca 5100cggcgcctgg gtcgcgctgc tcggcatgtg
catcttcttc gcgaccatga cggcgatccg 5160caagcactac gaccgggtcg
ccgaggagat cgcggccccg gaggaccccg aggaggcaca 5220gagcgacgac
atggtgcgcc cctcacgcgt tcactcggtg gtcctgatct ccaagatcca
5280ccgccccacg ctccgcgccc tcgcctacgc caagctgatg cgctccgaca
gcctggaggc 5340gctcagcgtc aacgtcgacc cggccgagac gaaggcgctg
cgcgaggagt gggagcgccg 5400cggcatcgcc gtaccgctga aggtcctgga
ctcgccgtac cgcgagatca cccggccggt 5460catcgagtac gtcaagagcc
tgcgcaagga gtccccgcgc gacgcggtct cggtgatcat 5520ccccgagtac
gtggtcggcc actggtacga gcacctgctg cacaaccaga gcgccctgcg
5580cctcaagggc cggctgctgt tcacgccggg cgtcatggtc acgtcggtcc
cgtaccagct 5640ggagtcctcc gaggccgcca ggcgccgggc gcgcaagcgc
caggactgga gcgcgccggg 5700tgcggtgcgg cgcggaccgg cccaccacca
ccaggaccgt gaccgtacga aggactcctc 5760ctcgtccacg tagactggac
ggctgttgtc cctgtcatcc ccccgttctc tggagtcacc 5820ccgccatgca
ggcagaaccg aagaagtcgc aggcggaaca gcgagcggtc gcggagccgg
5880tctcggagcc ggtctcgctg gtgggcgagg agtacgaggt cgaggtcggc
cccgtcgccc 5940acggcggcca ctgcatcgcc cgcacgtccg agggccaggt
gctgttcgtc cggcacacgc 6000tgcccggcga gcgggtcgtg gcccgggtga
cggagggcga ggagggtgcc cgcttcctgc 6060gggcggacgc ggtcgagatc
ctggacccct ccaaggaccg catcgaagcc ccctgcccct 6120tcgccggccc
cggccgctgc ggcggctgcg actggcagca cgccaagccg ggcgcccagc
6180gacgcctgaa gggcgaggtg gtcgccgagc agttgcagcg cctggcgggt
ctcaccccgg 6240aggaggccgg ctgggacggc acggtgatgc cggccgaggg
cgacaagctg ccggccggcc 6300aggtcccgtc gtggcgcacg cgcgtgcagt
tcgcggtgga cgccgacggt cgcgccggtc 6360tgcgccgcca ccgctcccac
gagatcgagc cgatcgacca ctgcatgatc gcggcggagg 6420gcgtcagcga
actgggcatc gagcgccgtg actggcccgg catggcgacg gtcgaggcga
6480tcgcggcgac gggctcccag gaccgccagg tcatcctgac cccgcgcccc
ggcgcccgcc 6540tccccatcgt cgaactggac cgcccggtct cggtcatgcg
cgtcggggag aaggacggcg 6600gcgtccaccg cgtccacggc cgccccttcg
tccgcgagcg cgccgacgac cgcacctacc 6660gcgtcggctc cggcggcttc
tggcaggtcc acccgaaggc cgccgacacc ctggtcaccg 6720cggtcatgca
gggcctgctg ccccgcaagg gcgacatggc cctggacctc tactgcggcg
6780tcggcctctt cgccggcgcc ctggccgacc gcgtcgggga ccagggagcg
gtcctcggca 6840tcgagtccgg caagcgcgcc gtcgaggacg cccgccacaa
cctcgccgcc ttcgaccgcg 6900tccgcatcga gcagggcaag gtcgagtccg
tcctgccccg caccggcatc gacgaggtcg 6960acctcatcgt cctcgacccg
ccccgcgccg gcgccggccg caagacggtc cagcacctct 7020cgaccctggg
cgcccgcagg atcgcctacg tggcctgcga cccggccgcg ctggcccggg
7080acctggggta cttccaggac ggggggtacc gggtgcggac gctgcgggtg
ttcgatctgt 7140tcccgatgac tgcgcacgtt gagtgcgtgg cgattttgga
gcccgccgca aaggggctct 7200gacctgcatt tttcttggct ggatcaggag
cggcctgttg cgctcgacct gttctccaaa 7260gcgcacgacg tagagcttgc
ggaccgctcg tgaaagccgc ctgacctggc gttgcacgag 7320cggtgccgcg
atgtcggcgt ggtcggccct tctcctggcg cgaagggaaa ccgaaggtct
7380tgacgctcgg gtgacgctat ttctgaaggg tcgtcaccga ctggggaggc
agggccctgc 7440ctctcgcgcc cgatgaagca ggttctctct gctccaggta
atcgtcgagg gtgccctgac 7500ggatcaggta gacggtcagg gaccgcaggg
tgcagggcgt cgacgcccag gtcgaggatc 7560atcagggcgc tgtcattggt
gatggcgaag gcgccgatca caccgtcgac ggaacaggtt 7620gcgttcagga
gtctgtgcgg gcgccgcacc ggcacggtct ggaagctcgg ctccgaccgc
7680agctcgccac cagtccgaga ggagccgata ctgtccggtg ccgggtgacc
cttcgtgcaa 7740gcgttgctgc ccccgctcgg cagaccgggg cagcaacgct
tgcacgatcg gccggtactc 7800aacgggatcg tgtggagttt cggaccggaa
cggcttggca ggacgtgccc gagcggtacg 7860gctcctgggc cacattgcac
acccgcttcc gtcgatgggt gaaaggcggc acctttcagt 7920gaaagggggt
tccgcccccc cccgggacct tgcgcccacc gtcgccgacc ggctgatgaa
7980ccggctccgc gctcccgcca ccaacctgac ccgacgtgag accgaagtcc
tctcaccggt 8040cgccgacgga ctgtccgacc aggccatcgg cgcacgcctc
cacttgaccg aaggcaccgt 8100cgatatcacc tggcctgcat ctatgccaac
ctcggaaccg actcgcgcac cgccgctgtg 8160gccactgtca ccgccatcga
cgacctcggg ctcatccgcc gctgaacagt atgtggtggg 8220cggtgtttcc
gttctccacg acttcagcgg cgtccggagt tgtggtgctg gctgggcttg
8280gtgcccgctc ctctgaaccc atgtgaacgc ccacggccag ttcgagccgg
acacgccccc 8340cggacctggc ctccgccgcg gccagaatgc ccggcccctg
cacctggtct gcctacctgt 8400acaggcgagg gcggtccctc ggagccactg
cctgtaactc cgaggggccg cccttggccg 8460actcggcggt caccgcggac
gcggtgcggt caggaggcac cgtctgcgtc atcaggcgcg 8520gctcggccga
gcgagttatt ccggcacccg tgggaccaga aatgtcagcc ctgcgtgacc
8580gcttcgaaga ccgtgacgcg gttgtcgtcg gagagcgagt gggtggggtc
ggcgtgcgcg 8640gcgcggaatg cctccgacga ggtgtaggcg gtgaatgcgg
cgtcgtcctc gaagttgagg 8700acggccaggt agccgtgtgc gcccttgcgg
ggacgcagca gccgtgcgtt gcgcagcccc 8760ggcacgttgg aaagggtggc
gcgcatgctg gcggtgcagt tgttctcgaa cgcgccctgg 8820gcgggcgcgg
cgacggtgaa ctcggtgacg gcggtgctca tttctgtctt ctctcggttg
8880ttggtgtgat gtcggtggct gtcccgcccg gccggggccc gcacggcgat
ggcgatgatg 8940tgcaccgcgt gtccgatcga gttccgttgc ggggtgcggt
tacagggctg gagttgggct 9000cgggtccgcg gtcggctgag ggagcctgcc
tgtgcggcgg gcccagattt cgaacgcgat 9060agtcatgaac gggggtacgg
cggccagcag ggcgaagatc gttgtccggc ccagccgcca 9120cttcagccgg
atcgcgacca ggacggtcag ggacacgtag acgatgaagg cggcgccgtg
9180gagggtgccg aagatccgta cgccgagttc ggtggtttcg gggatgtact
tgaggtacat 9240cccggccagc agacctgccc acgtgcacgc ttcgatgatc
gcgatccagg tgaacgcgcg 9300cagcagacgg ctggtgccgg tttcggcagc
ccgtgcggcc ggcgcgtcgg cggaaggcgg 9360ttcggaggtg ggcgtggagg
gtgtctgcgg ggtgcctggg cgtgcgggcc acagggcgcg 9420gttgccgagc
aggcggacga tggcggggac caggagcggg cggatgagga aggtgtccag
9480caggatgccg caggccatgg cgaagccgaa ctggaacagt tcgcggatcg
gctgggtcat 9540caggacggcg aaggtcgccg cgaggatgag gcccgcggag
gagatgacgc cgccggtgcg 9600tgtcagtgcg gcggtgatcg ccttcgctgg
gggctgggtg cgcagttcct gcttgaaccg 9660gctcatgatg aagatgttgt
agtcgacgcc gagcgcgacg aggaagacga agatgtacgc 9720ggtgacgcgg
ttgccgatgc cgtcgtcacc gaggacggtc acggtgaaga aggtggtggc
9780gcccagggtg gccaggaacg acaggagcag ggtcgcgacc aggtagagcg
gggcaaggag 9840cgagcggagc agcaggacga ggaccacggt gacgatggct
aggaccagca gcacgatgag 9900ggtcgtgtcg cggtcgaggg cggagcggat
gtcggcgttc tgcgcggtct cgccgccgat 9960gagcaccgtg gcgtcctgga
cgccggcggc ctgggctgcg gattgtgtgg cctgcttgag 10020gggaccgatc
gcgtcgagtg ccttggagct gtaggggtcg aggtcgagga tgacgtcgta
10080gaagacggtc ttgccgtcct tgcccatgcg ggggtctgcg acacggctga
cgtgatcggc 10140gtcggtgagc gcggtggcga tgtcggcggg tgcggggctg
gagcgcaggt tgtcctggga 10200atggacgacg acggtactgg gggcgatctc
gccgggcccg aattcctccc gaatgaggtg 10260ctgtccgtgc tccgactcgg
tggcggcgcg gaagccgctg agggtgttga agctctcctg 10320gtagccgagc
agtcccgcgc tcagtaccac caggagtgcg atcacggccg aggccacctt
10380gacgggggcc cgtgcgacca gggcggcgat gcggtgccag atgcctgcgc
cgcgactgcg 10440ttcggcggcc ttgtccacgc ccccgggcca gaagacgctc
ctgcccagca ggaggaccag 10500ggcggggatg aaggtgaacg ccaccagcgc
catgacggcc acgcccagag cgaggtacgg 10560tccgaagccg tgaagtgccg
gggagacggc cacgagcagg gcaaacatgg cgagcacgat 10620ggtcgaggcg
ctggcgagga cggactcggc ggtgcggcgc acggcggcct gcatcgcgcg
10680ggcgcggtct ggctcgtcga gcagggtctc gcggtagcgg gcggtgatga
tcagcgcgta 10740gtccgtgccc accccgaaca gcagcacggt catgatcgag
gcggtctggg agctgaccgt 10800gatgactccg gcgtccgcga gaatcgcgcc
gagagtctcc gccacgcgca tagccacgcc 10860cacggcaaga agcggcacga
gcgccatcag gggcgagcgg tagatcgcca gcaggatgat 10920caggacgagc
acgacggtgg ccagcagcag gactttgtca ccgccgctga agaccttcac
10980ggtgtcggtg gcgatcccgg cggggccggt caccgcgacg tcggcgggcc
cggcccggtc 11040ggacgcgagg gcacgcacct cgtcgaccgc attctggaag
gactcgtccg aggggctgcc 11100ctccatgggc acgatgacca gctgagcacc
gcggtcctgc gagaccaact cggccgcagc 11160gtcgggagcg gtcaccgtgg
agaccacgct cacgacatgg tcgggtcggc tggttccgga 11220aagggccgag
gtgatggcgg cgaccgattg cgtggcgctc ttcgcggcgt cggtgccctt
11280gccgcggacc acgatgatcg ccggcgtcgc gtcctggccc ggaagctggg
cgcggacgag 11340atcacgggcc ttcatggagt ccgaggcggc gggcggcagg
ttggcggagg cgttgtcctc 11400gacggattcc agggccgggg cgaccccggc
gaggaggccc gcgatcagga cccagaaggc 11460caccaccacg gcggcgcgct
tcttcgatcc caggagacat cgcagcagag cgggggagtt 11520catcggttgc
atcgggcagc cttcggcagg aagtacggac agaacttagc gacagggtgt
11580ctctaagttg cgtcaagcta acacgccccc tcggcctctc gggcgtgggg
gtaggttggc 11640gggagacggc acagcgtccg aggtgaagcg gagaaaatgc
ccaagattga agccggcagc 11700gtccgggagc accgggcgca gcggctcgcg
cagctgattg acgcggccga ggagctcctg 11760gaagagggcg gtgccgaagc
cctcacagcc ggagcggttg ccgcgcgagc cgggatcgcc 11820cgcaacagca
tctaccgcta cttcaactcc atcgacgacc tgctcgaact cgtcgtcacc
11880cgcgaattcc ccgcctggat cgacgcagtg gagcaggcca tcgcggccga
gaccacaccc 11940gccgcccagg ctgccgccta cgtcagggcc aacctcgaac
aggcagctcg cggcacccac 12000ggctggcggg ccgcgctcac gcgcgactcg
ctctccccgt cggcgcggga gcgggtgagg 12060aatctgcaca tctcgctaca
cgaggcgctc gcccgggtcg tgcgcgaact ggggcagcca 12120cagcccgagc
tgaccgtggc ggtggtccaa gcagtcgtcg atgcgtgcat ccgcagaatc
12180gaccaaggcg acgatctgac aaccgtgtcc gacttcgcgg ccggagcgac
gcgtcgactg 12240ctcgcggatg acgacttgcc acatcacccg tgacgcaccc
cgtccaggcg gctcgcaggc 12300ccgtcgacag cgaagccccg gcagaacgag
ccggatcttg agccgcaccg gagcgtgacg 12360cagaccgctg gtggctcatg
cctcgtctca tccgatcttg ccaccgggcg gccgaccggt 12420cagtgcccga
cgcccatcga ttacgacgtc cacgacccga accagcgcgt tcagtgcgtt
12480gacgttcgtg gtgcgctcat tggtcacccg gcctctgggg gtcaccagcg
cttttagggc 12540acgagactcg acggtggcgc gtgataccag gcaggcatca
tgaccttatg gcgatgacac 12600tccggcttcc cgacgacctg gacacgaagc
ttacggagcg ggctcgtggg gagggttgca 12660gcaagcagga acttgccatc
ggggccattc gtgatgcccg ggaccgggcc gagctgaagg 12720tcgatgacgt
tctggccggt ctgatggaca gcgatgcgga gattctggac tacctgaagt
12780gagcggcgtg cgctacctcc agatcgacga gatcctggcc atcgtgcgca
cggtcaacgg 12840tgccgagcac agcgtgcgtg acatgggcct ccttgtgtcg
gcgatcgaac ggccccggac 12900gaacgtcttc ggagccgagc tgtatcccac
cctgcacgag aagccgcggc actactgcac 12960tccgtcgccc gcaatcacgc
gctgatcgac ggcaacaagc gcaccgcctg gttcgccatg 13020cgcgtcttcc
tgcggttcaa cggcgccagc gccagtaccg tcccgcccca cgggcgccgg
13080cccgacggac ccgaggcccg tcacgcgctg ctcaccagca gccctctcct
cagcagcgca 13140ctgggaccgg cgctgctgat cgccctgtcc gccctggggg
ttctcgccct ggacacggcg 13200ttgtgggtct cggtggtcag tgaggtggcg
gcgccggccc ggtggggctt cgtgggcggg 13260ctgcgtgtcg gcgccgggcg
tctgggagcc ctgatcgccg gcgtactcaa cgccgtgatc 13320ggtcttggcg
tggtcgctgt caaactcatc gccgggcact gagagggcct gtggtggtgt
13380tcgcggagcg catacggtgg cagaccggtc ggaatcctcg gcgccgcggc
cggagcggtc 13440ccggcacccc ggcgaacagc cgcacgtccc cgtccggtcg
ggtcaggtcc gagccgtcag 13500atccaggtca gtcgccacag gcgcagaagc
ccggtgccgt ccaccgcgta ctggccgccg 13560cccacgtcct ccccggacac
cacgaagtcc ttggcctgcc acagcgggac gacgggcacg 13620tcgcgggcga
cgatccgctg aagggcttcg aggtcggctt cggcgtcgct ccggtcggcg
13680aagcgctgac tgctcgtgat cagccggtcg gcggccttgc tgccgtaccc
cgtcgccatg 13740gtgccgtccg tgccgacgag aggaccgccg aaggtgtcgg
gatcggggta gtcggcgacc 13800cagccgacgg cgtaggcgtc gagctctccc
tcggcccagc tcttctggaa ttcgtcccat 13860tcatatcctc tgagggtcac
cttgaacagc ccgtcggcct ctagttgctt tttcacctcc 13920gcagcctcct
cgtgggctga tccgcgtccc gccgcgtaac cgtaggtgaa agacagcggg
13980atttcctcac cggcctcgac gaggaggcgg cgtgcctttt cggcgtcctt
gtgagggtag 14040ttgtcgaaga aggaggtggt gtggcccgtg atgctcgtcg
ggatgaggga gtagagcggg 14100tccacggttc cgtcgtagac gtcgtaggaa
atccggtccc tgtctatcag ccaggccgcc 14160gcctgccgtg cgcgtctgtc
gtgaaacggc ttgccgcggc ggttgttgag gtacaggttt 14220cgagtctccg
cgctctgcgc ctccgtcacg cgaagccccg gatcgctcgg gttcagatcg
14280gcgagcattt cggggggaag ctgtctgagg gcgacatcga tgcggtggga
tatccaggcc 14340cgggcgagtg agtcgggggt gtcgtagaag tggagttcga
tcggccggcc ggtgttctcg 14400gcggcgccct tgtaccgagg gttgggcgag
agggagatct tctcgccctt cgcgtaggag 14460acgacgccgt acggtccggt
cccgtcgatc cggccgtccg agcgcaggga gtccgccggg 14520tacgtggtcg
agtcgacgat cgagcccgcc ccggtcgtca gcttgaaggg gaacgtggcg
14580tccggtgcgg tcagtcggaa agtgacggtc cggtcgcggg cgtccatcga
ctcgatggtg 14640tccaggaggg acgacggccc cacgtcggaa tctatcttct
tgacccgttc gaacgagaac 14700cggacgtcct tggctgtcat tctgcgtccg
ctggagaagg tgatgtcatc ccgcagccgg 14760catcgatagg tgcgtaggcc
ggaatcggtg aaggagcagc tttcggctgc gtcgggaacg 14820ggctccgcca
ctccgggctc cagggtcagc agtgtctgga agacattgct gtacagagtg
14880gtcgagccgg agtcgtagcc gccggccggg tcgagtgacg tcggcggttc
cgtcgtcccg 14940accttgatgg tggtgccctc ctggtcgtcc gtcgggtaaa
gcagcagacc ggctgccagt 15000gccgtggcga tcaccgtggg tgtgatgacg
gacgcacgga tgtgcgcacg aataggtctc 15060atgaggctcg tcctcgcaag
atcgagacga acaggaattt tcgtacccct gggtggagag 15120tgcgtcggcc
aagtatgcgc aggcgtcgct tccttcggag cccgacggca cttccggaac
15180gaagtcttat gactgacacg gtggaactgc tatgccccgt tcggcgagag
ggccgccagg 15240ggtcggcacc ccctctcagc agccgttccg cctcgtctcc
ggtggtcctg cggacccgct 15300tgcgcgggtc ccgcccacgg tctcactcct
cgatgccatt ccctgtgcaa tgtcacctgt 15360gccatgttcc gtgttgcagg
gcgtggccat gccaagtcgg gaggtcgttc gtcttccgtc 15420aggtggcagt
gcggtactcc gtttcccacg tcctctcccc cttcagtcgg ccgtgctccg
15480cacggccgga tccctcatgg gaggcgctgt gagaaagtca ctggtacggc
gaggtctggg 15540ggcggcgctg ccgctggccc tgaccgtcgc catgagcgtg
ggcctgctgt cgcagccggc 15600cggcgcagcc gggaacaccg ggtccgtcgt
gcacgtcgcg gcggacgacc cggagcacgc 15660gggacccccg cccgtcgcgc
agtcccccac cgccgagacg gagcacgtcg cgcagggacg 15720cacgagggcg
tccgagcttc cgcccgtggc cgcgagtaag gacgcgctca aggaggtgta
15780cggcaagacc gcgaaggcgc cggtccgtcc ctcgaagtcg acggacaagg
cggtcgccgg 15840caagaccggc aactcccgtg cgcgtgccgc cgcgtgcaac
gtctccgact tcaccagccg 15900gagcggcggc gcgctggtcc agcagatcaa
ggcgtccacg accgactgcg tcaacaccct 15960gttcaacctg accgggaacg
acgcctacta cgccttccgt gagtcgcaga tgacctcggt 16020cgcctacgcc
ctgcgcgacg gctcgacgtc ctacccgggc aacgcctcca ccggtatgcc
16080gcagctcgtg ctctacctgc gcgccggcta ctacgtgcac tactacaacg
ccggcacggt 16140gggcacctac ggcagcagcc tgcagaccgc gatacgcgcc
gggatcgacg ccttcttcgc 16200cagcccgcac tcccgcgacg tcaacgacgc
caacggcgag acgctcgccg aggccgtcac 16260gctcatcgac agcgccgagg
agaacgcccg ctacatccac gtcgtcaagc gactgctggc 16320ggactacgac
tccacctgga actcgtcgtg gtggatgctc aacgcggtca acaacgtgta
16380cacggtgacc ttccgcggtc accaggtgcc cgcgttcgtg agtgccgtgc
agtctgaccc 16440cggcctgatc gacgcgctct acaacttcgc gagcggccac
ctcgcgctgc tgggaacgga 16500ccagtcctac ctcacgtcga acgcgggacg
tgaactcggc cggttcctgc agcattccgc 16560actgcgctcc aaggtcagcc
ctctggccgg cggcctgctc aactccagct ccatcaaggg 16620ccggacggcc
ccgctgtggg tcggtgtcgc cgagatgacc gactactacg acaaggccaa
16680ctgctcctac tacggcacct gcgacctcca ggcacaactg gcccgctccg
tcctgacggt 16740gacctaccca tgcagctcca gcatcaccat caaggcgcag
cagatgacct cgggcgagct 16800gtcctccagc tgcagcagcc tgcgcaacca
ggacgcctac ttccacaacg tggtccgtga 16860caacggcccc gtcgcgaacg
acaacaacag caccatcgag gtcgtggtct tcgactccag 16920caccgactac
cagacctacg ccggcgcgat gtacgggatc gacaccaaca acggcggcat
16980gtacctggag gggaatccgt cggcggccgg caaccagccg cgcttcatcg
cctacgaggc 17040cgagtggctg cgtccggact tccagatctg gaacctcaac
cacgagtaca cccactacct 17100cgacggccgc ttcgacatgt acggcgactt
caacgccaac atcaccaccc cgaccatctg 17160gtgggtcgaa ggcttcgccg
agtacgtctc ctactcctac cgcggcgtcc cctacaccga 17220ggccacgacc
gaggcggggc gtcgcacgta cgcgctgagc accctgttcg acaccacgta
17280cagccacgac accacgcgca tctaccgctg gggctacctc gccgtgcggt
acatgctcga 17340aaaccaccgc gccgacatgg acaccgtcct cagccactac
cgcgcgggaa actggaacgc 17400cgcccgcagc tacctgaccg gcaccatcgg
cacccgctac gacaacgact ggtacacctg 17460gctggcggcc tgcgcggccg
gcaactgcgg tggcgggggc accaacccgc ccgggaacca 17520ggcgcccacc
gccgcgttca ccaccgccgt ccagggcctg aacgtcacct tcaccgacca
17580gtccaccgac gccgacggca ccatcgcctc ccgctcctgg agcttcggcg
acggcaccac 17640ctccacggcc accaaccccg tcaagacgta cgggtcggcc
gggtcctaca cggtcaagct 17700gaccgtcacc gacgacaagg gagccaccgc
caccgccacg aggacggtca ccgtcggcag 17760cggcggaggc ggcggcaccg
aatgcaacgg gaccgacacc cgggaactgg gccagaactg 17820ccaacgcggc
aaccagtccg ccaccaccgg caactacgcc tacctgtacc tctacgtccc
17880ggccggcacc acccagctga agatcaccac ctccggcggg acgggcgacg
cggacctgta 17940ctacagcacc agcggctggc ccggcaccac gagctacacg
cagcgggcca cgggagccgg 18000caacaaccac accctgacca tcaccaaccc
gccggccggc gccaactaca tcagcctgca 18060cgccgtcagc agcttcagcg
gcgtcaccgt gagttccgcc tactgaccca cggctccgca 18120ccaaggcacg
accctcacga cggcccgggg cggctctccc cgccccgggc ggcgtccggg
18180gcggcggcag gggggagacc tccgtcgccc cggaccgaga acacatcgcc
cgcccgcaca 18240cgggcatccc tacctcccag gaggcagagc gtgaagtcat
tacccgcacg caggcgacgc 18300cgcgccatgt ggtccctcat catgtccgtc
ggtctcacct gcgcactcgc cacacccgcc 18360gtcggcagcg gtgaccaggg
cacgtcacgg ctcagcgcct cgcaacaggc cgcggccggc 18420caactcgcag
cggaccagca catctccacc caggaggcac agcggcgcgt actgcggcag
18480gagcggctca ccggcgtcgc aacagcgctg cgtgagcgcc tgggttcccg
cttcgcagga 18540gcctggatcg accagaagca cggcggcagg ctgaccgtcg
ccgtcacccg gtcgacggcc 18600acggccctcg tcgaggcccg gtccgctcag
gctcaggcac ccgacacgac caccgtcgtc 18660gtcgaccgca gcctgcggca
actcgaccgc atgtccgcag gactggccca ccgtatcgcc 18720gcagcgaaca
agggcgccgc ccacggcctg cagtccgcgg tggtggtgca ggacaacaag
18780gttcgtctgg acctgccacg gggcaagacc ctcacccccg cccagcacgc
agtcgtggag 18840tgggcgaagc ggaccctcgg cgatggcctc gaggtcagca
cctacgcgca tgcctccgaa 18900cccttctact gcggcggcca gtactcgtgc
gaccccccgc tgcgctcggg cctggccatc 18960tacggcacga acgtccgctg
ctccagcgcc ttcatggcgt acagcggcag cagctactac 19020atgatgaccg
ccggccactg tgcggaggac agctcgtact gggaggtccc cacctacagc
19080tacggctacc agggggtcgg tcacgtcgcc gactacacct tcggctacta
cggcgactcc 19140gcgatcgtca gggtcgacga ccccggcttc tggcagccgc
gcggctgggt ctacccctcg 19200acccgcatca ccaactggga ctacgactac
gtcggccagt acgtgtgcaa gcagggctcc 19260acgaccggct acacctgcgg
gcagatcacc gagaccaacg caacggtgtc ctacccaggc 19320cgcaccctga
ccggcatgac ctggtccacc gcatgcgacg ctcccggtga cagcggcagc
19380ggcgtctacg acggctcaac ggcccacggc atcctcagcg gggggccgaa
cagcggatgc 19440ggcatgatcc acgaaccgat cagccgagca ctggcggacc
gcggggtcac gctgctggcc 19500ggctaagcag cccgggcgga ccgtgagtac
gccgccccgg tcacatcacg aggacgtcga 19560ccgccgcacg cgcggtcggc
gtctttcccc gtgctccgct ccgtccgcca cccagcggac 19620tgggggcggg
ggcgtggcac gtcgtgcacg ccgcagcgcg gtggaacccg tcggccgatt
19680agaccgtacc ggggagcgcc tttccggctc cgttcgtggg acgggcgggt
gcgtatgcgc 19740gcgtcaccca tttctggaag tgcggagcct gcgacagcag
ttgccagtgg gcgcgtacgg 19800catgatggtg caccacctcg acggccgacg
cctcgaccga atcccgccgc cagacgagca 19860gatgccgctg ccacagcgga
tcccccgcga ggggtttaac cagtactccg cccaccgggc 19920gcatggtggg
ctggacggcg gccaccccca gacccttggc gatcatcgac tgcagttggt
19980cgagcatgtg gaactcgtgg gtgacggcgg gcctgaatcc cgcggcccca
caagcgtcgt 20040agaaggcgcc gggccagccc accccgtcgt ccgcggagac
gaaccacgcg tcctccgaca 20100ggtcggccaa ggacacctcc agccggtgcg
ccagtgggtg atcggcaggg gtggccacga 20160acaccgggac ggttctgata
gctcggtggt ccagcttcgg agagtgtcga agaggcagcc 20220ctgggtagtc
gcaacccagg gcgacgtcga gctcgccggc ctctaggaga tcgatgagtt
20280ctccggtcgc gtacacactg ctgaccgaga cggtcagatc ggggcaggct
tcacggagga 20340cgtcgagcaa ggtgggtacc accggtgtgt tgatggcccc
gaggcgaagc cgacgtgtcg 20400ccccggacga gcggggaggc cgcagccgtg
cgagattgtc ggagagcgcc aggatctccc 20460gggcccggcc gacgacctgg
gcgccgtagg cggtgagctc cacgcccgcg ctgctgcgca 20520ggaagacccc
ctcgccgagc agtccctcga tgcggcgcag ttgggtactc atcgccggct
20580gggtgtatcc gagcgccgca gcagcccggc cgacgccccc cgcgtcggct
atcgcacaca 20640gcacgcgcaa gtggcgcagc tcaagttcca cgggggcacc
tcgctccggg cgaacagagt 20700tccattatgc gccaggagga aggcggtggg
gaatccggga cggcctgacg ccttcggtcg 20760accagtagcc cgagggttat
ggatgagccg gagcctctgg tatggcctgg ccggttgttc 20820ccgggtgacc
gccgtggaaa tctcggacct gcgtgttggt ccgcagaggc gactgcggaa
20880gcctgaagcg caccgccatc gaggagcgac atcatgcctc acacctgcat
cagcttcacc 20940gtcgaagcga ccggggccgc ggttcaccgc gcccgccacc
gcgtctccac cgcgctgagc 21000tggtggggag ggccggtcga ggaagagctc
cgcttcagcg cggaactcgt gacctccgag 21060ctcctcacca acgggctgcg
gcacgcgggc gggcccatga ccgtcgagtt gacgctggtg 21120cacgacatgg
tcgtcgtcgc ggtcctcgat gacagccggg agctgccgcg gcctcggcag
21180acggaggcgg acgacgagtg cgggcgggga ctcgccctga tcgaggacct
cagtctgata 21240cggggagtcg agaccacttc ccgcgggaag cgctgctggg
cggttctgcc gctgcggacg 21300ccacaggagc gggctatcga gtcggctccg
gctgaggagg cggaccacgg cttcgaggca 21360gaccgggaac gctggtcact
ggctccccaa ggaagcggac tactggcgag tctgtttccg 21420gcgatgtgag
ttcgtcctcc tcgggcggcc cagtagccga cccagggcag gcgggcgtgc
21480ctgagggcgt gatgacgctc gtctgacgct ctggccgctt tcaagctgca
cagcgagccg 21540agaaacagcc tttgacctgg ccttttctgc ggctgcctca
ggccgacatc tttccgatga 21600cgcaccacgt ggagtacgtg gcgattctgg
agcctgctgg caaggggttc tgacctgcgc 21660ttttgttctc ctgcggcggg
cgcggcaagc tcgtgcgggg cagttgggtt tcccgaaggc 21720cggtgctcgt
gtgtccggcc ggcgggtggg ctgccttcgt ttcagtgggt gcgagagggc
21780actcggacgc ctgagccgag atgcggttcg ttcggcacca tggggtccgc
aggatgaccc 21840ggtcagcgac cgctggcacc tgtggaagaa cctttgcgac
aaggccctgg ccgaggttcg 21900ctcccacagc gcctgctgga ccacagcgaa
cacaccccgc ccggtcggcg tccatgagca 21960gaccacccgc gaacgttggc
atcagctcca cgacctcctc ggcaagggtg tcggcttgct 22020cgaatgcgcc
cgccgcctga acctgtccct caacaccgtc aagcgctacc cgcgcacccg
22080cgatcctgaa gccctgcgcc ccgtgaagca gctgtttcgc gaggtccagg
agcagggctg 22140caccggcagc ttcaccctgc tctaccgcag cacccagggc
cgggcagaag gcgaccggcc 22200cgtcggaggg tcgcggcttg acgctcaccg
tatccatcac tggaacggcg acgtctgatc 22260ccgtctgccc ggggcttggg
tcccggctgc ggcccgtagg cccggctcac cccagcaccc 22320atcactgttc
gagagtgatt acctctccgc cggacacatg gaaatctgca tcggctggag
22380tagacattgg gcagcagtgt ggttatgttt ctcctgtaac ccagaaggac
cgcagggccc 22440ggcagagacg aactgccggg cagcagtacc cgcagttgca
ggacggtgcg gtggtggagt 22500gtcgaagcca ggatggtgca ggacggcgac
gggactgacg
accggaccgg gcggcccgca 22560gtggtcaggg gccgccaccg cagtgcagta
cccagcagcg aagtcagtga gcggtacctc 22620ggtgaaggcg tcggctgcgg
acgcgcgcgc cgggaggttc ggcagtggtg gttccaagcc 22680agagcagacg
caggacgggc aacggggccg actgtcggac agtggcgctg tcacaggtca
22740ctgagaggtt cgtgtcacca gcagtagagc agtaccagag gaaagaacgg
aggaaccaag 22800cgccatcagg atcgcccggg cgcagttttg ggcccgggta
ccgcaggaca tcgatagtga 22860ggtggtctcc ggtcaagaaa ccgcgatccc
cgcgcccccg gcagcaggca ggtcgggtcc 22920gcggacacag aaggccggtg
cagtatcagg gccggcagat ggtgtaggag ttccttcggg 22980gccctggtgc
cgcatggcac cagggcccct ccatgcgttc cgcagagagg tgcagatgac
23040agcagacgat tcgtacggcc gtctcgacga cgacgattac cccgcctaca
ccatggggcg 23100ggcggccgag atgctcggta cgacccccgc tttcctgcgg
gccgtcggag aagcccggct 23160gatcacgccg ctccgctcgg agggcggcca
ccgccgctac tcccgctacc agttgcgcat 23220cgcggcccgc gcccgcgaac
tcgtcgacca gggcactccc gtcgaggcgg cctgccgcat 23280cgtcatcctg
gaagaccagc tccaagaagc gcggcgtatc aacgaggaac tgcagaggcg
23340cccggccggc ctggtggaca aggccgaggg ctgaggccgc atctgccggc
cggtcctgtg 23400agggctcgcc tgccaagacg ggaagccctt gccgcaacga
gaagaggcaa ctgtccgcac 23460cgatgtgctg ggcccggtcc tggctaggac
tcccgtcttc ttgccggagc gatgcggctg 23520tggacgcgga accggacggc
agtgtcgtcg ggcgcggagc gcggggcgca cgtcgatggc 23580gacaggaccg
gcgaaggtgt attcgtgttc ggcggtgtga cggcgcacct ggccggcgag
23640ggcggcggcg caggtgtcac agggacatcg gttccgactt ccaccacccg
tccgggttcc 23700accagcgtgt catccacctg atccaggctg ccgcggtagg
tgctcgacgt cgggggtgta 23760cgggggcagt tgtaccgttc cgccgcgagg
agtgacccga ttgaccaccg gcctgtggcg 23820ctcaggaacg ggctggactg
tcgcagtccg ggccaactca agcccgacca tgaggccgac 23880cacggcgccg
cgcgaccccg accacagcta cacgcgtggc atgaccaagg cggcacatgc
23940ttcgaacgag ccatctcatg tgtgccggta tgaacgtgat cgacgtcccc
ggcactctgg 24000tgcggacgca agccgtctgg ggcgccaccc acgactggct
cgccgccccg cccccgcggc 24060gccaccgtcc gtcccgccct gcgtcgtcgc
ccgtgtggcg tcatgacggc gacagacttc 24120ctcgcgtatg ggccgaccat
acggccaacg ccagaggtaa agcgctgtcc atggtgagtt 24180ccctgaacag
aagggctggc gggacctcct ttccaagacc gtgctgcagg agtccgtcag
24240agcgcaggta atcccgtgct gtccgcgacc cagggctgtc ctccgtctgg
ccgagggtcc 24300tcgtcttctg ggcgacatcc ctttagcgtg ggcggtagcc
gccgaaggga ggcgccatgt 24360cggacgaatt gacgggcccg ttgggaacgg
caatgcggga ggtcacgttt ccggaccggt 24420ctcgcgggat catcttggtg
cgggctggaa caccgcaggc cgaggccgag gcaatggccg 24480cccgtatgtg
ggccgagatg ccggaaggct gacgtgcccg aacgcagaca acccgtaccg
24540tcctcacacg cattcccctg agccgtcggc catggaacgg aaccagccgt
acgaaccccg 24600gaggcgccgt tgcggtctct gcggcgaggc cggggccacg
cagggcgaag aggccgcgcc 24660gcgttctgcc gcctggcgcg gctgccggct
gttcacgaga acaccgaggg aggagtcgcc 24720cgcctcttgc ccggcgcgtt
gccgggtgga gagcaggtgg tgaaggactg gctcgctgaa 24780ggcggccgag
gcgacctcgt cggccggcct gaacggcttt cactgtccca gcggcggcag
24840gccgccgaca caggcatgct ttgccatctc cctcgctgtc tactgacccc
agcagcagga 24900tccagtacgg cgtcgcggcg ctgccgcctc actcgcgcat
cgatcgggga atgcggcatg 24960tggtgagggc ccggccggcg tgccggtcgg
gccctcacac tgttttggtg tcggcgcgtt 25020tgtcgtgtcg gtcagacgga
caggtggggg gcgccgagca tggcggaagc ccgctgcaac 25080ggactgtcgc
tgcgcgcggg ggcggcctcg ggaagggtgc ggcaggtgaa gcccagctgg
25140gccatggccc ttaggatttc gccggtgctg aagtcgcggc ggtcctggcg
ggtgatgacc 25200tggccgacct gcttggcggg gtagtggcgt cgtccgatga
tcacggactc gccggtgacc 25260ggttcgggtt tgacgccctt catcgattcc
agcacgccgc tcttggtcag gtcgaacggg 25320aagcgggcaa tgacacagcg
catgatgcct cacaggcagg agagttacgg ggccggccgc 25380cgtctggcgg
ttcagcggga gagagcgagg acgcccaggg cgctgccgtg ttcgtcgacc
25440acgggcacca gcccgagccg tccgaagggc accgcgtcct cggcttcctc
cctcgtggcc 25500gacggtgaga cgaagggctc gctgtcgtcg gtgatgtcac
cgaggcggag ccggtcggtg 25560tatcgggagc tgtcccggac ggcggtgagc
cgggcctggg tgaccaggcc gacgcaccgg 25620gcatcctcgt cgcagacgac
cagatgctcg gcacgggcgg cggccatcac ggacagcgcc 25680acctcgacgg
tcatgtcgta ccagacctgt ggcccggcgg cgtccatgac gtcggccacc
25740gtgccgcgca atgggagagc gcctacggag cgatcctgca actgtcctgg
cgtcaagggg 25800tgcctcctgc gcagacgggc ggggttcctg atcaggacgg
tcctaggcgg ccgcgccagc 25860cgtggacttg agtgcggggg tacgccgcgt
cgccgaggcg gggcggcgtc ggccgcgtga 25920ggtggcgccg cgcttcttgg
ggcgttcagt cgccggggcg gtgatgacga ccgggatgcc 25980ggtcggggcc
tgggctccgg tgatccggct gagggcctcg tcgcccgggc tgacctgggt
26040ggtctgcggc cggatcccgg cttccgacat gagacggacc atgccgcggc
gctggttcgg 26100ggtgacgagc gtgacgacgc tgccggactc gccggcgcgg
gccgtgcggc cgccccggtg 26160gaggtagtcc ttgtggtcgg tcggcgggtc
gacgttgacg acgaggtcga ggttgtcgac 26220gtggattccg cgtgccgcga
cgttggtcgc caccagcacg gtgacgtgcc cggtcttgaa 26280ctgcgccaga
gtgcgggtgc gctgcggctg ggacttgccg ccgtgcaggg cggcggcccg
26340taccccgctg ttgagcaggt cccgggtcag tctgtcgacg gcgtgcttgg
tgtcgaggaa 26400catgatcacg cggccgtcgc gtgcggcgat ctcggtggtg
gccgcgtgct tgtcggcgcc 26460gtggacatgg agtacgtggt gctccatcgt
ggtgacggcg ccggccgagg ggtcgacgga 26520gtgcacgacg gggtcgctga
ggtagcggcg tacgagcagg tcgacgttgc ggtcgagggt 26580ggcggagaac
agcatgcgct ggccttcggg acgcacctgg tcgagcagtg cggtgacctg
26640cggcatgaag cccatatcgg ccatctggtc ggcctcgtcg aggacggtga
cggagacctg 26700gttcaaccgg cagtcgccgc ggtcgatgag gtccttgaga
cgtcccggag tggcgacgac 26760gacctcggcg ccaccacgca gcgccgacgc
ctgcctgccg atcgacatcc cgcccaccac 26820cgtggccagc cgcagcttca
cagagcgggc gtacggggtg agcgcgtcgg tgacctgctg 26880cgccagctca
cgtgtcggta cgaggaccag ccccagcggc tgccgaggct cggcccgccg
26940gccggccgta cgggccagca gagccaggcc gaaggcgagg gtctttccgg
aaccggtgcg 27000cccgcggccc atgatgtcgc ggccggcgag ggagttcggc
agggtcgcgg cctggatcgg 27060gaacggcacg gtcacccctt gttggccgag
cgcggccagc agttccccgg gcatgtcgag 27120atcggcgaag ccctccgcag
cgggaagcgc gggggtgatc gtccggggga gggcgaactc 27180cccctgaacg
gcgccgggcc ggcggccgta accgccggag cggctgggtc cggccggccg
27240gcgcggcgcc ggcgaaccga agcggctgcc gccctttccg gagtcggcac
cgccatgacg 27300ggtgcgagcg aagcggtcgt tcgtgcgtgt gcggttcata
cggaaccttc ctcgatgcgg 27360cacatatcaa ggaatttccg aagcaatgag
cagcacggag aatcgcaaga atggaccggt 27420gggccttgcc agcggatctg
gccgacagaa aatctgtgcg gcacgtgcgc tggaatgatt 27480gggggtgctg
tgggctcgat attcgaagcg tccactgcac tgtagctatg aaggatgcgg
27540ctgcaccttc gaaggacgat ccgtgtgcgg taaacacacg ctgtccggag
cgtcgtccgc 27600aggtgaaatc actgcgggaa acgcatgtag ctggggcccg
caccccgaag gatgcgggcc 27660ccagctacaa gtacgtgaca gtcggcgtca
ggcgggaacg atgttctcgg ccgtcgggcc 27720cttctggccc tgcgcgatgt
cgaagttcac cttctggcct tcgagcagct cgcggaagcc 27780ctgggcggcg
atgttcgagt agtgggcgaa cacatcagcg ccgccaccgt cctgctcgat
27840gaagccgaag cccttttccg cgttgaacca cttcacggta ccagcagcca
tgtcatttct 27900ccttcggggc agtcgtacgg gatccgcacc gcgcggacct
cgtgtcgccg caatgatcac 27960cccgcccgga aaaagaccgg agatgtaaaa
gtgcttccag gggtactgag cccgaccgga 28020gcacttgaaa tttcgggaac
cacaactgca actgacatcg acagtagcac gccacagcag 28080ccactgtgcg
gtgaagaacg ccaccttgct tattgcggca gagaatctat ccgcatgctc
28140cgatgaaaac tcaaaccgcg cgcacagata ttgaccttcg cgcgacgcca
tatatcgcat 28200gccgcgctcg cgtgatccgg tcccccacca cgctctccgc
tactgcacgg gtcgcaccgc 28260cgcgggggca gacaggtccg gccatgacgc
cggccatgct cggggcgtag cggacgcctg 28320ccggtcgggt gtacgtctcg
cgcgcggcga gcactgcggg ggaggggccg gttgccagac 28380gtcttgcctg
gcaaccggct gtcggctcgg gctggttggt cagccgtggc aggtgatgtg
28440gttctgcgcg cccgcttccg tgaacgcgcc gcagccccgg ctgccttcta
ccaggccgac 28500cctcaggagg cgtgacccgg ggaagccgag gatcagcggt
agtcgtcagg ggaggcttcc 28560ttgccgccgt aggtgacgtc ctcgaagtat
gcccaggcat ccggccggct gccgtccacg 28620tccgtcaccc cgtatgccct
ggccagttcc ccgctggagg tggacttgcc gttccaccgc 28680ttcgcgcggt
ctgggtcggc ggccagcgcc gcgaccgtac gggccaggta gtgcggggac
28740tccgcgatcg cgaacgtcgg ctcttgggcg atcgcgtcac gccagttctc
ctcactcaca 28800ccgaagtggg agagcatctg ctccgaacgc aggaagcccg
gggacaccgc gaccgccgtg 28860ccctcgtact ccgccagctc ctgagccagc
ccgaacgcga ggcggatcgg ggcgttcttc 28920gccaggtcgt agtagatgtt
ctcgcggtag cggcggttgg agtgcgcggt accgtcggtg 28980acttccacat
gcagcggcgc gtcggagcgg atcagcagcg gaagcagcag cgccgccgtg
29040atcacgtgcg agcgcgcgcc cagctccagg atccgcaggc cgtcggcgag
cggtgtctcc 29100cagctcttct tcccgaacac cgaggtggcc agaaggtgct
cgccgcccca caggtcgttg 29160acgagaatgt cgagccgctc gtactcccgg
tcgatccgct cgacgagggc gcggacctgg 29220gcttcgtcga gatggtcggt
gggaactgcg attccggtgc cgcccgctgc ggtgacgagt 29280tcggcggtct
cctcgatggt ctcggtcgtc cggccgacct cgctggcccg ggcccgggtg
29340gttcggccgg tcacatacac ggtagcgccg gcccgcccca gttccacagc
ctgagctcgt 29400cccgccccgc gggtagcgcc cgccacgagg gcgatccgtc
ctgccagcgg acccttcgga 29460ccggcctgct cggtgttctc agtggtctgc
ctggtgatgt cctcgttgct catgtcatcc 29520atcgttcacg ctaaaaccga
cagaacacgt caccttttat gtggggggta ccgcgcatca 29580tcccggccat
agcgccaact acgtcctcgc actgagcgtt ttcagcgtgg gccaccgatc
29640gggtgacgcc ggtcaggtcg gggtaggggc cgcaacgcac aaggctcgcg
tgcacgacat 29700ggccaccgcg cgcatgatct cccagcggga gcccagccgt
ccccggcagc cccagccgct 29760gagaccagct cacccgggac acccggtccg
acaccgcaca cgatcaagta gtcgacctcc 29820agacgcgttc agcagcccac
atcccaggag ccgtctaccg tcccaggaac ccctgctccg 29880ggaccatcgg
gctcggcacc gggagtgcac agttgatcag taactggcaa cgagctcgtg
29940cacggtaagc ggtgaggtgt cgaggtccag atgggcggcg gcggtggtgc
ccccagcggt 30000cggccgaccg gcatgccgag cgggcagccc accggtgtgc
cgagcggcgg acccggcggc 30060ggcacgggca tgggcggcac ccccaccccg
cagcacctga agtcggtcag gaccggccgc 30120gtgacgggct tcgggtcaga
cctgtgcggg gaacagcagg cagtcgtccg ggcggatgat 30180caggttgatc
tcgccgtccg tgtgccggac ggggctctcg gcatggacgc gcacgtcgcc
30240gatgctgagc tcgtactcga atcgcgctcc ggtgtacgag cactgctcga
tcctcgcccg 30300gagcacgttg acggcaccgt cgtgcggggc gtcggcgcgg
tcggtgagcg tgatgcgttc 30360cgagcgcagg cccacggtgg cggacgaccc
cgcggagcag gcgccggcca ccctcaagcg 30420ctgaccggtc tcacccagtt
cgacctgtac ggctccgccc tcggtggcgc cgacgcgccc 30480ctccaggagg
ttgcagcggc cgatgaagcc ggcgacctcg ggagtggcgg gagtctcgta
30540gatctcggtc ggtgtgccca cctgctggag gtgtccgtgc atgaacacgg
cgatgcggtc 30600ggacagggac atggcctcga cctggtcgtg ggtgacgtac
acggtggtga tgccgacctc 30660ccgctggagg tccttgagcc agacgcgggc
ctggtcgcgc atcttcgcgt ccaggttgga 30720gagcggttcg tccaggagca
gcacgccggg ggagtagacg atgcctcggg cgagggcgac 30780gcgctgctgc
tgtccgccgg agagctggtg ggggtagcgg tcgcgcaggt gagccatgtc
30840gaccttggtg aggacgtcgt cgatgaggcg ccgttgctcg cccttggtga
ccttgcggag 30900cttcagcggc agtgcgaggt tgtcggcgac ggtcatgtgt
ggccagagcg cgtacgactg 30960gaagaccagg ccgagattgc ggccttcggg
gggcaccgtg ctgcgccggg tgccgtcgaa 31020gaagacctgg tcgccgacac
ggatggtgcc cgagtcgggg gtctccagac ccgcgacgca 31080cgacaaggtg
gtggacttgc cgcagcccga cgggccgagc agagtgaaga actccccgtc
31140cgcgacggtg aagttgacgt cctccaggac cgcggtcccg tggaaggact
tcttgatgtt 31200ctcgacgacc agctcaggca tgcttcttcc ccttcaggag
gagaccggcg aggccggcga 31260cgacggcggt gacggcgatc tggagggtgg
cgagggcggc cacggagccg gtctcaccct 31320gggtccacag atcgatggcg
gtggtgccga tgacctgtga ctcggctccg gcgaggaaca 31380tggcgggggc
gtactcgcgg atcatctggg tccagatgag caggaacgag gcgagcatcg
31440cgggcacgag gagacggagc atgatccggg acaccgtgcg ccaccagtcg
gcgccggcga 31500cgcgtgcggc gttgtcgagt tcggctccga gctgcatggt
cgccggggag atcgcgccgt 31560acgccgacgg gagtgcccgg atgccgaagg
cgatgatcag cgcgaagagc gtgccgcgca 31620ccgcgtcgcc gccgggtatc
caggtgaagg cccagaacag gccgatgccg acgatcaggc 31680ccgggaccgc
gtgcggtgac tgcgctgtcg tctccaggag acgggcgaag cggaagtcgg
31740agcggcgtgc cacgaggacg accaccgtgc cgaacagggt cacggccacc
gcccccacga 31800aggccacggt gatgctgttg acgatcgact cggtgtaggg
ggcgtagtcg aagatcagac 31860ggaagttgtc cagggtgagc aggtcgaacg
ggttcaccag cggagtgagc agcgaggtga 31920acgcgcgcag gatgagcgcg
agcatcggca gcagtgcgcc gaagacgacg tacagaccga 31980cgaaggcgaa
gcccagccac ttccaggcac cgatgtcgag caggtcggag cgggtcgcct
32040tgccgcgcac cgacacgaac cgctgggcgt gccccagcag ccgcgtctgg
aacacgacca 32100gggcgatggt ggtgagcagc atgaaggtgg acgccgcgcc
cagcaggccg tagtccggat 32160tgatcgagtc gatgccctgc tcgtagagga
agttggagaa gagggtgatg ccggcgggct 32220cgcccaggat gagcgggatg
gacagggtct cgatcgccgt gccgaagatc agcagacccg 32280cgtagagcat
cggcgggcgc agcatcggca ccacgaccga gcgcaggacg cgcagaggcc
32340ccgcgccgac gctgcgggcc gcgttctcca gagaggtgtc ggaggcggcc
agcgcgttgg 32400cgcagaacag gtaggcgatg gggacctggg cgacggcctc
gacgaacgcc ataccgggca 32460gtgagtacag gttccagggc acccagccga
agccctcgcg caccgcgccg gtcaggaagc 32520cggccgggcc gtagacgacg
atccacccga aggccaggac gagcggggag atgtagatgg 32580gccagcgcag
cacctgcccg aacaggcggg cggcggggaa gcgggtgcgc tccagcagaa
32640tcgccatcgg caccgcgatg gcgagcgcga acacggtcgt caggacggcg
aagaggaggg 32700tgtcgaggac gatcgaaccg aagcccgccg acgtgaacag
gtgggtgtag ttcgagaggg 32760tgaaggcgcc gccggccgcg tacaggggct
ggttgcggac cgactggtag aggatcggta 32820cgacgggggc gaggacgagc
acggcggtga cgaggaacgt cagccagtgg atggtgacct 32880cacgtccggc
gccgaacagg cgccggtact ggggcgtgcc cagctcgccc gcgcgcggga
32940tgcgggacgg cgcgggtggc gccgggggtg tctggatggc catgacgact
ccgtacgaac 33000ggggtgggga caggggcgtt gggcgggcgg gggcggctca
gccggccgcc ttctcccagc 33060gcgcgacgta cgcctcccgc acgcgctccg
gcacccgcac gggccggtac agatggacgc 33120ggtccgcgcc gagcctgcgc
cgcatgtcct gcagactgtc catggcgtcc tggcgcacgt 33180ccggccggta
cggcaccagg ccgccctcgg cgaccgccgc ctgcccttcg gcggagagca
33240ggaagtccag gaagagacgg gccgcgttcg ggtgcggggc ggtcttcacg
acggacagcg 33300cgcgcggcat gacgacggtg ccctccgcgt agtagctcca
ccccagcagt cccccgctgt 33360gctcggcggc gggtatcgcg acgccatcac
gtacggcagg atcccgccgt gccgcaggta 33420ggccagttcc tggcgcgagt
ggaggcgcag cctgagacgg accgtggcgc ggggtgcgtc 33480gggccggacg
agggacaggg cgacggggtt cgtgccgacg cacaggtcgg cgaggccgtc
33540gaaggtgaat tcctcctctc cggtgaaggc gtgggccgat gcggtgtcgc
cctcctcgaa 33600ctccaggggc agtacaccca tgccgatcag gttgttgcgg
tggatgcgct cgaaggactc 33660ggctatcacc gcccgcactc ccagcagcgc
ctgtgccttg gcggcccagt cgcggctgga 33720gccggcgccg tagttgcggc
ccgcgaccac gacgagatcg tggcccgcgg cgcggtaggt 33780cgccgcggct
tcgtggacgg gccccatccg cagttgcgtg ccccgatggc cgccctgcgc
33840atggacgatg cggccgggtc ccttcgaggg cctggccgcc gggtcgttcc
tgacccgagg 33900cgccgatcag gacgacgccg tgcaacggac gcgaggacag
cgtcagcttc gtccgcggca 33960acagcgacga ccccggtgac ttccgatgac
gcgcacgccc ccgccgcccg aacccgagct 34020gaccgtcgac cgcgccgcct
gctctgggtc accctcccgc tccgcctgcg agatcagatc 34080gccgacgcgc
cgccgggcac cgtcgtccac gtcgtcgcca ccgacccccg cggcaccgct
34140cgacctgccc acctggtgcc acatgacagg tcacacctgt ctcggcacgc
ccccggcgaa 34200cggccggtgt acgccccgaa gctcaccgcc gacgcgcgcg
ccacccgccc ggacgcaccc 34260tggcacccgc tccggcggcg gcaggagcag
ccccggaacc ggtgacgcat ctcgtcggcc 34320ggccgtttcg agtggaccgc
ggacgcggaa cgtcacggcg tccggaaacc ccggaaggtg 34380accggcctgc
gtgtcttgaa gccgagccgt tcgtacaagg cgatcgcgcc ggtgttcgcc
34440tcggccacgt gcaggaaggg acgatcaccg cgcgccgaga tgcgctcggt
gagagcgcgg 34500acgaggcggg cggcataacc ctgcccgcgc gcctcgggag
cggcgcagac ggcgctgatc 34560tcggtccagc ccggaggacg caggcgttcc
ccggccatcg ccaccagggt gccgtcgacc 34620cggacaccca ggtaggtgcc
gagttcatgg gtacggggcc agaacggccc cggctcggtc 34680cgcgcggcga
gatccagcat ctcaggcacg ctgtccgcgc ccagctcgac cacgtcggtg
34740tcggacgcgg agcgagttcg gccggggcgg ccgtcgccgg gccaggtcat
ctgacggccc 34800tcaagactga aaaccggctc ccaacccggc ggcggaacgg
ccggggagct gaacatgtcg 34860gcgaaggcgc cgggaccgag taggccggcc
aggtcggccc agtcctccgc gtccgggtcg 34920acggacacgg aggagaaggt
cgccacgtcg gtgagatagg tggctgctcg accgaaccgt 34980cgggcgagat
gagcgtgccg accactgagc gactgaccta ccgggtcgtc gagtgcgggg
35040tcgtcgtcgt tcatcatcgt gccgtttcct tcctggtgag cgcggtggtc
gaagggtggc 35100cgcggtaggc gaaaagtcgg cggcggggcc cgtggcccga
tagtcgtagc ccttgtcacc 35160gtgcagtttg ccgggtcgcc tgaggacttc
cggctggagg ccaatgccaa agcgctctcg 35220tgccggcgga ggcacgcctt
ctgacgtgcc tccaccggca ccactcagtt caggcagatt 35280gagcttgagc
gatgcagcgc cgccggaagt cgagcgcctc aatgcatcga gcggcgactt
35340cctcgttctg ggtgagagca gtcctgcctt gtcgagtgat gcggcgttcg
gaccgtcacc 35400ccggcgaagg ccaggacctg tcccacggag tggctcatcc
acctccccct cctcggccca 35460cagcttcagg cccgacgtag ggggaggggc
gactcggaac ccggcgtccc gctcgcgaag 35520gtcggtcaga cctgttcgaa
gtggaacgcc ttgatgaagc agtcccgggg ttgggcgacg 35580gcgaagagga
tgcactccac gagggactgc gcggtgaggg cgtcctcggc ttcgcgtgaa
35640gtggtcgccc actcctcgga gagcgggtcg gcgttgtcga agtcgggcgg
gtagagcgag 35700atcacccgga ctccttgggc gcgcaggcgc ttggagagga
tttcggtgaa ccctgcctgg 35760gcgctcttgg ccgcgtagaa ggcgtcgtgt
gcgtccgagc ggtggtggcc cggtgttccg 35820caggcggaga ccatcgtcac
gacgtcgggt gtgtccgagt tgagcaggag ggggaggaaa 35880ctcctcgtgg
tcaggaccgt gccggtggct ccggaggcga tggtgtccac gacgtcggcg
35940tcggttgccg acagcaggtc cggcccggtg aggtagcggg agccgttgtt
gacgagtacg 36000tcgacgcggt cggtgtgttc cgcgacgccg gaggcgaagt
cgcggatcga ggcaggatcc 36060gtcaggtcgc aggcgaaggc gtgcacccgc
tggtgtccgc ggtcgcggat ctcgtcgcgg 36120acccgttggg cggcggcgag
ccggcgtgcc gagaggaaga cctccgcgcc gaggtccgcg 36180aggcggatgg
ccagggttcg tccgaagtcc cggccggcgg ccgtgatgac gacgcggtgg
36240ttgtcccatc tcatggtgtc gttccccagt cgccgtttcg tggatcgggt
ggtgccgtgc 36300accgcgtctc tacgctatcg gtcatggtcg ctcacgaacg
gtcgttcacg gtcaatgatg 36360atgttgaggt gcccaacccc ggtgcggacg
aggtctggac cgtcggcgcg gtcatcctca 36420atcgggaagg tcgtgccttt
gcccagaagc ggagccggga ccgtcgcctg ttccccgggg 36480cctgggacat
cgtgggcggt catgtcgagg agggcgagac gcttctggag gccctcgcgc
36540gtgaagtcga ggaggagacc ggctggcgcc tgacccgtgt gcggcggttc
ctcggcacca 36600cgacctggac gggggacgac ggcggcggcc tgcgtcacga
ggccgactac ctggtcgagg 36660tggacggcga cctggaccac ccgaggctgg
aatggtccaa gcactccgcc tacgactggt 36720tcggccccgg cgatctcacc
cgcctcaagg agaaccgcgg accaggggag tacctgatcc 36780acgacctcat
agccggtgcc gttgccgact cgcctttcga cttgctccgg gcggacgccc
36840tcaccagccc ggaccggctg cgcgagctct acccgcagcc gaacccgaac
tcgctgcgca 36900aggagaccga ccgcctgacc gaggagaccc gggcgctgat
cggctgttcg tcactggtgt 36960tcatcggcag cgcggaccgc gagggccggg
cggacgtgac gccacgtggc ggcccggccg 37020ggttcgtctc ggtgctggac
gagcagaccc tggtgatccc cgacgcgacc ggcaacaaac 37080ggctcgacac
cctgcacaac gtgctggaga ccggacgcct ggggctgctc ttcctcgtcc
37140ccggccgccc gaccacgctg cggatcaacg gacgcgcctg tgtttcggcc
cgcccggagc 37200tgctcgcccg cctcactccc gtcggaaagc cgccggtcac
cgcgctggtg gtgcaggtcg 37260agcaggtgta tccgcactgc ccgaagtcac
tgatgcgcgc cgacgcctgg cgacccgagc 37320agtggatgcc cgccgacgcc
cagccgagca gcgccgaggt gacccttgcg cagctgaacc 37380tgcccggcct
gaccctggac cggatcgagg atgccgaacg ggagtcgctg cgcctgcggt
37440acgaatgacg acgagtcgat gagcgccgat gagccgatga gacccgacgg
gatccgacgg 37500gtcggcgtcc gcggcgagca gaccggtcgc gaaggtcacc
gcccgcacgg cggcgaccct 37560cgcgacggtc agtactgtcc ggtcaggtgc
gggtccagcg
ttggttgctg ccgttggagc 37620aggtgtacag ctggatcagg gtgccgttgg
ccgtgccgtt cccgacggcg tcgaggcaga 37680ggccggactg gacgccgacg
acggacccgt cggagttgag gcgccacttc tggttgtcgc 37740cgccccagca
gctgtagatc tggaccttgg agccgttgcc ggtgcctgcg gcgtccaggc
37800acttgtcgcc gtagaccctg agctcgcccg cgtcagtggc ggcccactgc
tggttggtgc 37860cgctgtggca gtcccacagc tggagctggg tgccgtcgga
ggtgctggcg tcgggcacgt 37920cgaggcagcg gcccgaaccg acgcccttga
tctgtccccc gtccgcgggg ggctccgagg 37980agtcgccgcc gttgagtgcg
tcgaggacgg cggtgtacgc ggccttcttg ctgccgtcgt 38040tgttgaacag
caacggcgtc tgctccgacc gccaggagtc gctgtcgcgc acaccccaga
38100cggtgatgcc gaggcagcgc gagacggcca ggcagtcgtt ggtcacgttg
gcgtaggtcg 38160aggccggggc gccctggatg tccagctcgg tgatggccac
gtcgacgccg agggcggcga 38220agttctgcag tgtggtgcgg aagttgctgt
tgtaggggct gccgctgttg aagtgcgact 38280ggaagccgac gcagtcgatc
ggcacgccgc gctgcttgaa gtcccgcacc atgttgtaca 38340tggcctgggt
cttggcccag gtccagttct cgacgttgta gtcgttgtag cagagcttgg
38400cggacgggtc ggcggcgcgc gcggtgcgga aggcgacctc gatccagtcg
ttgccgctgc 38460gttgcaggtt ggagtcccgc cgcgctcccg aactgccgtc
ggcgaaggcc tcgttcacga 38520cgtcccactg gacgatcttg cccttgtagt
gggccatcac gccgttgatg tggtcgatca 38580tcgcctggcg cagcgcgctg
ccgctgaggc tctgcatcca gccgggctgc tgggagtgcc 38640aggccagggt
gtggccgcgc acctgcttgc cgttctgcac cgcccagttg tagacgcggt
38700cggcggagct gaagttgaac tggccccgct gcgg
38734313331DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number D86074 31tcggatctcc ccacacaaca tagatagagg
atatccgcct gggttcacaa tgaagttact 60ggtggttctc accaccctcg tgggctttag
ctcagcacta agtttcggtt gtaattacag 120accagtatta ggcttcaatt
cacagtatat gctgggagga ctaagacttt tctgtatgcc 180tgccatggtt
tatgatccat gggcatgtgg ttgcgtttcg gcatggagca gtgcaggtct
240ttacggtgtc ggagggggcg gaggcgcctg gggagctggc ggtgctggag
gagccgacgg 300cggacgcggc ggcggcggtg gagattggga atatgactat
gatgacgaca gcgatgacga 360tgatgaatgg gactgggatg atgacggtgg
aatgggagct ggcgccggag gtggtgctgg 420tggtggtgcc ggaggtggtg
ctggtgctgg tgctggagca ggcgcaggag caggagcagg 480tgctggactc
ggacttggat tgggcggagg tctcggaggt ggacttggcg gacttggagg
540tcttggcgga cttggcggtg gagacgattt atttgattta gatttcgatg
atcttggtgc 600agctcttgcc ctcggtggag ctggtggagc tggaggtgct
gctgctgctg ctgcagctgc 660cgctgctgcc gccgggggtg gagttggtgg
agctgctgcc gcagccgcag ccgctgctgc 720cgctgcagga ggaggcgcag
gtagacttgg aggagctgct gctgcagccg cagccgctgc 780tgccgctgca
ggaggcgcag gtggacttgg aggactcggt ggcggacttg gaggactcgg
840tggcggactt ggaggcctcg gaggtcttgg tggcctcgga ggatatggag
gatctgctgc 900tgccgctgct gctgctgccg ccgctgctgc cggaggtgga
ggactcggtg gtgttggttt 960ctacggtgga cgaggaggta gacgcggtcg
aggaagagga ggccgcagac gtgctgctgc 1020tgccgctgct gcagctgccg
ccgcagccgc tggtggtggc ggaggaggtg gaggtggtgg 1080aggaggaggc
ggaggcgctg gtgctgccgc tgccgctgca gccgctgctg catctgcttc
1140agcttctaga caaatgagtg gtataaggga cgcattagga gacattaaag
accttctcag 1200gagtaatgga gcctctgcaa aagcctctgc taaagcatca
gcagtagcaa gcacaaaatc 1260tcaaattgac gatttgaagg atgtcttaaa
ggatcttgca ggtctattga aaagctcagc 1320atctgcttca gcatctgcat
ctgcatcagc ttcagctgga ggtggaggcg gtggtggtaa 1380cggaggtggt
aacggaggag gaggcggcgg tggagctgga gctctagctg ctgctctcgc
1440tgctgcagga gccggaggtg gacttggagg tggaggcgga ggcggagctt
tagccgctgc 1500actagctgct gctggtgcag gtggaggagg ttttggtgga
cttggaggac taggcggtct 1560tggtggggga tctgccgcag ctgctgcagc
cgctgccgct gctgcatcag gtggtggagg 1620aagagcactt agaagggctt
tgagaagaca aatgcgtgga ggtggatccg ctgctgccgc 1680tgctgctgct
gctgcagctg ctgctggagg tggatgggga ggtggaatgg gtggaggatt
1740cggagtaggt ctcggtggag gattcggagg aggatttggt ggtggatcat
cagcagcagc 1800tgctgccgct gctgcagccg ccgctggatt tggtggaggt
ggacgaagag gtagaggtag 1860aggacgtgga ggcgatggcg acggtaacgg
agctagtgct gtagctgcag ccgccgccgc 1920tgctgctgct gctggaggat
ctgctgctga tgttgccgct gccgctgctg cagccgcagc 1980tatgtacggt
gacggtgctg atggacctga tttcgataat ggattcggtg gtggaaacgg
2040aaatggaggt ggcggatctg gtggtggcgg atccggcgga ggtggatccg
gtggcggatc 2100tggaggtggc ggtggatctg gtggatcagg cggtggcggc
ggatctggtg gttcaggcgg 2160tggcggatca ggcggcggtg gaaacaatgg
atggggaaat aacggcaaca ataaatatga 2220cgatgatgac tgtgatgaat
atggtaaccc tattagaagg gggtaaatta tttgacatta 2280tccgccattt
gactcatttt tcttagttct ctatgtttta tacttcacct tagattgttt
2340tagtttgatt gaataaatta tgttttcgat ataaattttt tttaaattaa
attaaacttt 2400attagttgac ctgtaaactt tttcatggag ttataatcta
aggaacaaaa aacatacata 2460atatgttcag tattgtggta aagcacctgt
accgcaaaca caatcacctc tatacatgta 2520tacaaaatca gtaatgctga
caaaatcttc tacactctca cctacacact cgcacacagt 2580cctcttacat
acacagcact ataatatcct gaacatgaag tttgtgttga taaaaagttc
2640agaaaaatct cccctacatc acctgatctt tcactgaaaa tttacgacaa
gtattgaaaa 2700tagcagaaag aaaacgggaa attgagaagt tttctataaa
aaacaatcgg aacaatgact 2760ggaatgacaa ggatgaaaat aatgataact
tacattaatt aaggccccaa taatctctct 2820attttcaaac ttttttttca
aatgttctct ctaactcact tgcatctatg tggaaattca 2880catactatac
taaattacca caagtatcaa ggtttcacaa cctctcatgc cttcatggca
2940gaccatgctg ggtatttgtc taacaatgcc tcataaatac ataaaactaa
ctaacaaaat 3000aggtcagtct gtaacaaatt attaatgcac cattattgca
ttttctaaaa caaagcatac 3060actggatatt ggcagacaaa atgttgttat
tggatacctt tccattctat ctagacactt 3120gctttccaca agtcatcata
aataaatccc ccctatccca aatgtcaatg gaatgcccca 3180acccttcccc
cataatttta aaacctagaa taaattaaaa catctatagt tcgtcatgat
3240catctttctt atcatcctct tcttcttcct cctcctcctt cttcttcttc
ctcctcctca 3300ggttcttggc tgcctgctcc ttccttgcca a
3331325224DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number L05390 32ggatcccctg ctcgacgccg gcggcccggt
acacctccat cgtgcggacg ttgttcccgc 60gcccgcgagg gtggacggag gtgccggcgt
gacgctccac cagcatgtgc cgcaccccga 120gccggcccag gaacacggac
gtcgacaggc ccacgagcga tccgccgacg acgaggaccg 180gaaccctgtg
gaccgtgtcc ccggcccgat cggctcttgc gttcatcttt ctcctccagc
240gcgtgatgtc cgcccactcg gccggtttcg gccggggtca tgcatgcccc
gcgaggctgg 300agcgcggtgc gccggggacc acacttcacc cgcttaaccc
gctgcgttcg cgcaggggca 360cggcacgccc gacgatcgtg ctcacgggcc
gacgcaccgt catgtgacgc gtcggccgcc 420ttaccgttcc tccaggaaga
ggtgcgcctc aatgacggtc tctgccgctg tgtccacggt 480cccggaccgt
gtccccctca ccgtgttcga cggttcccgg gtgcgggtcg tgctgatgct
540ggacatccgc gacgggacgc aagcggaggt cctggacgcc tacgagcgga
tgtccgaccg 600ggtcgccgcc gtgccggggc acatcagcga ccagctgtgc
cagtcgctgg agaaccccac 660ccagtggctc atcaccagcg agtgggagag
cgcaccggag ttcctcgcct gggccaacag 720cgaggaacac ctggagatgg
tccgtcccct ggagccctac gtccgcggca cccactcgat 780gcgctactcg
gtgctgcgcg agacggccga ggagcgggcc ggggcgggtg cggcggcccg
840gggcgcgctg cagccccggc cgcgcatcgg cgacaacgtg gtccggcacg
ccgtcaccta 900caccgtcaag cccgacagcg tcaccgaggt cgtgaagatc
ctctccgcct acacctcgcc 960cgaggtgcgc gtggacgaca ccacgcggct
cgtgcgcacc tccctcttcc tgtacggcaa 1020ccgggtcgtc cgggcgatcg
aggtgcgggg cgacctgcag gccgccctgc gccacgtggc 1080ccggcagccg
gaggtgcgcg ccgtcgagga agccctcacc ccgcacatcg aacaggaccg
1140ggacctcacc gacccgcggt ccgcccggct gttcttcacc cgggccgcgc
tgccggccgt 1200ccaccacgtg gtgtccgggc gcgggacggg cggcgacacg
cagcggtgcg cgctgtacta 1260cccggcccac cccggcgccg gaccggcgct
cgcccggctg ctggcgcggc agggcgaggc 1320caccgtgggc gacccgggca
gtccggtcgt cgcctgcacc gtcttccacc gcgacgacct 1380cgtcgtacgg
ctcgtcgaca cggcgggcgc accggagcgc gcgcccgggg ccgtcctggc
1440cctgcacgag ccggacgccc tcgccgaggc cgggcggctg ctggacgccg
ccgcgctcgg 1500cgccgacggc cccccggacg accgggcgct gccgacgttc
ctcgcgcacg cccggatgcg 1560gcctctgaca gaccgtcagt cgccggcctc
ctgacccccc gctcgcccga cctcagggag 1620tgaccgacat gacagaacag
caggcacgca tcgtcgcctt cgacgacgtc ccgcccaacc 1680ggcggcgcgg
cggcgacgtc cgggccctgc tcacgcccac gaccgcgggg gcgaccagcg
1740gcttcatggg cgtggccgtc gtacggcccg gagaacgcat ctccgagcac
taccacccgt 1800actccgagga gttcgtgtac gtcaccgccg gcgccttcga
ggtggacctg gacgacgtgc 1860cgcatcccct gcgcaccggg cagggcctgc
tcatccccaa ggacgtgcgc caccgcttcc 1920gcaacaccgg cgacgtcgag
gcgcgcctcg tcttccacct gggtccgctg gccccccggc 1980cggacctcgg
gcacgtcgac accgaggaga ccgacgagac cgcgccggcc ggggtggtgt
2040catgagccgc cgggtcgtcg tcaccggcat aggcgtcgtc gccccgggcg
gcatcggcgc 2100ggcccggttc tgggacctgc tggccggcgg gcgtacggcg
acgcgccgga tctccctgtt 2160cgacccggcg cgcctgcgct cgcagatcgc
cgccgagtgc gacttcgacc cgtccgcgca 2220cggcctggac gacgagacgg
tccggcggtg cgaccggtac gtgcagttcg cgctggtcgc 2280caccgccgag
gcggtccgcg acgcgggcct ggacaccacg cgcgaggacc cctggcgcat
2340gggggccgtc ctcggcacgg cggtcggcgg caccacccgc ctggagcacg
actacgtcct 2400ggtcagcgag ggcggctcgc gctgggacgt ggaccaccgg
cgggccgagc cgcacctgca 2460ccgcgccttc gcccccagca cgctcgcctc
caccgtcgcc gagaccttcg gcgcgcaggg 2520cccggtgcag accgtctcca
ccggctgcac gtccgggctg gacgcggtgg ggtacgccta 2580ccacgccatc
gccgagggcc gtgccgacgt gtgcctggcg ggcgcctcgg actcgccgat
2640atcgccgatc accatggcgt gcttcgacgc catcaaggcg acctcgccca
gcaacgacga 2700cccggagcac gcctcccgcc ccttcgacgc ccgccgcaac
gggttcgtga tgggcgaggg 2760cggcgcggtg ctcgtgctgg aggagctgga
gcacgcccgg gcccgcggcg cggacgtcta 2820ctgcgagctc gccggctacg
ccaccttcgg caacgcccac cacatgaccg ggctcacccg 2880ggagggcctg
gagatggcgc gggccatcga caccgcgctg gacatggccc gcctggacgg
2940cacggacatc gactacgtca acgcgcacgg ctccggcacc cagcagaacg
accggcacga 3000gaccgcggcg gtcaagcggt cgctgggcga gcacgcgtac
cggaccccga tgagctcgat 3060caagtcgatg gtgggccact cgctcggcgc
gatcggctcg atcgaggtcg tcgcctgcgt 3120cctcgccctg gcgcaccagg
tggtgccgcc cacggccaac tacgagacac cggaccccga 3180gtgcgacctg
gactacgtgc cgcgcgaggc acgcgagcgg gagctgcgca gcgtgctgtc
3240ggtgggcagc ggcttcggcg gcttccagtc cgcggtcgtg ctgaccggac
cggagaggag 3300gctgagatga gcgcaccccg gcgagccgtc gtcaccggac
tcggagtggt ggcaccccac 3360ggcatcggtg ccgagacgtt ctggaagacg
gccgtggacg gcaccagcag cctggcccgg 3420atcgaccggg agggctgcgg
ccacctgccc ctgaagatcg ccggccaggt ccccgacttc 3480gacccggccg
ccctgatcga ggacacctac ctcgtccaga ccgaccgctt cacccacttc
3540gcgatggcgg ccacccagct cgccctcgac gacgcccggc tctcccgcgc
cgacatcgac 3600tcgccgtact cggtgggcgt ggtgacggcc gcgggctccg
gcggcggcga gttcggccag 3660cgcgagctgc agaaactgtg gggccagggc
tcgaagtacg tcggccccta ccagtcgatc 3720gcctggttct acgcggcgag
caccggccag atctccatcc gcggcggctt caagggcccc 3780tgcggcgtgg
tggccgccga cgaggccggc ggcctggacg ccctcgcgca cgccgcgctg
3840gcggtacggc gcggcaccgc caccgtcgtc gccggcgcga ccgaggcccc
gctggccccg 3900tactcgatgg tctgccagct gggttacccg gagctcagcc
gcagcgccga cccgggccgg 3960gcctaccgtc ccttcacctc cgccgcctgc
gggttcgtgc ccgccgaggg cggggcgatg 4020ttcgtcctgg aggaggaggg
cgcggcacgc gagcgcggcg ccgacgcgcg ggcgacggtg 4080gccggccacg
cggccacgtt caccggcgcc tcccgctggg aggagtccag ggccggcctg
4140gcgcacgcga tcggcacggc gctggcgcgg gccggctgcc gtccgcagga
cgtggacgtc 4200gtgttcgccg acgccctcgg cgtgccggag gccgaccggg
ccgaggccct ggccctggcc 4260gacgcgctcg gcccgcacgc gcggcgggtc
cccgtcaccg ccccgaaggc gggcatcggc 4320cgggcgttct gcgcggccgc
ggtgctcgac gtggcgaccg cgctgctcgc catggagcac 4380gagctgatcc
cgcccacccc ccatgtgctc gacgtctgcc acgacctgga cctggtggtc
4440ggccgggcgc gtcccgcccg gccgcgcacc gcgctggtgc tcagccgcgg
actcatgggc 4500aacaactcgg cgctcgtcct gcgcaggggc gccgcgccgt
tccccgagta agtaccccga 4560acaggtgtct cacgtcccct tcgggcgcgg
gcacccgagt caaggagctc aaccacatga 4620ccgacatgac cgaacgcgtg
ggcacccagg tgaccttcga ggaactgtcc gccctgatga 4680agcgcaccgc
gggcgtgcac gtggaaccgc ctgacctgcg ggcgcgggcc gaggagggct
4740tcgacggctt cggcctggac tccctgggcc tgctgggcat cgtggccgag
ctggagaaga 4800agcacggcgt gggactgccg gagcaggtgg agcgctgcaa
gacgcccgcg gagttcctcg 4860cgcaggtgaa cgccaccctc aggacggcgg
tgtgacatgg ccgggcacac cgagaacgag 4920atcgtcatcg ccgcgccgct
ggacctggtc tgggacatga ccaacgacgt cgagaactgg 4980ccgcggctgt
tcagcgagta cgcctccgcc gagatcctgg agcgcgaggg cgaccgcgtc
5040cgcttccggc tcaccatgca cccggacgac gagggccggg tgtggagctg
ggtctccgaa 5100cgcgtcgccg accgcgcctc cctgacggtc cgcgcccacc
gcgtggagac cggccccttc 5160cagttcatgg acatccagtg ggtgtacgag
cagacgcccg agggcgtgct gatgcgctgg 5220atcc
52243330601DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number Z69361 33gatcttagac cttattcact tgatacgtgt
aatagttatt acgatagtat gtttttggcc 60gattcctccg cgtcttcttt cgacgacgtg
gaggtggagg caaaagcgaa gtagttgtgg 120aagaataaga attatgatta
tcatgattat tattcaaatt aactctattg ttacgtaccg 180cgctccatgc
agacgtttgc caggagacga cgggtggaag gataggaagc gaagaagcgg
240aagcggaaga cgtcgtattt gaattcgaag atgataatga tgtcattgat
gctgatgatg 300ttttgttgtg ataatgagat cggcatggag gcatttcaca
atctctttgt tcgcgaggct 360taatctctga gcactcgata tcgtcttgtt
tacgaccgga aagcacgtct tcacaccaga 420ttttgcggcg ttgaactccc
ccgccacacg atacagaaca ctggaattat tattagaagc 480ttcaatgatg
ttctaagaac ttacgtgagt ccatggagat atctgccaag aattatttgt
540gagtggtgga catagttctt cattgcattt atcaaacatc tttggttttc
tggtttcatc 600gcaagaagac gaagtgcaag atacactgcg acgacgccat
cccccaccac aagaagcaga 660gcactgaaca atcgtacatt agtaaattct
aaatctgaaa attatatatc ccacttttga 720ccaatctcca ataatccagg
atccaatatg ttcttctccc tttggacagg gttcaagtcg 780gcaatttctt
gcacttgttg gtctcttttg cacatcacaa tcaacatctt tcaaaatcgt
840ccgaccaccg tcttccgcac tgacgcatgt aacatttcta ctttgttgaa
catgagttcc 900acaagtagct ggacactctt cccattccgc cattttccag
tatgaacaat cacgaaggcg 960acaatttctt gttgatactt ccttatccaa
atgattgcaa tactcatcag gaagatcacg 1020aacatgatct cggcacttga
gaagacgacg ttgagtacca ttaccacaag ttgctgaaca 1080ggctgtccat
ggtccggttg cccatcggat tggtggtacg tcggcttgaa gtttttgaag
1140tactctgggc ccatcacaag tatctttttc acaagtcttt tttaggcgtg
gacgagtatt 1200ctgaaatgat attttcgttc aagattaaaa gcaaactgaa
acgtactcga tcacaaaaat 1260attcatcaac aatagttcct tcagatccac
gagtacacga aacacttcta gtctgaattc 1320ctgatccaca agtgactgag
caaggggacc aatgacttgg tttccaagat gtacatggca 1380gaagatggca
agtttgacta gtttctggca ttttggtatc tccacaaaaa gaagcatcaa
1440cagattgttc gcggtatatg cattcggttg tacgttcccg atgaccgatt
ccacaagata 1500cagaacactg aaacgtattt atggtattga caacagcaat
tctggagtat ttgaataaac 1560ttacagcact ccaatcagta tttctccaaa
atgggcaagt gcctaaatta catgtctgat 1620gagaagcagg ccgatcagat
gcagtaccac aaagtgacat atcgacttca gttccatttc 1680cagaaacaca
tgaaactctt cttgacgacc atccatcctc acaagaaaca ctacactgag
1740accattctcc aagtttatat tttggacatg attctctatg acatggtttt
gtaattatct 1800tttccatttt aaggcaacga tgttccggta gtactgatct
atgacgatcg gtacaattag 1860cgtctcgata ctgtacacca tctccacact
tagctgagca gtcagaccag actccgaact 1920gccaccaagt acaagcatgt
tcattacaat gttcttgtgt ctgtgctgga ccacatctgg 1980atgtatgtgt
ttcccgatcg gctgcatcca aacattgagc atgacgcatt ttgactccac
2040catcacaact tcgagagcac tctgaccaat gcccataaac ccatcttgga
catggaattc 2100tgttacattc ccgttctgtc gcctctttct gttctctgcc
gcacaatgac tcatcaactc 2160gacgattcga atcatcaacg caatatgact
tccgatgcat ttttccattc gatccgcaag 2220tttcagaaca tgaagtccat
tctccatagt tccattttct tccagagcag tcaatgtaac 2280aactggcaat
atcggatggt tttgaattac gatcacatag atgttcggat gctggagttt
2340gacgatcacc ctccattttt acgcaagaaa ctcgttgacg tttctgtcca
gatccacatt 2400tggcactaca actagacaca tcttcagtga tccatctgta
aatataaaaa tttattatag 2460aaatctaatg aaaatatgta gtttaccttg
tagaacaatc tatattgcac attcgtgttg 2520cttgttttgg tttgagaaca
ttttgacaat ttctatcatg actttgacga tgagtcgaca 2580tgtccagaca
cattaatttt tgcgattgct gtccacgaca ggctctatca cattctgtcc
2640aagtatccgt aactctccac aaatacaatg cactggatat tggccgaatt
acagcatttg 2700gaacagccgc agtcatgtac tcatatgaga tgtcgggtgg
atgactacca acagaaagaa 2760catgaacata aatgtcactt ctaatcggac
cagttccatt tatccgttca ataattgcat 2820cagaaccaga atattcgaga
acagtgtctt ggaatgcaat ttgttggcga gccagtgata 2880cttggaaatg
accgttaagt aggaattcac cattggcggc acggagagct gaaggttaaa
2940ataaagattt tcatgggtat tgataacaca agggtgagtg atgaaaaaag
taaatgttcc 3000aaaaacactt tgtatagaaa ctcacaaaga taattgtcat
cttctttcat attattatat 3060cctttctgcc ggatatcaat atttgcagaa
ccagctggaa tcttcattac ttcgttataa 3120ccaaaggttc cttgctcatt
aaatgttcct ttgacaacct tacaggaaga atcatcccca 3180ccgcaaacac
cacatttgtc tcttcggaga gttgaatgaa gttgatgatc acagcctgaa
3240aatccactta ttttcaattt tcttttgaaa tcagatcaat gttacctgct
ggcatacaag 3300ctccagctac acaaatatcg tctccatttc tatcacatgg
tgttccatca acaactttat 3360ctcgaagcag atagaacgct gcagatccac
tgagccgaca atacagcttg caacgttcat 3420ttggtgcaac attcgcatat
tttggaaccc agtgagtatt cgttgaagcg acaccttgga 3480ttccaatatc
tttattgttg aattcagaac attgaacttc acggtatggt tgagtatccc
3540atgggcattc ttgtgtatta catgaccgat aacgttctcg ttgaccaaca
cagtactttc 3600caccatttcg aggtctaaag taatatggga aaatgtcatt
ttaatattga taggaaagct 3660tagccagtgt ggcctaaaag ctggaagttt
ttttaaagat gcgttttcta tcaatttaag 3720ataaccggct acttcaggtg
attctataaa ttttataaag cttggaagct aggtaaatct 3780gaaaagcctt
aaactatctc gaagcggccc gaaagcccag aaaagcagag acggacaaac
3840atttaagagt gatcagaagc actccatacc ttgatgttac atttgatttt
agtgtttcca 3900cctcgttttc acttctgaac tcgccgattg aaaatatttt
gattgaatat attatttgct 3960ttcagactat ttgatatcat ttcgtttggc
agtttaactc actttgggct gtcacaatct 4020cttaatcctt tttgaacacc
accaccacaa gtacgactgc attctcccca tgatcgccag 4080tcaccccatt
gtccgtcaat tttggtaagg gattcggggg ctagacgaac acaggctcca
4140tgatgacaga actaaatatc caagttttta tgagtttctt ttgtgattaa
tttctgagat 4200actcaccatg cttcttgatt cgtcacaagg agttccgtcg
gcccatggca tatgctgagt 4260tcgacagccc atctggcttc cgtagaatgt
tgcacaccaa agacggcggc atgtcggctg 4320taaaatatca atgtttcatc
ttaaagaata tatttaggca aactaaccat ataagggcac 4380aactcagaag
ctggtccaaa tacaaacttg cactgttgat gagcatcgta tttctttcct
4440ggttcatcac gtacaaagac atcctcgtag taacgacgtt cgaccggctg
atcgaataga 4500cattgagttt gacctcgatt atttctgaca aattgacaat
taaatagaat caaaatttta 4560atagctatct tactcgagga atcgttcgag
cattccagct gaacatggcg accaactcca 4620tggatgagtg ttatattcca
acgttggtgc cattatgtgg aagttgttct gaaactgcgt 4680tttatcaaat
ttagtgcttt ggaacttgca aaccttatta accggcatgt aggtagagca
4740ttttcgttcg tcatcatgag gaatcgaaaa cacatgaccc aattcatgag
caattgtgaa 4800tgcagcactc aatccattgt cttctatgat tgcacaactt
ttttgcatat cacacattgt 4860tccaagttca gcaagtccaa gtgtatcgca
ttttccttgt gatcgacaaa tatctttacg 4920cgtcaaaagg attgcaacgt
catgatgttg gacactcgaa tcatctggat cattgtaata 4980ctgctgccat
ctacagaaat cttgaagtgt ttgttgagcg ttctgagtga ttcgtggtcc
5040agcgttttcc gttttcaaaa cgatcaactt gacaacaacg acattgatag
atgcacgaag 5100ggattggtga cgatagatgg aggcaactgt ggagaagaga
gtgagaacgt agtcttcaag 5160agatcttccg tgatattcgt acatttttgt
atccgccacc acaaggactt caacatagtg 5220atcccaagag ttggcagctc
ttcgggatct tgctttgcgt tctataatta aatccttttg 5280tttcataaaa
ttatttaaac atttttttac tgtatccttc ctattaatct tgcaccccag
5340agctccactt tgacctatct ttgttgtcat tgactctatc aaaaactgtt
caactatgaa 5400aatggggatg caagactaat aaaaggtatt ggtaactggt
tccagtagag ctttttttac 5460tatctgtttc attgattcaa ttttcagatg
tttatataac catcttaacc gttcaaatct 5520cataacatag aacagcctgg
cagcccgtga aaaggtgctg aaatcccagt aatttcaatg 5580gcattcgacc
acacacaagt gatccattat cctttgctct tttacttcgt taactaccat
5640tagctatagg ggacccacga gcaaaattct atagtttctg tgtgtgttag
ggtgttttaa 5700tgggctatta cacaacaccc gatgggatca gcagaatctg
agatcttttg ggaaccggaa 5760aaaaatattg tgataacttc tcttttttct
acatttttta cagaactagc aggtaaactt 5820tcagattgaa atctcgaaaa
atgcatccgc ctactcaaaa agtcgttttt aaaatgattg 5880tttctttgtg
tttgtcctct ttttcccgga cgtacgcaac acaaaaccgc ttgcgcgagg
5940atgtacacaa aacgtacgtt ctgcgcaatc ttttccctgc agctctctct
ctctcacttt 6000ttctactcca taaatcagtt ctctgtctgt ctcccaccac
ctaaatcatc atcagcatca 6060tcacagtccc cccaccaagt tcttgtgtct
tctctgacct ttacacgtcg actagggaaa 6120agctctcaag cagacactcg
agcgccagtt gaaaaaaata gtgtgtccaa atgagcagtt 6180tcgaatttga
accgtttgtt cttgttctga cataaaccca aaaaaacgaa ctaggcggca
6240aaagagatct ggataatcta aagaatctag acaaatttca gaagttctta
ccaataacat 6300cttcccactg atcttgccac gtggcaaccg tcgtctccgt
ctcgttgaca ctggtcgagt 6360taagatggtc aaacgatttg aagtgcattg
gatcgaactt tcggacgaga tgttgcctat 6420ggcgacttgc tccgtcgtgc
tctagatgtt taaagtgtca gagaaagtga ttacaaagtt 6480tctacctgtt
ccgtttccac taataattgg ctcaaccgta tggattccgc tgggtagtgc
6540aagcattccg tactgaaaaa ggcttttatt caccaaaatt cgaacttata
caaaccaatc 6600cgtcttccga gtcgcataaa ttgacgatgc tgtgctgatg
tacaccttta acgtgtgcac 6660ggtagataca atcgggatct gttcgagaca
ttccacctct aacctcctcc tccgagtcca 6720aatataagac catcggcgcg
aaatttgaat tggaaaagtg gggaacactt ctggaattga 6780aaattaatac
gactgttata ataaaattga aatctcatac ttgttatgtg agtccggtat
6840ttgattccat ctgtgcaaat gaacgatgta gacggcatca tctgatcgta
atcgtaagtg 6900acaagcatgt ccacagtctc tggcaactcc ttggagtcga
cgtcgccgat ctgttgacgt 6960gacatcacgt tttccacgac gtccataaga
atctcttcgg acgatgtgat ggctgtcgat 7020gacgtgtata ccggcgtctt
gacgccatcg actgtgatgc actggcacac ctgaaactta 7080gaacattatt
tcacttcaaa actttttgga ttgttacctg agtacttggc cctggagaac
7140agcacatctg atgagaattc tgagatcgtg ccactccctg ctgaaaggaa
catttggtta 7200aaaacaaaag ctgataaatt aaaataatta gataaaaacg
aacattgcaa cgcataaacg 7260aggcagacga cgaggagtat gagagcggcg
acgacgggct gcagcagatg gaatgagccg 7320ccgatggagc gcataccaac
agctccgtga tgatgattat gattgtgtgg agagcagcaa 7380agaaaaaaga
gatggaaaga agcagaagct ccgataaagt tcgtccgtct cttctgaaac
7440cttccaaaaa ctacctgctc gaggtgaagg gaagtcgtct gattgaactg
ctactgcttc 7500tgatcttttg ataatctccc gagtttgtgt tttcgtttag
tcgaattaaa attgtagatt 7560gtggaatgag cacttgcaat agggaacaga
gcatcacaga ctgaaaaatt aaaaattatc 7620tagaatgcaa gcaattttta
aatttgtttt aaaatcactt attctgacgc catcttcttt 7680tccgatttgc
gcagaataaa taaaaacttg actgtaatat tgggaaaatt tcgaaaaaaa
7740acaccgttaa gtctgagccc acctttcgcc tttttttgtt gacgaaaaaa
accaaacaag 7800ctttaaattc ataaaattcc caattttaaa aacatctaaa
gtcaattcct cccaataatg 7860catttgtata tgaacaaaag tctgttgacc
ataagtcgtt atattactac aagcaattgg 7920tcatcaacaa acctcataaa
aatcagtttt gaacgggagc aatttatata aactctgtgt 7980gctcttttgc
tctttttctt atttcttagt tgtcttctag ttccgccacc actttcgctg
8040ctcttgacga aatctgtaaa ttgttcgtca tttttgattt ataagatttg
tttggctctc 8100ggtaggagct ctcaagctgc taatagtcct atagtaaagt
actaaaaaca caaagaagca 8160gatgaaggtg tcataaaaca ctgataagaa
tcatcatgat taggttggtg cagagaaaag 8220aagaagaaga aaaaggagat
ttagagaaga gaaacaagaa taaaaatgca aaaataaaaa 8280aaatagtaat
aacaatgaac gcagagtctt ccatgttgga gaaggaacag gacccatgtt
8340gatgtgtatc tgaggggatc caatgtgtag tgatggtagt aaacacttga
gagggaactt 8400ccacccccga ctagatgatt ggaagcaatt gatgatagat
gtagagccaa agaattggga 8460cctactaatg atctagtcaa gattcttctg
ataagagaaa aagacaagga agaacatgaa 8520aatgactggt gattgaaaaa
taaaacggtt tatgaagtcg gggtgtacta aagatgcaag 8580gtctcttgtg
acgtattttt tcttccaggc acgttcgcgt tattcacgat tttatgcaaa
8640caaggtaagg agtgttttga attttgaata taaaaattta aaagaaatta
aagttagaca 8700tttgaaaaat tagacaccct catgggaaaa attatagggc
gaggagaggc ggtgagaggc 8760gccctaattt ctgctcggtc gggtagaatg
tctaatctaa atcctacctc atgtttggct 8820ccttcttaaa tcaaaagctt
aaggtcatct ctgaaacgtg cagttgacaa gttcaatggt 8880aagaacaggg
agcaagcatt tacaacaaaa aagtaaacaa aaattgcatt tgtcgcagtt
8940caaaatggaa caactcactc ccactcgaga acgttttgaa ggggagagga
agaagaggaa 9000aatcatcaca caggcacatg gaacttctgg gacacaaaac
aatacaaact gggtgccgtg 9060aatctcagta cacacacaca aaaatcaaaa
aagacggaaa ttaggagcag atgtggtaaa 9120gggtggttca atgctgatgg
gagagagagg gagaaacttc aaaaaaagaa gtttagattt 9180atgttggcta
tttcaatcct aaatttatct aaacaattct aaaaatgctg gttttggaag
9240gttatctggt aatggtgaag ttttataaac aaaacaagac aaacaattct
tgagatctta 9300aaaatcttag cgactacaac aatatttagg tattttttaa
tggaaaaaag tattgattgt 9360tgacttggga aattgaacag caattttttg
tacttttaaa tcagttatat tttaactttt 9420tagagcacat ttcgtagaca
aaagggaaaa cgattggtcc aacatgtgaa gatgatgatg 9480tcaacaagtt
ttggatcgga gccaaaaaag aaacaaaaca ttcataccat gatgggaaac
9540aagaggtgca gcaacaactt ttatcaatat tttgtttatg ttttgattat
ttttctggca 9600cccagccagt aattcttttc cgtagagttg acctagaaaa
tgttggaggc ggagtcttag 9660gatcaagaga cgcagactat caaagtaaaa
tgagtaaaag gaagtgatat aaacttagga 9720aacggaggaa aaaaggacga
tgataagaga ttgaagactt ggaagagtgt gctctttgcg 9780ggagagcata
ttcttttgag aaaaatggga cctaggggca actgacgcaa ttgaaacatg
9840gtcgagcggt cggcgggaag acaaaaagtg aagaaggatg ggcaagaaga
agcaagagaa 9900atggcaccca ccgtggaaca tgatcatgat gattgagagt
gaaaattgga aatctcgaac 9960ttttttgcaa cggcgcgttt tggaaaacta
acaaagttga ccaaaaaatt attttacatg 10020tataccggga tgtctaagaa
ttgtaaaatt gagtgatcct ttctgtgaca taatttaaag 10080caatttattt
tggttatttc taagcgcctt tttatactag catgttatat tgttaatttt
10140attatctaaa ctgccgttct tcctatattt attattgcac cccctttgtt
cattctgaca 10200gactatacct cgattaatca taaaaatgtc acaaaagaat
aaaaacaact aaaattaaga 10260aaatacaaga aatttatcaa ttgccaaaaa
ttcggccaat cggaaaaatg cttggttgcc 10320aatttgtcaa aaatttagtc
aattggaatt tgtcgatttt ccgaaatgat atgaaagttt 10380gaatgatgca
gctaattttg cagtttaagt ttacattttc aagtttactg taatttttcc
10440aaaatatgaa gaagagtttt acgaaattaa aagataataa aaaagcaatg
caaacatagc 10500tatgaaatct gatcccgact aagtttgatg gacataggat
taataatatt agtctaactt 10560tctatagaac actaaataaa tacattcact
ctcgaaactc tcccttttct gccatcaact 10620accgtactca cttttgactc
aatgacccgc aactgtcaag atgagttagt ttcaagattc 10680tctgaaacag
caataatcta acaagagaaa ctgaaaaaat agagtaaaac taataataat
10740accacataaa ttgacatgca tgatagatga ttttccggtt ttcaacaaga
aaaacaacaa 10800tttccgagaa atcctcatag tttttggtaa gaaaaaataa
attgatagtg atacggtatg 10860actattactt ctaaagactt acctgattag
aaacgtgtag taattgaaga agaaaagttg 10920aatttgagaa gttgaatcga
gtttacgatg tctgaaaaaa acatagatat tatggtaaga 10980tcaagcatag
aaaaaatgga aaaatacaag aaaatagaga ctagagattg cataggtttt
11040gcggtggcga aaccgcacac atttttgtct gtgttatctc taattttacg
ctctcggtgt 11100tctctattta ctgtccagaa gaatgaagaa tatgggggaa
aagtgcgcgg gaaaattgag 11160agaccgagtg atgagagccg cagttttgca
aaactttttc gggcaataat ccgccggcga 11220gtactacgag aagcacacac
acatacgaaa actgttgagt taaaacctaa aaaattgttt 11280cgacatattt
aattttcgaa ctaaagttta gagggtctgt gcgtgcattt ttgaattttc
11340caaacaactt tcagttttgc ggaagaaaat tacagcgatt ttttcgaata
tttctgaaaa 11400caacactatt gcgtatcaaa aatttttcga tttgccaaaa
ttcagactaa gttttggtgg 11460ttttggtttg caaacattta aaagaactca
aaaaacattt ttagatgttc gaaaccgtac 11520aattgtagga tacaaatagc
tacagaacaa ttagaatata aaatagagtt gtcaaacatg 11580tttaactaat
acaaaaacac agaaactttg aaactcgaaa tttttatatc aaaattgaaa
11640aagcttgtaa aatttaaata tggatacagt acaaacaata taatcataga
tcaaatagtt 11700catttattta tatatcttgg caaatcaaat cgtatccctt
acccactcat attcgatgag 11760tctacaatta aatcagttgt tttttcatcc
tcccggacta ttagtttaac ttccacttga 11820acaagggcaa agagtacatt
aggaagagtt tatgatgaca ggaaaaaagc tatgtaaaat 11880gacctctttg
gattgaaaaa gcgaacgaat tgaggtttag gacccccgga aaatgaagaa
11940ttcgtggcct cgagaatagc aaattggcgg aattaattat ccgtaagagt
gtgaattgga 12000aacaaccggg acgaatggat tactgaatca aaaatgaaag
aaagaagaga tgaaaatacg 12060tgtgaatcgg atgaaatgtg atgattttag
aataacctaa atgcaacaaa acgacgtaaa 12120gacgcggaag aacaggaatg
atcaaggggt acatcttata ggggaaaaat gcactttttg 12180tgctccaaat
gtgagagata atcaggtagg aagagacgta gaataggaac aggaaacggt
12240aacgatagtg cgcaggtgct tgatttctgt gcttttgcat gtgttccgat
ggaatttttg 12300gaacttttca aggggtttcg gaaagggttc gagatttcgc
atgtgagctt tggaagaatt 12360ttggaagaac tttcaggata acatcgctca
agcttgtttg ttagatttca gacttcaaag 12420tatataccga ttattgaaac
attttaatcg tttcttacta ttagtaaagt ttaatcacag 12480tttgaaaaaa
aaatcacaat tttttcaatt atttagacca aactaattat ggtacagaaa
12540ataacttgca acccgggtat ttcattctaa tttttttcat ttggaaccac
tagtttttga 12600aatagaaact cgttaggatt cttcacatat tatcataact
atcagtattt tgttgcacat 12660cagatctaag ttcagtctaa ttagaatcgc
aaatttgacc atcacacttt aaaacaaatt 12720tacttaggca cagggcatcc
ttctaacttt tttgtccccg acaaaatgat gacaaaaatg 12780acgtgaggaa
tcaaggagaa aaaggaaaag aacaggaagc gaaaagtagg agaagctctt
12840gatttctgtg ctcattcctt gttcggatga gctcactgtt tgcaacattg
gcgttggtgc 12900gcgggaatcg ccattgccga actttttcaa gagacagaga
gagagagaaa gagaaggaaa 12960acgttccgat ttttaaaatg gaaaaaaatg
aaagaggaag atgatgaaaa aatgaactct 13020gcgtgacatt tgttaatatg
gaaaaagcat gattacttca aaattgtaca ctaatcccca 13080cagcacacat
tttgaagact tttttacaaa aacaatggtt taagcaagct ttaaaaaatt
13140gatagtatcc ttaatgctta atcatatcca agtttagttt taagttttga
tttcaaaaat 13200ttctacatca aaaaatcata cttagtgatt atatgcaaaa
caatttttaa attcaaggac 13260atatttttga tttttggaag gatgataact
tttttgtgat tccgaaaaag attaaagtag 13320gtttaaaacc tctgaccttc
tacagaaaaa acattacctc tatgaatttt ttttcatctc 13380gttcagaact
tgtctcgggt caagccatga agacatgaga tagggtgtaa aacgttccga
13440agagaggttt atgactatta ttgtagttga agagaaaaat gatatctcaa
tggatttcat 13500acagatggtc ggatttcatt cataaaatat cataagaaaa
ggtacgttta tgactgtcta 13560ggtcaactgg ttttaggttt cttggaattg
tttcaaacat ttttaggaaa tattttcttg 13620caaatatcta ctaaattgaa
gtttgttatt gtttttgaca tattgtagat tttagagaag 13680aatcactcag
agcaaaaatg ttgggaaaac gtgagaaaaa tccaagagac aaaagaatgg
13740tcttactatt agtagatcaa aaaaccagac caattattca tattcctact
attcaatata 13800tattcaaaaa tgagcaaacc aagaaattgc acctaattta
tcatcccaca tatattccga 13860cgaaacattc gctctacctt ctttttttct
gtctaggaat tataaagggc cataattata 13920atttcagtca aggttttgga
aaattgttcg actaaccatt atgaaagtta aaaaccaatc 13980agtcaaaaca
cacaatagga atataaaatt cgtagaagaa aagctttttt tttggtcgaa
14040agcaaaatca aattctggaa ctgcgacttt tttagtgcaa ttatccattc
aacgcaagtt 14100gtctttcaaa atttaaattc cagaagagtt ataacaaaac
agacaggtgt acaagtaaaa 14160gaaaaataca agttttatcg taaaaactga
tacgaatcta gatacacctg ttaaaaaagg 14220ctttctcgaa acccagatgc
cgtacgaagt aagcagcagc caactaaaca ttttgagtaa 14280acatatggca
agtgttttgg cgcaaattgt aaagattttc cgtgtgggta actagaattt
14340gaaactgtaa gtatgacgac ttaaccacac aaaatcaaat ttcaaaagat
cttaaaatgt 14400tcgaactttc aaaactttta agctctctcg catctaccgt
agtcttctaa taacaacagt 14460cgtaagagaa agctcaaaat ttttcaaact
ttttctgaat gacagaatca gttgtataca 14520aaaaaaaccc ccaaaatgcg
agccccatga acctgacaac cagacaagtc gaaattgtaa 14580aatcgtatag
atcttggttc acgacatgaa gagcaccgcg ggggcacacg agagcaacta
14640ctgcaagcgc tcctgaagag aagaaacatc ttttttccag gaccactggc
cagtagtgct 14700cccccagatc actttctttt ttcttgcttc atctgatttg
tgtctgcgtc gtctgatctc 14760tttagaacct atccttcttc ttcttctttt
tgatacttcg acatcagaac aacatcgaca 14820tgtatcatct tttctctttt
ttttttgtta tctattcatt cattcacttt tcatttagtt 14880tgattaatag
gtgacatgaa ctcttgtcac ttttcaattt caacttctta aatcttaaac
14940tcacagtgat tccagatatg agcaactcca atgaggtgtt gagtagaaac
ctaaatataa 15000cattttggat gttttgataa tgttggaaca aataaattga
aacaaacaag acttgaaata 15060gagacaacgt gcagaataat gtctaccagc
tggtttcagt ggcatattgt accacgaacg 15120tccgacagaa cgaataacat
aaagatcaag aaaaactgtt tgggagcaga caaacaatca 15180gaacacagtt
ttgttgaggg gaccaaatca taattaatga ctaaatttta acgaagaaag
15240tgctcgaaaa gaacagaatt tagaagttga tgaacaatat ttttactttt
agattaacaa 15300ttatgcttta caaatgacat ccaatctaaa gcatctggta
atctgaaatt tgtcaaaaca 15360gctttcaaga ctagtttcaa atttgtcgat
tcaatggatc aagtgtgtaa ttgatccaat 15420aaaaaagagt ataaagtgag
aaggaagaaa gtgtgaaaaa agaagaacgt gaaacgtgca 15480gaagatacga
aatgagtttg aagactgcac ttttcgagcc tcgatggtca gtcacttggt
15540cagttgcgaa aaagctgtga aaatgataca ttgtgtcggc tctcgtagag
aagaaagcca 15600catggtcagg atgactccaa ctgggatatt cagttgtaaa
gaacacaatt gatatttttg 15660catctttttt aactagtttt tacaatatga
gaaattgttc tgtgcgaaaa atatgacttc 15720ttccttgttg ccgaagtgta
tttccctgga aattccagta aatacctaat gtaaaaaatc 15780tcagcagaat
gtgttcttac attttgttgt aataataatg tattaaaatt gcattaatta
15840aaaatttctt caaaatgttc ctacgtcttc tatgcacatt atttaggtca
cagtttcatg 15900gagcacaaaa cacctgccga cgcctctaaa atagttataa
ctgcgcatga aatcaggtag 15960aaaaaactac aaaataacca atacaaattg
agtagggcga tggagaggtg ggcggttgga 16020gaggcgggca acaagcgtcc
tcatgacgcc ttgttcattt agaatgtgtt tgctttgaat 16080tacatacaag
tttctaaaat ttaacttaca aaatttaaaa aaagtcacaa caataataaa
16140agttgtggca atgaaatgtt ttaaaaatct aaatattgag ttttaaataa
atgatttttg 16200aaaattcaca aagaaatgtt acaatctgtg aatgaagacg
aacaatgaaa aagtgaggaa 16260cggacgcgga tattacacat tcagtcacac
aataaacgtt cggacactac cacacatttc 16320tctcatcatt tttttccaaa
gtttattcta aagttcaata ttttagtttg attattttgg 16380acactattct
taaaattaat gtataatagt ttagaaaata ttttgaaaca tgaaactttt
16440ttgttgataa aatagtgcca aacatcctta tgttacgcag ttatccaacc
acatttttct 16500catttttcca ccaaaaaaca ctgaaatggt ccataaaacc
tattcaaatg gatatgagaa 16560tattactttt ttgacatgaa attttcaatg
atgtaatgta aaacaaagaa aaatattgcg 16620ggaaaaattg aacggcgtat
tgcaaaaatc ggtgtgcgga ggaggagaag gaaaaggaag 16680agcaggagaa
gcggaccgaa gaattcagaa gcttttaaaa taagaacggc gactttcaga
16740caaacaatgg actgttgtat aaaaataaag cggaggcggt agagagtcaa
agctttcaga 16800aatgtattag aataggtttc actacctgtt gttgaactca
aaaaggtgtg aaaaagtgaa 16860agtttgtctg aagtttatga cgggaagtgt
ccatcaaata actttcaaaa tttgacttat 16920cagtgagaaa aacacgtcat
tttggaacgt taaaatgggt ggcaccgcaa aatgttcaca 16980atgtgaagtg
aattacgtaa taaaatcagt tttattaagc ttattaaact aacccttccg
17040gactatttgt ggaatgaaac aattgggggg gttttttttt ccaattttcg
attttttttt 17100gaatttataa ttaccggaac aaaaatatct ttaaattatt
aagatttgag tgatgtttga 17160aattttgaac ctgcaaaaca taagcacaaa
ataatggagt ttttgtttta aaatatcaat 17220aggtgttttt tcacagaact
ttaaacaaca aatactcata atttgaatga aaacagtaga 17280tcccacaata
ttttgaaaac ttatctatat atatatatat atatatataa ttacgaaaaa
17340aaaacaaaaa gaaaaaaaca aataatttgt cagttgataa tttttagata
tgagttgcca 17400aaattgggca atatggtgaa gaaatacggt agttcgtcgc
actgtcagac taattttcaa 17460gtgttcctag tggaatgaaa ctaacagaag
ctatacggta tataatatta ggaacacaat 17520taaaacgaac agcggaagaa
aagatctagt ggtcacttcc gatttctcag ctgacttttg 17580aatgggcacc
tatcatcatc tcacttgttt atttgaacag tctcgacttt ttccaattgt
17640tggcttctag ttcaagaaac gaaaaaaaga gcaataacgg aacagaaaat
tcagaaagtg 17700gaagagaaat atgagaaaat gatgatgata ataataataa
gttagaagag ggttatcgat 17760gaggaacgga aacgttatct ctgatcgcca
tctcattatt attatgagac acaaagatgt 17820aagttatggt atctttgaaa
gaaaagaaaa caggaaatta tacagaacac acacaatttc 17880ggagatttca
ttcgaagaac ctaacccaat ttgaactcac tcccacttcc tcttgtctat
17940aaaacagtca atcacaggaa caggtgtctg tcttttcaaa atgtatacgt
tttccgaata 18000atgacacaca atatcacaga caaaatgatc aatgaggttg
cagaaaagaa tgcaaaaaaa 18060tatagaaaga gagggtgaac aggagataga
gaatcaaaat ttgcatagat aaatatgcaa 18120tagaaaataa caatttttga
acaacaaaga aataatttag tggcatataa tatagcgatg 18180gaacttgcaa
atttttagaa ttatcatata aaaataacaa tgtttctata ttttatgccc
18240tataagtctt gcagtatttc ttaaatttaa cagttcattt cttggtaatc
tttattttta 18300tcaagaagtg ttcaggaaat tttaggacat caaattttta
tttattttct aaatctactt 18360ttatcaaaat tttagaggtc tagtacacat
ctacccaaaa agaagacttt ggagctctca 18420aaaaccacct agtgtatggt
aaagtacatg agaagtgacg tgtctttggg cagctggcca 18480tctttgtcga
tatgcgggtg atggtgtttc tgtgagcagt aacaggaaat tctggacacc
18540tgctagggtg tcaaaccaaa tttatttcaa cccattcttg cttcaaaaaa
cccccaacta 18600aattattcaa attctcgtaa tttaatgaat cactcagtaa
ctgtaacgtt ttttttttca 18660gagacaatga tcgaaagtta acaaaaaaaa
ctgaggatta aacgttattt ggtatctaca 18720gctgacattg gaacatatca
aaaagtggta agtgaaagtg aaacgaaaag tgcaacattt 18780gaaattgaga
gtagaaaaga tcattgaagc agaaatatgg aagtgaattg aaagccgtgg
18840cgccaaaacg acggtcaggc gccattgaga aaattaatga gagttcggaa
ggttgaaaca 18900acacaaagac aacgtgaaaa attagtttgg agaagataaa
aaatgtctgg agatggacga 18960tttcttagtt agctgagaat agtttacatt
gattttcggg aaaacgcaga atgttagaaa 19020aatggaaaca tgtctagact
tcagataaat ttgtagaatt tatatttgta gcaaaagcac 19080actaacaaag
gttacaaagc tattaggaaa aatacggaat gtatttttga aaatttttga
19140tttctctaaa ataataacac cattaatttg ctatatttgc tatatatgct
atatagtatg 19200ttcgcattac tgagcacaaa acttggaaaa agtttaaaaa
aaaaggaaac ttgttttctg 19260gagaaatcat taaaaacagt acaatttcag
acagaaataa atctttcagt gaaagctttt 19320ttttgagtaa gactaagtat
gcactcacaa cttttctgag tgttccaaaa atgtttaaag 19380aaaatactag
taaaaatgag catttcgaaa agcaatatat catacaacta cacaaacatt
19440tcaattaaag gaatcaattt tataatagtt ctaggcaatc ccacttttag
attcaatttt 19500ctagcacagg gagcattgga agatataaaa acataaagat
aaaggtgata aaagatccat 19560taaacacatc atatctatca aaccatcact
tccatcaaat ccacagattt atcacaaatc 19620agtgtgtgac aaatataccg
taatattaag ttcaaatggt ggaaaagacg cagacaaagc 19680ttttgcataa
atactaaata attgaaagaa acgcagagaa tgtaagagaa aaatatacaa
19740tatgtgtatt atcaaccatc aacagttttt gattaaaacc atggagaagc
gatatacagg 19800agcaaattag gagacgcaga ttgagaaaaa atgagaaaat
aatgaaagta cggaagggtt 19860attgtacaat aagacaggta gcatctctca
aagaacctat tgtcaagcag tttaaacatt 19920caacaacgtt catttatttt
ttagccttca ttatgatatc tcattggttc tataattgga 19980ttttttaaat
tcagatttct cattcatgta caagtaaagt tgttaattgg ttattatgcc
20040caaagtttaa
ttatttgagc gcagaaaatt tgaatggaaa tttcagaaaa ctgattcatg
20100ctaacttcaa aaaatcctga ataaatacca attcttttcc aagtatgatt
ctcgagcctg 20160tttacgtgcc tgcctacggt ctattttcta atttttttaa
tgataaaatt ttagagtaga 20220tcttcaaaaa tcttccttaa aaaatctcca
aaaaaatcaa gttcaggaaa actaaagtac 20280tccaataaaa tactcttatg
caaaaacccc ccattcattt tgcagaaaaa gacaaacaag 20340aattaaagat
aaaaagttat gatagacagg aagctgattt attagatcaa tgaatcgact
20400tttagttttt cttgaactct aatttgaaat agtattcgaa tgagaaaatt
gaaaatatac 20460aaagatcaaa agttataatt gaaaatcaac aaattgatag
tgtttgtata ggattaaatt 20520aaaatgtgcg gtacatgaga cagtagtagt
agtagccata gtacgtattg gtggctccac 20580tcggctactg ataatttcct
tttttactga taatttgatg tcatttcgta attttatttg 20640tgtttccaaa
aattgtgggc gtggtttatg aattggtcaa gacatgaatt aaaggaattg
20700taaagtaaag aagaaaatga cagaggagaa attattttcg tttgctttgg
aaattgcaaa 20760ataaattaga ttattaaaga taatagttac ggtttaaaat
aaataggtga taaaaaaata 20820tccaaaagtt caagtcctaa gaatcttgct
attttgcaaa aaaaaagcat gagcttttgg 20880cctaaaaatg gcggacagct
gtcgggacac tatccaagaa ttcgtgataa acgggtgaag 20940caccgtctct
tatcatcatg ccatttttcg aattttaaac tcagactttg ataaagaaaa
21000ttaaaaagag agagtgtgag aaataagagt acacatggaa aatgcaagat
ttgaatttgt 21060ttccaatttt taaaatgtat ttaaaagagt taccgttcca
tttttgatta gctttataag 21120tggaaaaatc gtttttggat tattttttga
ggaatatttt tgaatgcgct ttcaattttc 21180ctataaaaaa ctttgtgttc
acttttttat cccgttttta tttttatttt tacaactttc 21240aaatttttat
gaatgtttta ttgtaaaatc ataaaaaggt gcgaaacatc taaattgcct
21300ggattgcatt taaaagtgca ttagcagaaa tgtattccta tggaatgttt
tttgtgcaac 21360gagatccaga agctcgaaaa acatccaaat ttcttccaag
aaagttgatg ttccaaaaat 21420aaaaaagatt ttagcccaat caactaaaaa
aaaactctcg tttttttcat atttcacatt 21480ttctggtcac tttgaaggaa
acactaatcc caaactgaga accgaacatg gattaaacca 21540tcccatttac
tatttcttgt tgtcttcaaa aagtcttaga attgtgcaaa aaatagaatg
21600tttcgaaata ttgcggtttt cgttaaaacc ttttttgagt agattgaggg
tccattagaa 21660ttcccaagag aacttgatga ccttcatcat caaaattagt
ggtcattgaa tgtttgatca 21720gacaaaaatg gaaatgactg aatcggaaag
agcaagaaaa tcgaaaaaaa aagtatttgg 21780aaattctgga aaacttttta
aaatttaaga agggcaacga taagaaacag gaaattaggg 21840attttttagt
gatggagaag tacgtgataa ggttaaggtg gaacactagt gcacacgttt
21900tgaatacact acgtgttttt atttatggta gaatatagca cttaaagaac
gtttttaata 21960caaactgaaa taaaaatacg gaaatgtaat tttttttttt
gaaagaatcc gcctgaaact 22020gaattttcac atcaaacggt agtgattctc
tttatgcgtt gggtgatatg tatttacgct 22080gtcttaaagt tttcgactat
aatttaagta atatgtttgt caaaaatcat catggtgctg 22140tgtcctatgt
agccttttct acacttgaaa aatgataatt tttatttgaa aatggtattt
22200aaattcaagt agaaagttat ttagtcttgt gtgccaagca ataaacacat
agtctattag 22260gcaataaaaa gtcagctact gtttgattta aaaacttaga
ctactggtgt gcctgtgcaa 22320gttactcccg tagtacggat acagagtgaa
aactagtgat tgtactttag atcggctgat 22380agtgaattta cagagaaata
attataaaac ttaaaatttt tagcagctca gtcttcaggc 22440tgcacagcca
tattgttaca cttggagtta caaattctgc aaaccatcta ggattgaatg
22500caaaaactct gaaagtcaca tcaagaaatt ccaacaaaaa acacattaga
tgccaactca 22560ttgaattgca ttgattccca agagaaatag tagtaaaagt
gacccctatc cattcctccg 22620ttacatacaa atatacacac aaaaaagagt
gtagacctct tccttctaac ccaaccaaca 22680cacaacaata tcgttccctt
ttatctctaa ttctctgcgt ctccataagc tttgagagct 22740cttcggagca
tcttgtgctt gctccttgta cggcggtaca gtttcctccc tctgctccct
22800tatgtgtgtt taggtgttgt ttgaacaaat aagtttttgg ccatccacct
ccttctcaaa 22860acctttttct tatgcttctt cttgttttgt gcacattttg
gctcttgctt gtctgctcga 22920gccatagaca aggcggcgac atttttgaaa
aaattatatt agtactgtta tatagtactt 22980aatacaacga tcacaacaac
aacacaacga aatgaaaaca tgagatcaaa agacaaattg 23040ttaggaggag
ttggagtttc tacaatcatg aaatgtttat ctagttatta taaaactgaa
23100attgctcata aaattgtgat accatgaaga ccgaaaaact ctatgcaact
gcatactgca 23160catacttaca acctttattc tgacttgaat ttcagttttt
ggtgtttgca gttattctat 23220tttgtttaaa agaaaattca attaggaaat
aagcaataaa ttttggcatg tatttcgata 23280gaaggcacgt gtaaatgcca
cccggaaatt agaaaaaata agatttctca aactgaaaat 23340gattgtgaat
tgaaaattta agagaatcat tgcaaaagta cacaaatgaa tcatttttca
23400gattgaacag gaaagtgcag aaatatcaga ttaccgtccc aacagaaacc
ggaaataaca 23460cttttcaggt aaagaattat acagaaatcg taataaattt
aaaacaaaag agagttatga 23520cacattgcag aacggtctct gtggaaaata
ggaggaggtg ctgcaaaaac tccttagaca 23580tggtcatact tacaaaaaaa
acagagttta actaaaaatt aaattaagtg agaaaatgaa 23640gaaaatggag
gtctttcgcg gattcatttt acttcttctt ttttccactt ttcgttgcaa
23700gctttggttt aaaagtttcg caaacaaata aacaatgaac attgtgttga
gaagacaagc 23760caagtgaaag gaaaccattg agagcaaaaa caacaatcaa
ttgaaataaa gagtaaagtt 23820tattgaatat actgatatgt gaatactgga
aaaataatta gtctctataa ttggtaccgc 23880ctggaagatt catttctgat
tcccttgtgt ctttgaccaa aactttattt ttttcagttc 23940aaaattacaa
aaaataaata ctcatcttca tcgattcagt ggtgttttaa actcctacgt
24000ttttctttta caataaaggt aatgtaaacg ttccgagcgt gtagttttct
ctgaaaattt 24060tttaaaaata acaactttat ggtatttttc ttaaagtctt
aaactgaaac cgaaacattt 24120ttgataggaa aactatttta acattttggg
aactcggcaa aagctctgca ggcttgccga 24180acaactctca tttgaaagta
ataaatatga aaataaatta tcgaagtttt tttttttgat 24240attttatgaa
tacggctctt ggtagttttt gacgagaaaa ttacatgttg cataaatttc
24300aagagttata actcatggag accctaattt ctggtttcac tagaaaatca
aaaaatcaag 24360cgtttgagca gaagactgta ggaagagcac acgtcataaa
aattagggga tcaacgatcc 24420gaaacgggga attgaaatac gatatgcgat
gagttttggt tcgaaccggc tttgtcccaa 24480aaaacaacag aacgatggtc
tcaggctcac ttgactcatc tcggtgggaa caatttttat 24540ttgtttttat
tccgtacgca cagaaacttt ttttgaggta tttttgatcg tgggtgggtg
24600gaatggtagc acccaatttc aaatagtgtt tgatttgaag agacaatgaa
agaaacaagt 24660gggagataat ggaaatgacg tgatgaaatg gaacggagga
aaactggtat aaatatcgtt 24720gactatcaaa actacaataa tactaatgga
gaaaagttca ggattcttga agattttaca 24780ttatgatagt tgggatttac
tggtttcaag ttcaaatgtc aaacatctgg aagaaaaacg 24840tataagatta
catcaaaata aaactaaaat ttgaaggata aagtaaaaca gcataatata
24900gtgttttaca tctcatgtag gaaacgaaca aaatctttga acacctagat
aacttcaaac 24960ggaagttggg tgaagaaaag aataggggcc agaatagaag
gtcattttga caaagtgaac 25020agacaaagac attcctaact cggaggtatt
ccaaaaactg ttccaatatt gaagaatgac 25080actatttgat tttatatcat
aacattatta atcacatggc ttttttctta ggaaatttat 25140atcgcaaaat
aaaaagtggc cttgatgagt cattcattca aaacatgcct aaaaaccttc
25200ataattaatt ataaaaatgc tgatacttga ggacccgttt ttttatattt
ataaacagtt 25260gttttcttta ttccgttctc actttgagtt tttttctgaa
aatactaaaa aaattaacaa 25320agttcggcgt tttttgtcga taattccatc
tgattatttt cggttttttt acctaattat 25380caaatatttt agccagagtg
aaatttatta tcttattaat atgtttttca atttgttttg 25440gtattattct
gttgaaggaa catgttgcat tttaaatctg ttgttaatac agcggccaca
25500tgtttagaac tttataacct cgtttaaaca taaattgtat gccatattta
ttgcaagtac 25560tacatgagtt tgaaacagta tcagatacta tattttaaac
aaaaatacac attttccccg 25620ctatgagaga ttctgataca ttggtttcca
atttttttaa aaacttgaaa ttcctcaagt 25680ctcccactga attacagatt
tctgttctag atacctccaa agacacctag attcgacttc 25740ggcatcttcc
tcatttttat cttcagtttc atcttttgtc taattttccg tacatttctt
25800tgcatcctta ccatctctcc ctctctcact cactcttctt gttcactaaa
tctcaattca 25860aaatgttttc tgccacgtca tcatcatcat caatgccacc
ttctcagagc ccattcgaaa 25920aattaccacg gcatcaaaat attcgatatc
acgaaaaatg cttctcaatt ccacttcata 25980cacttaacta ttttctatgc
gttattattt tttatttctt tgttttcact atattttatc 26040acgaacgtta
tggtggaaaa cctgaaaatg ttcaagttac atcagcaatt tatgattcaa
26100attcaaacga actgtcatta atctttctat ttgattcttc aattcgtcga
cgggaaatat 26160tccttggatt tggtccaaat gactcaaaaa catcaagaaa
tgaaactcaa attgagctta 26220aaccaccacc cggatttgtt gataactcac
aaatttcagt aagtttagga ttttttttca 26280aaaaaacttg atatgaagtg
ttgaaaaatt gataattggg ccgggcttac atcagagtat 26340ctagttatct
tgtatttcaa atattaatat tcaaacattg tagagattcg aaatgcgaca
26400gtacttcagt aattaccacc cacattttga ctgtcaaaaa agttcccaaa
aattgtcgaa 26460aacttttatt aggatgtttt ctcattttgg cacgattgga
gtgttttttt aacaaatccc 26520ttttatgcat caaattaata tctaattttt
aaatcaataa tttggattaa ttcaacttgt 26580tttataagat tttctcgcta
ttaaattagc aaaaaaaaac tatcttcaaa caattagcgt 26640gctttaaaac
tactaggcct ttgttggcaa cgtcttttca cattttggca caaaactata
26700aactatgctc agaatttggt aatgtttgaa aatgttttgg gcaagcatat
agttattcca 26760attctaaagt aagattagtc atctattttc cattccattt
ttccattttt cacctatttt 26820ttccattatt taacaaccaa gactgagcaa
acattttcct gttttaattt tcatatatga 26880aaagacataa gcaaaagctg
gatcaaagct tgggcaaatc ctattcaaag tattttccaa 26940cgtttccatt
ccctcgtttg taaagtacaa ttggtaatct taaggcttaa ttaattattg
27000tgggagattc ataatgtgaa aactaaatgt taagatttgg tcatcaattg
aaaaggaaaa 27060accccagtct ttaactgtga atgcagaaca tccaaagtca
ttgcttttac gagatcacac 27120aggacatcca tatttagaag taagttcaaa
tcagaaatcc ccaatccatt ttttcttgta 27180gttaccactt caagaaccat
actccgattt tcgcgacatt gttagttgtt tcagtccaat 27240ttatggagat
tttgagatgg ttttaacagg tttaacaaat taatttggtt tcttttttaa
27300aacatttaat ttttatagct ttaacatcat ccatatcaat gggatcattt
gttagtatac 27360catatgaaga gcttactgga gagctttaca agtttctacg
tgtatttgaa aaaacgggac 27420atgtcaggtt aactgcattt ccaatgatac
gtcatcagcc tcgcttcgat tcggaaaatg 27480aaaattatca tttgaaaatg
atcaaactta aaacagattt aacgcatttg cattgttggc 27540taatgcataa
aaaccgggcc aaattcatga tcttccaaaa ctctgctgaa attgttttac
27600cgatttcctc gacgctggaa aatcccaatt acgcctctga atttacacga
atatttgaaa 27660caccacgagt tgaaggatat gatattttag aatataatgt
caaaatttca acggataaac 27720gcttaggcga cttttcggat ttctccatca
ggcagacaat tgaagcagca aaagcagaag 27780aattaaccgg aaattctaaa
acattaatca tgagaatggt atcacttttt ttcaaaataa 27840tttactgttt
ctattttggc atttatttca gcattctcca actccacaga atctcttaaa
27900acgcggtaaa atgtatccat ttttcaaaaa tttcccatct ccaccacaag
ttattccaaa 27960gaaaacattg gacaaattgg atacaataac agaaataatt
gaagaatctg atgcattctg 28020gacacttatc aaagaatgtt cagaaaattc
gaaatcttgg aaatgctcgt caagaaaatg 28080tgtaagacca tcagttagac
atcgatctct tcatggatgg tattcatatg atattcattt 28140ttctaaattt
ttgaatgttg aaagtttttt ttgttcagat tttcaataaa cttttaagaa
28200aagaataatt ttaaattcta taattcctga atttccaact atgtttatca
tttcccaaag 28260tacattcgaa aaagctcaat aagcaaaacg accacgaaat
aacagtatta aaaaaaaaga 28320tgttgtcatt tgaagttctg gagtgcgatg
aaaagtctct cacctcggac tttctgtaat 28380ttatttagca tacaacatga
atttgaccaa ctcgaaataa ggttaagact gaaaattttt 28440cacaaaaatt
ggaacacttg cgaagcgaat tcaagacttt tcgaagttat taaacaagct
28500ttcaaattct cagtaaaact gaacgttttt tttatgctct ccaaatcatt
ttaatatggc 28560tgctcgcgtc gctgaagtat tttctagagt atgtttaata
aaactaatat gtaaatgaaa 28620aaccaaaaac tcagataaag agcataactt
ttataacgca ttttcagaac tcttcaagct 28680ttttcagatc acttctatca
gcagtattct tcttttttcc aaagacacca agaactgaaa 28740aggttgaagg
agcatcaccg gaaatagagg atgactgctt attgttcttc tttttctgaa
28800taaaatcaaa ttaaacaccg aaaatatgaa acatattcac taacctgaac
agctttcagg 28860tttgatttat tctgattttc cgccgctgat ctgctctgac
ttttgaaacc gggacttgga 28920gagttaccat tgcgtatgcg agttcgaact
ggacgccgat tcttctttct gaataaacga 28980attatacaaa tttgtatttg
aaaacggaca acatacactc cttcttccgc cgaattgctc 29040atcgattttc
tcatttcttg tgttttttcc tggcgttcag gttcaaaagg tggagcaact
29100ggtttggaca tatacggaag aatgttcgag acttgaatct tttttggttg
ctcaatattc 29160tccattggaa tatgatcggg aagttcaaag tagctgttgg
atcctggagc ttgatcaaat 29220ccttcgagag ttaaaagttc acgaactgct
tcactcattg tgaccctttc ctcttcggca 29280ccagcacaga ttctatactg
aaattgcttg ttgtgttgtt ttactcaaaa gaatagtgaa 29340caaaattttc
tcaccgtaat gaatctgaca atggctggtg ggacgttagc ttcaaatggc
29400attcggtatc cgttctgaac acgtggtaaa acctcagcaa ctttcattcc
cggataaggt 29460tcgattccat catggtacac ttcccaacac atgactccat
aagcgaaaac atcagtcttt 29520ggagtataga acccagttct tggaacttct
ggagccaacc atctaatagg aactctgaaa 29580aatttgaaaa ggttggaatt
tttgacgttc tctaactttt tgtgaggatt catccgatag 29640ctatagcctt
ctcgtgacag tccaaagtcg gatatcttta cttgtccatt cccgtagaga
29700caatttctgg acgcaatatc gcgatgaatt atttgaagtg aatgaagata
ttcaagacca 29760agaccagctt gaagaaccat cgtatgtttc ttggaaattg
gcaatgaacc aatgttcttc 29820tttagatatg aatccaaagc tccattgtca
gcctaaaata atttacataa gacatttttt 29880cttagtaaaa taaaattaat
cagttaatta attaacatac caactccatt atgaccatca 29940aaggttcctg
tcctgcagcc acaccataaa aagtgacgac attcggatgt ttgaactttc
30000tcatcaatct ggcttcgtgc atgatttctt tgatctgctc ttttgtcaaa
gattccaact 30060ttgccagctt gattgcagct tttttgacgg tatttcctat
gcgaatttct cccaattgaa 30120cctctccaaa tgctccttct cctaatttct
tgattaatgt cacgtcagaa tgttgctttt 30180cccacggttc acgaccaatt
ggacggatga ttacagtttc tgggccctaa aagcaaacaa 30240atgaaaataa
gtttactcac ttaatttgta agatcacccc agcaacaggt tctttagaac
30300gatgatagta attgagaaga tctgcgatac tagaaaacca ttttttatca
actgcaaact 30360tgttattgtg ctctcgaatt acataatgac gaatctgaaa
taatattctt aaaaattatg 30420agcaatcgtt ttacgtacgt cctcaattac
tccaacatag acagagagaa caaatttcct 30480tggctctccc acttttggat
cagtaaatcg aactagaaaa tcgcctcgtt gagtgagcaa 30540ctgtttcata
tcctcacgtg gcaataagcc atggtaccag ggttcttttg caagtacttg 30600c
30601348009DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number X99599 34ggatccttgg ccacgccatg ggcgatgaaa
ttgaccgcgt cgtaacgggt catgtcctgc 60tcctgcagga agaaggccgc gttcgattcc
cgttccgcaa agatcgcgac aaggacattc 120gcccccgtca cctcggtccg
gcccgagctt tgcacatgga tcgcggcgcg ctggatcacc 180cgctggaagg
cggcggtcgg cacggcttcc gagccttcga cttcggtgat cagcgtcgag
240agatcatcgt cgatgaactc ggtcagggtg gtgcgcaact cgccaagatc
gacgccgcag 300gcgcgcatca cgcggctggc gtcgggctcg tcgatcagcg
cgacgagaag atgttcgagc 360gtcgccagtt catgtttgcg cgtgttggcc
agcgccagtg cggcgtgaat tgcttgctcg 420agcgtggtcg aaaacgaagg
catgcggcgc tcctttcctc gggtctcccg atactggcct 480catgtgatta
agtttcggtg gatttcgccg cgcttcaagg cccggacgcg tgttttttcc
540acctctcgcc gctctgttgc aaaactgacc agcgcggcgg gctttcgcgc
gatccgcagg 600cagcgcgcga aagtgctttc agaaccggtc cttgcgcgcc
cgcgccgcgg tgaagacggc 660gagcggcgcg gcatccccgg gcaggccgag
cgcggcgcgc aacgcagcgt catcggcgcg 720caaaaaggga ttcgttgccc
gttcctcgcc caaagtcacc ggcaaactgg gttccccggc 780cagccgcaag
gccgtcaccc ggtccatccg gtcgtgcagc cgaccgttcc ccggttccag
840gctgagcgcg aaccggccgt tcgcggcggt gtattcatgc cccgaacaga
cccgggtttc 900gggcggcagc gcggccagac gggtcagcgt gtcgaacatc
tgcgcggggg tcccctcgaa 960gagacgcccg cagccccagc tcatcaggct
gtcgccggaa aagagcagcc ccgccccggg 1020cagataccag gcgatatggc
cgagcgtatg gccgtcggcc gcgatcacct gcgcggcctc 1080catccccaga
tgcagcacgt cgcccggggc caccggatga tcgagcggcg gcagccggtg
1140ggcatcggcc gcggcccccg ccaccttggc cccggtcgcc tgcgccagcg
cctcgacccc 1200cgcgatgtga tcggcgtggt gatgggtgat caggatgtgg
tgcagctgcc agcgccggtc 1260ggtcagcacc ttcagcaccg gggccgcctc
ggggacatcg accaccacca cggtatcggt 1320ggcggtgtcg tgccagagcc
aggcgtaatt gtcggtcagg caggggatcg gggtcagttc 1380gagggtcatg
gccttttgcg catctttcgc tatcctgacc cagcttcgcc caaggaaggc
1440caacctgcaa tgcatctcga cgtgctcgac ctgcgtgatt tctactaccg
cacccaattg 1500gggcgcacgg cgcaaaaggc gatccgcgac aaggtggtcg
aactctggcc ggacacccag 1560tccggcatgg ccgggctgac ggtggcgggc
tacggcttcg cggtgccgct gttgcgcccc 1620tatctgggcc gggcgcggcg
ggtgatcggg ctgatgcccg cgcagcaggg cgtgatgccc 1680tggcccgccg
gagagcccaa tgtctcggtg ctctgtgccg aaaccagctg gccgctggag
1740accgggatga tcgaccggct ggtggtgctg cacgggcttg aagtctccga
cgaccccgat 1800gcgctgatgg aggaatgctg gcgcacgctg ggccccggcg
ggcgggcgct gttcatcgtg 1860ccgaaccggg tcgggctttg ggcgccgcgc
gaaaccacgc ccttcggctt tggccgcccc 1920tatacgatgg gccagctcga
ggcgcaggca cgacgggtgg ggtttgcccc cgaacgtcag 1980gcggcggcgc
tgtacattcc gccctcgcag cggcggttct ggctgcgctc ctccgagatg
2040tgggaacggc tgggcacaag ggcggcgggc tatctggcgg cgggggtggt
gatgcttgag 2100gtgatcaagc aggtgcattc ggtgcgccgc tcggggcttg
gcgcggcggt gcgcaagccg 2160ctctcgatcc ttgaaggggc gcccaagccg
gtggtcgggc ggatgtgagc cgcccgcggc 2220cgcaagaatc gcccggccgg
aaaagcccgt ttccgcggca cttcgccctg cggcggggaa 2280acgcagcggg
gcgggcttcg accctttgcg ctaacactcc gtgccggtgc agaaaatgtg
2340ccagcctgat gcggattcct gccgccaaga tggttgcgag ggtcttgatg
ctctgctaga 2400cgcaaccccg aatgcggcgt gcgagatcat tttgggcgcc
gaggggggcc tctgaatcgg 2460tgacggaacg attggttccg gtgtccgcgt
gcggaggcaa aagcatcgga agggtggacg 2520tgtccgaacc agcttcgatt
tccgcagcca ttgccgggcg ttatgccacg gccatcttcg 2580acctcgcgca
ggaggccaag ggcatcgacg cgctctcggc cgacgtggac gcgctgacgg
2640ccgccttggc cggttcggcc gagctgcgtg acctgatttc ctcgccggtc
tacacccgcg 2700aggagcaggg ggacgcgatc gccgcggtgg ctgcgaagat
gggcctgtcg gcgccgcttg 2760ccaacggtct gaaactgatg gcgacgaagc
gccgtctgtt cgcgctgccg cagctgctca 2820agggcctggc cgccgcgatc
gccgaagcca agggcgagat gaccgcggat gtcacctcgg 2880ccaccgcgct
gagcgcggcg caggccgaga agctggcggc gacgctggcg aaacagacgg
2940gcaagaccgt caaactgaac gtcgccgtcg atgaaagcct catcggtggc
atgatcgtca 3000agctgggttc gcgcatgatc gacaccacgg tcaaagccaa
actcgcttcc cttcagaacg 3060ccatgaaaga ggtcggataa atgggcatcc
aagcagctga gatttctgcg atcctcaagg 3120agcagatcaa gaacttcggg
caggatgccc aggtcgccga agtgggccgc gtgctctcgg 3180tcggtgacgg
gatcgcgcgc gtgcacgggc tcgacaacgt ccaggcgggc gagatggtcg
3240aattccccgg cggcatccgc gggatggcgc tgaaccttga agtcgacaac
gtcgggatcg 3300tgatcttcgg gtcggaccgc gacatcaagg aaggcgacac
cgtcaagcgc accaacgcca 3360tcgtggacgt tccggcgggc gaaggcctgc
tgggccgcgt cgtggacggc cttggcaacc 3420cgatcgacgg caagggcccg
atcgtggcga aagagcgtcg catcgccgac gtcaaagccc 3480cgggcatcat
tccgcggaaa tcggtgcatg agccgatggc gaccggcctc aagtcggtcg
3540acgcgatgat cccgatcggc cgcggccagc gcgagctgat catcggcgac
cgtcagaccg 3600gcaagaccgc gatcgcgctc gacaccattc tgaaccagaa
gtcgtacaac gacgccaacc 3660cgggcaacaa gctgcactgc ttctatgtcg
ccatcgggca gaagcgctcg accgtggcgc 3720agctggtgaa gaagctcgaa
gaagccggcg cgatggaata caccaccgtc gtcgccgcga 3780ccgcttcgga
cccggcgccg atgcagttcc ttgcccccta ttcggcgacc gcgatggcgg
3840aatacttccg cgacaacggc atgcacgcgc tgatcatcta tgatgacctc
tcgaagcaag 3900ccgtggccta tcgtcagatg tcgctgctgc tgcgccgtcc
gccggggcgt gaagcctatc 3960cgggcgacgt gttctatctg cactcgcgcc
tgctggaacg ttcggcgaaa ctgaacgagg 4020atttcggttc gggctcgctg
accgcgctgc cggtcatcga aacccagggc ggcgacgtgt 4080cggccttcat
cccgaccaac gtgatctcga tcaccgacgg tcagatcttc ctggaaaccg
4140aactgttcta ccagggcatc cgcccggccg tgaacaccgg tctctcggtg
tcgcgcgtcg 4200gttcgtcggc ccagaccaac tcgatgaagt cggttgccgg
tccggtgaaa ctggagcttg 4260cgcagtatcg cgaaatggcc gcctttgcgc
agttcggttc cgaccttgac gccgcgacgc 4320aaaagctgct gaaccgcggt
gcccgtctga ccgagctgat
gaaacagccg caatattcgc 4380cgctgaccaa cgccgaaatc gtggcggtga
tctttgcggg caccaacggc ttcctcgatg 4440ccgttccggt gaaggaagtc
ggccggttcg agaaaggcct gctggcctat ctgcgctcga 4500cccgcaagga
cgtgcttgag tggctcacca aggaagaccc caagatcaag ggcgacgccg
4560agaagaagct caaagacgcg atcgccgagt tcgccaagac cttcgcttga
cggcctgaaa 4620ggacagggag atgcccagcc ttaaggacct caagaaccgg
atcgtgagtg tcaagaacac 4680tcgcaagatc acgaaagcga tgcagatggt
cgcggcggcg aacattcgcc gcgcccagga 4740aagcgccgaa gctgcccggc
cctatgccga gcggatgaac gccgtgatgt cgagccttgc 4800cggtgcggtg
ggctcgaccg acggtgcgcc gcgcctactt gcgggcacgg gctccgacaa
4860ggtccatctc ctcgtcatca tgacgggcga gcgcgggctt tgcggcggct
tcaacgccaa 4920tatcgcgaaa ctcgcgaagg cgaaggcgat ggaactgctg
gcccagggca agacggtgaa 4980gatcctcacc gtcggcaaga aaggtcgcga
cgcgctgcgt cgtgatctgg gccagtatta 5040catcgatcac atcgacctga
gcgacgtgaa gaaactgagc tacccggtgg cgcagaagat 5100ttcgcaaaac
atcatcgacc gcttcgaggc gggcgaatac gatgtggcga cgatcttctt
5160ctcggtcttc cagagcgtga tcagccaggt gccgaccgcc aagcaggtga
tcccggcgca 5220gttcgaaacc gatgcggcct cggcctcggc ggtttacgac
tacgaaccgg gcgatcagga 5280aatcctgacc gcgctgctgc cgcgtgcggt
ggccacggcg atctttgccg cgctgctgga 5340aaacaacgcg tccttcaacg
gggcgcagat gtcggccatg gacaacgcca cccgcaacgc 5400gggtgacatg
atcgatcgct tgaccatcga gtataaccgc tcgcgtcagg ccgccatcac
5460caaagagctc atcgaaatca tctcgggcgc cgaggcgctc tgacggaacc
ggagatagaa 5520gagaatggca agcaaaggca aagtgaccca ggtcatcggc
gccgtcgtcg acgtgcagtt 5580cgaagacggc ctcccggcga ttctgaacgc
ccttgaaacc accaacaacg gcaagcgcct 5640cgttctcgaa gtggcgcagc
acctgggcga gaacaccgtc cgcaccatcg cgatggacgc 5700gaccgagggt
ctcgtgcgcg gcgcggccgt gtccgacacc ggcggcccga tcaccgttcc
5760ggtgggcaac gccaccctgg gccgcatcct gaacgtcatc ggcgagccgg
tggacgaacg 5820cggtgacgtg tcgaaagccg aagcccgggc gatccaccag
cccgcgcccg atttcgcggc 5880gcagtcgacg gaaagccaga tcctcgtcac
cggcatcaag gtgatcgacc tgctcgcccc 5940ctattccaag ggcggcaaga
tcggtctctt cggcggcgcc ggtgtgggca agaccgttct 6000gatcatggaa
ctgatcaaca acatcgcgaa agtgcactcg ggcttctcgg tgttcgcggg
6060cgttggcgaa cggacccgtg agggcaacga cctttaccac gagatgatcg
aatcgggcgt 6120tatcaacctc gagaagctcg aagaatcgaa agtggcgctg
gtctacggcc agatgaacga 6180acccccgggg gcccgtgccc gcgtggcgct
gaccggcctg accctggcgg aacagttccg 6240cgaccagtcg ggcaccgacg
tgctgttctt cgtcgacaac atcttccgct tcacccaggc 6300cggttcggaa
gtgtcggcgc tccttggccg tatcccctcg gccgtgggct accagccgac
6360gctggccacc gacatgggcg cgctgcaaga acgcatcacc tcgaccaaag
ccggttcgat 6420cacctcggtt caggccatct acgttccggc cgacgacctt
accgacccgg ccccggccac 6480gtcctttgcc cacctcgacg ccacgaccgt
tctgtcgcgt gcgatctcgg aactcgggat 6540ctacccggcc gtcgacccgc
tcgactccac ctcgcggatc cttgacccgc aagtcgtcgg 6600cgaagagcac
tatcaggtcg cccgtgacgt ccaagggatg ctgcaacgct acaagtcgct
6660gcaggacatc atcgccatcc tcggcatgga cgaactgtcg gaagaagaca
agctgacggt 6720ggcccgcgcc cggaagatcc agcgcttcct gtcgcagccc
ttcgacgtgg cgaaagtctt 6780caccggctcg gacggcgtgc aggttccgct
cgaagacacc atcaagtcgt tcaaggcggt 6840ggttgcgggc gaatacgacc
acctgccgga agcggccttc tacatggtcg gcggcatcga 6900tgacgtgatc
gcgaaagccc agcgcctcgc cgctgcggcg taagggggaa ccatggccga
6960taccatgcag ttcgatctcg tgtcgccgga acggcggctt gcctccgttg
ccgcgagcga 7020ggtccgtctt cccggcgtgg aaggcgatct gacggcgatg
ccgggccatg cgcccgtcat 7080cctctcgctg cgtcccggca tcctgaccgt
ggtcagcgcc gcgggcacgg ccgaatacgc 7140cgtgaccggc ggcttcgccg
aggtttcggg cgagaaggtg accgttctgg ccgagcgcgg 7200tctgacccgg
gcggaactga ccgccgcggt tcatgccgag atgctggccg aggccaagaa
7260agtcgcggac gccgcgcatc cgtcggtggc cgatgccgcc gcgaagatgc
tggccgacat 7320ggaagcgctt ggctcgcaca tcaatctctg acgggacatc
ccgccggata tctcgggccc 7380cggtcatcgc gccggggccc ttgctttttg
cttttgtctt gccgcgccgc atattagcgt 7440gaaggtgcag gcagccggag
tgagcgacag gaacggatga agaagttttc ctcgacccgg 7500atcggcgtgg
cccagggatc gctggtgctg ttttcggatt atctggacgg cggcgtgatg
7560tggacgggcg agggcccgcg cgaattgcgc aggctggtgg tgttcgacga
agccttccgc 7620gagatcccgg cggtgcaggt gtcgctgtcg atgtgggaca
tcgaccagaa gcacaatccg 7680cgcatggaca tttccgccga catggtgacg
gccgagggct tcgtgatcgt ctttcgcacc 7740tggggcgaca cccgcgtcgc
ccgcgtccgc gcggactggc tggcgatcgg cggctgcgcc 7800aatgacgacg
actgggacgt ggcctgatcc cgcccggctt gactttccgc ccccccgcgc
7860cgatggtgcg cgcgactttc ccatccaacg aggcccgccc gtgcaacaag
atgccccccg 7920ctggcagctc gtggtgatcc tgtgggggac gaaatatccg
gtcgccgaac tcaacgccct 7980gatcgagacc gtgtggcccg ggcctcgag
8009359810DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number AF018073 35gatatcgggc ttgtcatttt cgattgcgac
ggggttctgg ttgattcgga agttctggcc 60gtggccgtcc tcatcgcaga actggaccgg
gcgggcgtgc gggtcgacga ggccttcgtg 120catcggcatt ttctgggccg
gagcttcccg gctgttcagg aggtcgtgca gcgccagttc 180ggcgtgaccc
tgcccgagac cttccaggtc gaggaacgtg cccggctgct gtcagccttc
240gagaccggcc tgcgggccat gctcggggcc gcggagaccg tccgcgcgct
gtcggtgccc 300tactgcctcg ccacgtcgag cacgccggcc cggctcacgc
gctcgctgga gatcacgggc 360cttgcggccc tcttcgaggg acgctgcttc
accgcgagcc aggtggcgcg cggcaagccc 420gcgcccgatc tgttcctgct
cgccgcggcc gagatgggcg tcgcgcccga acgctgcctc 480gtgatcgagg
ataccgagcc cggcgtgcgc gcaggcctcg cggccgggat gcaggtctgg
540cgcttcaccg gcggtagcca tttcgcgaac cgatcccccg aggatgcgcc
cgatgccctg 600ccgcaccggc ggttcgacag cttcgaccgt ttctacgaga
ccctgcccgg cctgcgccgg 660gccaagtgcg agaccctgac atgatcgacc
ggcccgagag cgagccgacg cccctcgacg 720atgccgcgcg cgcgggctgg
ctctattatg tcgcaggcct gactcaggat cagatcgcgc 780gggagctcgg
cacctcgcgt cagcgggcgc agcggctggt gagccgggcc atctccgaac
840ggctgatcca cgtccggctc gagcaccggg tctcgggctg cctgcatctg
gaagccgcgc 900tcctccggcg cttcgggttg aagctggccc gcgtggcgcc
gagtctcggg tccgaggtgg 960atcccctgcc ctccatcgcc cccaccgccg
ccgccgaggt ggagcgggtg ctgcgctcgg 1020agcggccgat ggtggtggcc
ttcggcaccg gccggtcgct gcgcgccacc gtcgaggaga 1080tgacctcgat
ggtctgcgaa cagcacaaga tcgtgtcgct caacggaaat atttctgcgg
1140atggctcggc ctcctactac gatgtgatct tccgcatcgc cgaccgtgtg
cgtgcgccgc 1200actatccgat gccgatgccg gtcatcgcgc aggatgcggc
ggagcgggag ctgtttcatg 1260cgctaaagcc cgtgcagtcg gtgctgcggc
ttgcgcgcaa tgccgatgtg accttcgtcg 1320ggctgggaca gatgggcgag
gacgcgccgc tcctgaagga cgggttcatc acgcccgagg 1380agctgaccga
gatgcaggat ctgggcgccg tcggagaggt ggcgggatgg gtcttcgact
1440cggagggtcg ctacctcgaa accagcatca atcagcgggt tgcgggcgtc
cgtgtcgaac 1500tttccgagga tcggacggtg gtcgccatcg ccggtggcag
gcgcaagctc gcggcgctgc 1560acgcaggctt aaggggccgt cttttcaacg
gcctgatcac cgacgagttc acggcgcagg 1620cacttctgtc ctgaagccgc
cgaaaggcgc ggcaaaaagt atttgacagg ctggcacccc 1680tcggtgagta
attattcgcc gcacgaaata atgctcaccg tgcaggccag ggaggatact
1740gatgaccgca agatttcgcg ccctgatggg cgcgtgcgcc gtggctgcgc
tctcgtccgc 1800cgccggcgcc gaaaccatca ccgtggcgac tgtcaacaac
ggcgacatga tccgcatgca 1860ggggctcatg tccgagttca acgcgcagca
ccccgacatc accgtcgagt gggtgacgct 1920cgaggaaaac gtgctgcgcc
agaaggtcac gaccgacatc gccaccaagg gcgggcagtt 1980cgacgtgctg
accatcggca cctacgaggt tccgatctgg ggcaagcagg gctggctcgt
2040gagcctgaac gacctgccgc cggagtatga tgccgacgac atcctgcccg
cgatccgcaa 2100cggcctgacc gtcgacggcg agctctatgc cgcgcccttc
tacggcgaga gctcgatgat 2160catgtatcgc aaggacctga tggagaaggc
ggggctgacc atgcccgacg cccccacctg 2220ggacttcgtg aaggaagcgg
cgcagaagat gaccgacaag gatgccgagg tctacggcat 2280ctgcctgcgc
ggcaaggccg gctggggcga gaacatggcc ttcctcagcg ccatggccaa
2340cagctacggc gcgcgctggt tcgacgagaa ctggcagccg cagttcgacg
gcgaggcctg 2400gaaggccacg ctgaccgact atctcgacat gatgacgaac
tacggcccgc ccggcgcctc 2460gaaaaacggc ttcaacgaga acctcgcgct
gttccagcag ggcaagtgcg gcatgtggat 2520cgacgcgacg gtggccgcct
ccttcgtgac caaccccgag gaatccacgg tggccgacaa 2580ggtgggcttc
gcgctcgccc ccgataccgg caagggcaag cgggccaact ggctcggggc
2640ctggaacctc gcgatcccgg cgggctcgca gaaggtcgat gccgccaagc
agttcatcgc 2700ctgggcgacc tcgaaggact atgccgagct ggtggcctcg
aaggaaggct gggccaacgt 2760gcctccgggg acgcggacgt cgctctacga
gaacccggaa tatcagaagg tgccgttcgc 2820gaagatgacg ctcgacagca
tcaacgcggc tgacccgacc cacccggcgg tcgatccggt 2880gccttacgtc
ggtgtgcagt tcgtggcaat ccccgagttc cagggcatcg gcaccgccgt
2940gggccagcag ttctcggcag ccctcgcggg ctcgatgtcg gccgagcagg
cgcttcaggc 3000ggcccagcag ttcacgacgc gcgaaatgac ccgcgcgggc
tacatcaagt gagcccttcc 3060gcgggccggc cctgagcggc cggcccgcac
cgcttgccgc ttccggccgt atccgccgga 3120ggcctttccg ccccatcagc
cccgaggcct ccatggcgac ccagcattca aagactgcgg 3180cgcgtctgat
gatttccccg gccgtgatcc tcctgttcct gtggatgatc gtgccgctgt
3240cgatgacgct ctacttcagc ttcctgcgct acaacctcct catgccgggg
atggagagct 3300tcaccggctg ggacaattac tattacttcc tgaccgatcc
ggccttctcc gcggccctga 3360ccaacacgat cctcctcgtg gtcggcgtcc
ttctcatcac cgtggtgggc ggggtcctgc 3420tcgcgctcct gctcgaccag
cccttctggg ggcagggcat cgtgcgcgtg ctggtgatcg 3480ctcccttctt
cgtcatgccc accgtctcgg cgctggtctg gaagaacatg ttcatgaacc
3540ccgtgaacgg gatgttcgcc catatcgccc gcgggctcgg ccttccgccg
ttcgacttcc 3600tgtcgcaggc gccgctggcc tcgatcatcg gcatcgtggc
ctggcagtgg ctgcccttcg 3660ccacgctgat ccttctgacg gcgctccagt
cgctcgaccg cgagcagatg gaggcggccg 3720agatggacgg cgcctcggcg
ctcgaccggt tcatccacat caccgtgccg cacctgacgc 3780gtgccatcac
cgtggtggtg ctgatccaga ccatcttcct tctgggcgtc ttcgccgaga
3840tcctcgtcac gacgaacggt ggacccggca ccgcctcgac caacatcacc
tacctcgtct 3900atgcgcagtc gctcctgaat tacgacgtgg ggggcgggtc
ggccggcggc atcgtcgccg 3960tggtgctcgc caatatcgtg gcgatcttcc
tgatgcgcat gatcggcaag aatctggacg 4020cctgacatgt cacgccgcac
ctcaacccgc cgcacgctga tcgtcacgct cgccgcctgg 4080acgatagcct
tcctcatctt cttcccgatc ctctggacgg tgctgatgag cttcaaatcg
4140gaaggagacg ccatcaaggc gcccttcgcc atgctcttct cggactggac
cctgcaatcc 4200tacgccgatg tgcaggaacg gtcgaactac gcccgccact
tcatgaattc ggtggtgatc 4260tcgctgggct cgaccctcgt ggcgctcgcc
atcgcgatcc ccgccgcctg ggccatggcc 4320ttcgtgccgg gccggcggac
gaaggacgtg ctgatgtgga tgctgtcgac caagatgatg 4380ccggcggtgg
gcgtgctcat cccgctctat ctgatcttcc gcgacacggg ccttctcgac
4440acgcggatcg gcctcgtgat cgtgctcacg ctcatcaacc tgccgatcgt
ggtctggatg 4500ctctacacct acttcaagga gatcccgggc gagatcctcg
aggcggcgcg gatggacggg 4560gcgacgctcg gctccgagat cctctatatc
ctcacgccga tggccgtgcc gggcatcgcc 4620tcgacgctgc ttctgaacgt
gatcctcgcc tggaacgagg ccttctggac gctgcagctg 4680accacctcgc
gggcggcccc gctcacgcag ttcatcgcga gctattccag ccccgagggc
4740ctcttctacg ccaaactgtc ggcggcctcg accatggcca tcgcgccgat
cctgatcctt 4800ggctggttca gccagaaaca actcgtccgc ggcctgacct
tcggcgcggt gaagtgagga 4860ccacatgggc aagataaccc tgcgcaacgt
ccagaagcgg ttcggtgagg cggtcgtcat 4920cccctcgctc gacctcgaca
tcgaggatgg cgagttcgtc gtcttcgtcg gcccctcggg 4980ctgcggcaaa
tccacgctcc tgcgcctgat cgcgggcctc gaggatgtgt cggacggcca
5040gatcatgatc gacgggcgcg acgccaccga gatgccgccc gcgaagcgcg
gcctcgccat 5100ggtgtttcag agctacgcgc tctatccgca catgacggtg
aagaagaaca tcgccttccc 5160gctgcggatg gcgaagatgg agccacagga
gatcgagcgg cgcgtgtcga acgcggccaa 5220gatcctgaac ctcaccaact
atctcgaccg ccgccccggc cagctctcgg gcgggcaacg 5280gcagcgggtg
gccatcgggc gcgccatcgt gcgcgagccg gcggccttcc tgttcgacga
5340gccgctctcg aacctcgatg cggcgctgcg ggtcaacatg cggctcgaga
tcaccgagct 5400gcaccagtcg ctcgagacca cgatgatcta tgtcacccac
gatcaggtcg aggccatgac 5460catggccgac aagatcgtgg tgctgaacgc
gggccggatc gagcaggtgg gctcgcccct 5520caccctctac cgcaatccgg
cgaacctctt cgtggcgggc ttcatcggca gcccgaagat 5580gaacctgatc
gaggggcccg aggccgccaa gcacggcgcc accaccatcg ggatccgccc
5640cgaacatatc gacctgtcgc gcgaggcggg ggcgtgggag ggcgaggtcg
gcgtctcgga 5700acatctcggc tcggacacgt tcctgcatgt gcatgtcgcg
gggatgccca ccctcaccgt 5760gcggacgggc ggagagttcg gcgtccatca
cggcgaccgg gtctggctca cgccgcaggc 5820cgacaagatc caccgcttcg
gcgccgacgg aaaggcgctc tgacatgcgg ctcgacggca 5880agaccgccct
catcaccggc tcggcgcgcg gcataggccg cgccttcgcc gaggcctatg
5940tgcgtgaagg cgcgcgcgtg gccatcgccg acatcaacct cgaggcagcc
cgcgccaccg 6000cggccgagat cggccccgcg gcctgcgcca tcgccctcga
cgtgaccgat caggccagca 6060tcgaccgctg cgtggccgag cttctcgacc
gctggggcag catcgacatc ctcgtgaaca 6120atgcggccct cttcgatctg
gcgcccatcg tcgagatcac ccgcgagagc tacgaccggc 6180tgttcgcgat
caacgtctcg ggcacgctct tcatgatgca ggcggtggca cgggcgatga
6240tcgcgggcgg ccggggcggc aagatcatca acatggcaag ccaggccggc
cgccgcggcg 6300aggcgctggt gggcgtctat tgcgcgacca aggccgccgt
catctcgctc acccagagcg 6360cggggctgaa cctcatccgc cacgggatca
acgtcaatgc catcgccccg ggcgtggtgg 6420acggcgagca ctgggacggg
gtggatgcga agttcgccga ctacgagaac ctgccccgcg 6480gcgagaagaa
gcgtcaggtc ggcgcggcgg tgcccttcgg ccgcatgggc cgcgccgagg
6540acctgaccgg catggcgatc ttcctcgcca cgcccgaggc cgactacatc
gtggcccaga 6600cctacaacgt ggacggcggc aactggatga gctgaggccc
aaggcccggc cctccccccg 6660tcgaacgcgc cccctatccg aggtaatccc
atgacccgct ccgtcacccg tccctcctat 6720gaccgcaagg cgctcactcc
cggcatcgtc catatcggcg tcggcaactt ccaccgggcg 6780catcaggcgg
tctatctcga cgatctcttc gcgctgggcg agggccacga ctgggccatc
6840ctcggcgcgg gcgtccgccc gaccgatgcg cggatgcgcg aggctctggc
cgcgcaggac 6900aatctctcga cggtgatcga gctcgatccg gcgggccacc
gggcccggca ggtgggggcg 6960atggtgggct tcctgccggt cgaggccgac
aatgcggccc tgatcgaggc catgtcggat 7020ccgcgcatcc gcatcgtctc
gctgaccgtg accgagggcg gctattatgt cgatgcctcg 7080ggcgccttcg
atccgacgca tcccgatatc gtggccgatg cggcccatcc tgcgcggccc
7140gcgaccgcct tcggcgcgat cctcgccgcc ctccgcgccc gccgcgacgc
gggggttaca 7200cccttcaccg tgatgtcctg cgacaacctc cccggcaacg
gccatgtcac ccgcaacgcc 7260gtggtgggcc tggccgagct ctacgacgcc
gagcttgcgg gctgggtgaa ggcgcaggtg 7320gccttcccga acggcatggt
cgaccgcatc acccccgcca ccggcccgca cgagcgcgaa 7380ctggcgcagg
gcttcggcct cgccgatccg gtgcccgtca cctgcgagcc gttccggcag
7440tgggtgatcg aggatcattt ccccgccgga cgccccgcgc tcgagaaggt
gggcgtgacc 7500ttcaccccgc atgtccatgc ctacgaggcg atgaagatcc
gcatcctgaa cgggggccat 7560gcggtgatcg cctatccgtc ggcgctcatg
gacatccagc tcgtgcacgc ggccatggcc 7620catccgctga tcgcggcctt
cctgcacaag gtcgaggtcg aggagatcct gccccatgtc 7680ccgcccgtgc
ccgacaccag catccccgac tatcttaccc tgatcgagag ccgcttctcg
7740aaccccgaga tcgccgacac gacgcgcagg ctctgcctcg acggttcgaa
ccggcagccg 7800aagttcatcg tgccgtcgct gcgcgacaat ctggcggcgg
gcacggtgcc gaaggggctg 7860gtgctgctct cggcgctctg gtgccgctac
tgcttcggca cgacggactc gggcgttgtg 7920gtcgagccga acgatccgaa
ctggacggcg ctgcaggacc gggcgcggcg ggcgaaggag 7980acgccggccg
agtggctggc gatgaccgaa gtctacggcg atctggcgca gaacgatctt
8040ctggcggccg agttcgcggc agccctcgag gcggtctggc gcgacggggc
cgaggcggtg 8100ctgcggcgct tcctcgcggc ctgatccgca gggcccagcc
gctcggagca ccgaagcgga 8160gcccctgccc cttgcggcgc accgtgaggc
gaaacgaccg ggccaccccg gggccaccgc 8220ctcggtaaca ccatggtatc
gcgcaagaat gccggcgcct ctgccgaacg ggcccggctg 8280ccgggcgagg
cgccggactt gtcaaggcgg cggccctcgg gtagagaggg cgggcgtggc
8340cccgttagca cagtggtagt gcagcgctct tgtaaagcga aggtcgttcg
ttcaaatcgg 8400acacggggca cgcgatcctc cctccgcatc ggcgctcgcc
cccggtctgg actgcctctt 8460cggaaggcac ctgcccgctt gtgcgccgcg
ccctttcctc gcttcccaag cgtctgtcac 8520ggcttgcgga aagccgtgcg
cctcggttct ggacagccgc cccttgcggt gtaatctgcc 8580ctcagcgcgc
agccggcgga cagaagccgg cccgccacgt ccacaaggga ggaatgccat
8640ggatcgtcgt tcattcatca ccaaggccgc cgtgggaggg gccgccgcga
gcgccctcgc 8700cgcgccggcg cttgcccagt ccgcgcccaa ggtcacctgg
aggctcgcct cctccttccc 8760gaaatcgctc gacacgatct tcggcggcgc
cgaagtgctg tcgaagatgc tctccgaggc 8820caccgacggc aacttccaga
tccaggtctt ctcggcgggc gagctggtgc cgggcctgca 8880ggccgccgac
gccgtgaccg agggcaccgt cgaatgctgc cacacggtcg gctactatta
8940ctggggcaag gatcccacat tcgcgctggc cgcggccgtg cccttctcgc
tgtcggcgcg 9000cggcatcaac gcctggcact accatggcgg cgggatcgac
ctctacaacg atttcctcgc 9060gcagcacaac atcgtggcct tcccgggcgg
caacaccggc gtgcagatgg gcggctggtt 9120ccggcgcgag atcaacaccg
tggccgacat gcagggcctg aagatgcggg tcggcggctt 9180tgcggggaag
gtgatggagc gtctgggcgt cgtgccgcag cagatcgcgg gcggcgacat
9240ctatccggcg ctggagaagg ggacgatcga cgcgaccgaa tgggtcggcc
cctatgacga 9300cgagaagctc ggcttcttca aggtggcgcc ctactactac
tatcccggct ggtgggaagg 9360cggcccgacc gtccatttca tgttcaacaa
gagcgcctac gaggggctga ccccggccta 9420tcagtcgctg ctgcgcaccg
cctgccacgc ggccgatgcg aacatgctcc agctctacga 9480ctggaagaac
ccgacggcga tcaagtcgct ggtggcgcag ggaacccagc tcaggccctt
9540cagccccgag atcctgcagg cctgtttcga ggccgcgaac gaggtctatg
ccgagatgga 9600agcctcgaac cccgccttca agaagatctg ggactcgatc
aaggccttcc gctccgagca 9660ctacacctgg gcgcagatcg ccgaatacaa
ctacgacacc ttcatgatgg tgcagcagaa 9720cgccggcaag ctctgagccc
gagcgccgcg cgaaagagga ccccggagcc gcgttccggg 9780gtcttttcat
gggcgacagg ggccggcgcg 9810361886DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number L23760 36tgagtgtcta
ttttttttcg ggttttttta agtgtgaatc acatggttag gagcagttgt 60cttcaatgtg
accaaccatc ccaaggctct aattcaacgt ttgggtgtgg gggcccgctg
120gcagctgtgt gtgccactgg gctgttggtg ttggtgcttt actccccctc
atcgcaaacg 180gctaattggt cggcacaggg tatttccaca aaggcgctgt
atccggcagt gcctgtgcct 240tccactctgc tgcctggaag cgcgcctgcc
aaacaccagc tgcatgtttg gagggcacat 300gcgatgtcgg aggccacaac
aaacaattca ttcaaacagt cattatttgg gtacaatgcc 360atctcctcca
tttggcttca actggctggt gtggccgcca ctttctttgc atttggagct
420ttgatggcag ctgtaacgca acgcaaggag atcgccgtct tctccgcctc
gggtcaggct 480gctgagccgg agggggcgga gcccctgaag cggccttttc
cgtctcctgc tgccaaacct 540aagccgctct tctccacccc ggcaaattcc
ttcagcaaca tcttccaggc gcctccatcg 600ctgcgcacgg actccaccta
tggccgaggc ccgcgctcga ccagcttcac cgacatcagc 660aactggccct
ccaacaacgc actccgcaac ccccagtcgg tgattgacat cgggggagga
720gtcgacttcc tgggggacag aagccctgga aacccgttca cgcggctgcg
ggggtccccg 780agctccaccc tcagcaacct cggcatgggc ctaggcctgg
ggctgggcaa gggcaagggc 840ttcggcaagg gcttcggcaa aggccggggg
ttccccgtgg aggaggaggt ggaggaggag 900caggaggtgc tgtcgtgggc
cgaccgccgg cgggcgctgg cggaccccga cgccccgccg 960atgaacgagg
acatcaagta cccgcagctg cggctggtgc gggccgtgcc gggcggccgg
1020gacgagaagc tcggtgtgat gtcgaggcag gaggcgctgg agctggcgga
ggcggaagac 1080atcgacctcg tcctcgtcag catcgacacc gaccccccgg
tggccaagct agtcaattac 1140tcgaagttga agtacgagtc cgagaagaag
aagaaggaca gccacaagaa ggggaaggtg 1200aaggaggtga aggagctgaa
ggtgtcccat aagatcggcc agcacgacta cgacgtccgc 1260gtgaagcagg
cccgaaagtt cctggagggc ggccaccgca tcaaggtgtc gatggagttc
1320aaggggcgcg agaaccagtt cgtggagatc ggccgcgcgg
tgatgaagcg cttccagaac 1380gacctggcgg acatgggcaa ggcggacgcc
gtgcccaaga agctcggcac ccggctgatc 1440ctgaacctgg ccccggccgg
ggaggcgctg aaggtgattg cggagcggag ggcagagcgc 1500gacaggaaag
ccgcggctga ggaggagggg gagggcgacg acctcgactt cgtggacgag
1560aacgaggacg aggatgtgga gggggagggc gaggaggaag aggccgagga
gctggaggag 1620gagacagcgg aggggacgga ggtgccaacc cgcagctgat
cgccgatccg cgggggacag 1680ccacctcccc cccggcctcc ctgccggggg
ccggcaccat ccgtcgttgc ggtgcggcgc 1740tgccatcaac ggccgtcctt
gagcttaatg ctcccgccct ccgttggccc gcggcggtcg 1800ccaggttgct
ggcctggctg cccgcagctc ctcccctccc cgactgacac agtgtggatg
1860accgtgatgt gcgccttttc gccttc 1886373015DNAUnknownDescription of
Unknown Organism GenBank Clone Accession Number Z46970 37ccgctcatct
ccaggcctcc ctgagtgcgt acccgagagc ggcaagtaga gaaaggaaca 60cagatacagc
accatggcct ctaggctcgt ccgtgtgctg gcggccgcca tgctggttgc
120agcggccgtg tcggtcgacg cgcgcttcgt ggtgcgcatg gtgcaggtgg
tacaccgcca 180cggtgcgcgc agcgcactca tcgacgacaa cacgacggag
atttgtggca ccctgtaccc 240gtgcggtgag ctgaccggcg agggtgtcga
gatggtccgt gctatcggcg agtttgcccg 300cagccgctac aacaacctct
cattggtgga gagccctctc ttcccgtcga cgcggtacaa 360ctcctctgtc
gtgcacacac gctccaccca cacccagcgc accatccaga gcgcgaccgc
420ctttctgcgc ggcctcttcc aggacgacta cttctacccg gtggtgtact
cgaccaacag 480aacgaccgaa acgctgctca gcactgacgc ggtgccgtcc
gtggtgggcc gtagctggct 540cgacaacccg gcgctgcacg ccgccctcaa
cccggtgatc gatgagcacc tcagctggga 600cgccatccag agcgctgcca
aggacgcatg ggtcgagggc ctgtgcgcgg actacaacgc 660ccgcaccaac
tgcgtcctcg acatgtacga cgtggccgcc gccttcgagg ccgccgggcg
720tcttgacaat gccaccaatc tcaaggcggt gtatcccggc cttcaggagg
tgaacgccgc 780ctggttcaag tatgtcttca gctggaacca cacgagcaag
ctcgatctca cgcagggctc 840cgcctcgcag aaccttgcgc agacggtgct
ggccaacatc aacgcccacc gcctctctcc 900gtcgtacaac atgttccagt
acagcgctca cgacacaacg gtgactccct tggctgtcac 960gttcggtgac
cagggcgaga cgacgatgcg tccgcccttc gcggttacca tcttcgtgga
1020gctgctccag gacaccgcag atgccagtgg ctggtacgtg cgcctcatcc
gcggcaaccc 1080tgtgaaggca gccgacggca cctatgtctt ccaggagtct
ggtatcaagg catactgcat 1140cgatgaagcc gggaacaagt acctcgcaca
caccggcatc tgcccgctga atagcttccg 1200ccgcatggtc gactactcgc
gccccgccgt ggctgacggt cactgcgcca tgacacagac 1260tcagtacagc
aacatggatt gcccgcgcac tatcgcggac aacaagccgg tgccgtcgcg
1320ctgctggctc taccgccacg tttgccctag caaggcatgc ccggacagct
acattctctc 1380cgcggtcgac caccagtgct accccgggcc cgacgttacg
aaccccacca gcagcagcag 1440cagcgagggt accaccacca gcagcagcga
gggtaccgcc accagcagca gcgacgttac 1500caccaccagc agcagcgagg
gtaccgccac cagcagcagc gacgctacca ccagcagcag 1560cgagggtacc
gccaccagca gcagcgacgc taccaccagc agcagcagcg acgctaccac
1620caccagcagc agcgagggta ccaccagcag cagcagcgac gctaccacca
gcagcagcga 1680cgctaccacc accagtagca gcgagggtac cgccaccagc
agcagcgacg ctaccaccac 1740cagcagcgag ggtaccgcca ccagcagcag
cgacgttacc accaccagca gcgagggtac 1800cgccaccagc agcagcgacg
ctaccaccac cagcagcagc gagggtacca ccagcagcag 1860cagcgacgct
accaccagca gcagcgaggg taccgccacc accagcagcg acgctaccac
1920cagcagcagc agcgagggta ccaccagcag cagcagcgac gctaccacca
gcagcagcga 1980cgttaccacc accagcagca gcagcgaggg taccgccacc
agcagcagcg acgctaccac 2040cagcagcagc gagggtaccg ccaccaccag
cagcgacgct accaccagca gcagcagcga 2100gggtaccacc agcagcagca
gcgacgctac caccagcagc agcgagggta ccgccaccac 2160cagcagcgac
gctaccacca gcagcagcag cgagggtacc accagcagca gaagtgacgc
2220taccaccagc agcagcgagg gtaccgccac caccagcagc gacgctacca
ccagcagcag 2280cagcgagggt accaccagca gcagcagcga cgctaccacc
agcagcagcg agggtaccgc 2340caccaccagc agcgacgcta ccaccagcag
cagcagcgag ggtaccacca gcagcagcag 2400cgacgctacc accaccagca
gcgacgttac caccaccagc agcagcagcg agggtaccgc 2460caccagcagc
agcgacgcta ccaccaccag cagcgacgtt accaccacca gcagcagcag
2520cgagggtacc accaccagca gcagcagcag cagcagcaaa agcacaagtt
catcggatgt 2580cccttccttc aaaaagcccg cgaactggag cccgcgcgtt
ctctcgccgg aaaggggccg 2640ccacattgcc ggggacatca tccgccgcgt
gacgaacggt gttacgatcg gtgcgggtgt 2700ccgaaagcac gatgagtaca
gccggcaccg ccaacagtag cacaacggca tgtaactctt 2760ttgtgcatgt
ttgaatggag aggaggcttc tgtacagcgt acattgtttc gagaaggtat
2820cacaaccgct cgtttcaccc ccgtcatctt ttcattttga tctccgtcgt
ctcatactgc 2880ctttgtgggc tctctctggg tgtgggcgct tgtgcgtgtg
tcgctgtaaa gtcgttgacg 2940ccatcgctct tacctgtggg ctattttttt
aattatggtt tattattact tccctctctg 3000cgcgtccctc tgcag
30153838186DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number AC004449 38gatccttcct gcctcttccg gcgtctgggg
ctccaggcgc ccttggcttg cagattgatc 60tccctgatct ctgcctccat ctgctcacag
ccttctcccc tgtgtgtctc tgtctcttct 120tgtaaattca tccgtcgttg
gatcagggcc cacccggttc ctcgtggcct cgccttaact 180gggccatgtc
tgcagagacc ctatttccac ataaggtcct attcacagga accgggggtc
240aggatgtcag cctgtctttc tgggagatgt agttcaaccc acaacacaca
tcaaacagtt 300attgagcgcc gactgcgtgc cctgccgtgt gcttgaaggt
cccaccctca ggaagcgggg 360cctagggatg gcggccgtga tcacgcaggc
agcagagagc agctctggga agcggggagg 420gacgaggacg gggaggcgac
atcagcaagg ccgtgtgtga gccaggcagg gtgtccccgg 480tgtagcacct
ggctcgggca gaggccccga ggaggggctg gaggagctgg gcgaggaggc
540gggcaggacg ggcctgacac tagggacctc gggccccggg aatgcctctg
ggggggcgtg 600tacacccgtt gctcccagga ggcacacact gcggttcgct
tcgccaagaa tgtttaattg 660catttgatga ctacggtttc cattcattca
tttgtagaga tataacactc agaccacaaa 720atgcataaaa tgcggtggct
tttagtatta acagagtgct gcacccgata ccacagcctc 780actccagaac
attctcatgg gcccaaaagg agacctgggg tgttagtcac cagctcactc
840cccgtcccca gcccctggca acccacgcta cttagtcatt atttaggtgt
ttaggagttg 900caaagtcaaa tctttaaacc cacatatggc caggcgtggt
ggctcacgcc tgtaatccca 960gcactttcag aggccgagac gggcagatca
cctgaggtca ggagttcgag accagcctgg 1020ccaacatggt gaagccccgt
ctccactaaa aatacaaaat tagccgggcg tggtggtggg 1080cgcctgtaat
cccagctact ctggaggctg agacaggaga atcgcttgaa cccaggaggc
1140ggcggttgca gtgagccgag attgtgccac tgcactccag cctggacaac
agagcgagac 1200tccgtctcaa aaaaaaaaaa agtaccaaaa agtgccccag
gtcataaggg cacagctcga 1260tagctggtcc ctaaagggaa cgtggtgtaa
ccaccacaca gaacgaagct ggaacgttcc 1320tgccgtcctt agaagctgcc
tttgctaagg ggaattgccc tgacttccca caccattgat 1380tcatctccag
acccttggtt ttcatgttga tttttcaaaa atcacctgat agtctgaccg
1440aatgtagctt tccactggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
tgagagagag 1500atggagtctc gctctgtcac ccgggctcca gtgcagttgt
gtgatcttgg ttcactgtaa 1560cctcctcctc ccgggttcaa gagactcgtg
cctcagcctc ccgagtagct gggattacag 1620gcacccgcca ccacacccag
ctaatttttt gtatttttag tagagatggg gtttcaccat 1680gttggccagg
ctggtctcga actcctgaca tcaggcgatc cacccacctt ggcctcccag
1740agtgctggga ttacaggtgt gagccaccac gcccggcctt atttttcccc
cattttcttt 1800tttttttttt ttgagtcagg gtcttgttct gcgctcaggc
tggagggcag tggtgtgggg 1860atcacggctc actgcagcct cgacttcctg
caccaccacg cctggctgtt tttttttttt 1920ccggtagaga cgggggtctt
accgtgttgc ccaggctggt ctagaactcc tgggctcaag 1980cgatcctccc
gcctcggcct ccgcaaatgc tgagatcaca cgcgtgagcc cccgcacccg
2040gcctcctttc caccgctctt gtctacagcc gcccctcctg gtccgattgt
attggcagat 2100gtcgccaata cggtgtcaaa cggcgaaggg gcactgagcg
ttttttcttt ctcccgtcct 2160tggcggcagc agctcggttc cggctacggg
gctgagcccg tctctcagac gaggaaactg 2220gggtccgaga ggtgagccgg
tcccagaggc agggcgaggg ggaagcggga gtggggtccg 2280cagcggaccc
agccctgcct cccccctgca ggagatcgtc aacttcaact gccggaagct
2340ggtggcctcc atgccgctgt tcgccaacgc cgaccccaac ttcgtcacgg
ccatgctgac 2400caagctcaag ttcgaggtct tccagccggg tgactacatc
atccgcgaag gcaccatcgg 2460gaagaagatg tacttcatcc agcacggcgt
ggtcagcgtg ctcactaagg gcaacaagga 2520gatgaagctg tccgatggct
cctacttcgg gggtgagctt gaggggggcg cgcctggagg 2580gggagggggc
acgcgacccc cgcggtgtgc agagccaggg ggccggggcc ggggccgggg
2640ccggggatgg ggatggggat ggggatgggg ccggggatgg ggatggggat
ggggatgggg 2700ccggggatgg ggatggggat ggggccgggg atggggatgg
ggccggggat ggggccgggg 2760atggggccgg ggatggggcc ggggccggca
ccagggagag cctgggtggg aagcgcccac 2820gctggccaag gtgcagaggc
cgggccgtgt gcctgggcgg ggagggccgc ggcgcccgcc 2880tcgtccagca
acccccccct gcgcgccacg tgcagagatc tgcctgctca cccggggccg
2940ccgcacggcg agcgtgcggg ctgacaccta ctgccgcctc tattcgctga
gcgtggacaa 3000cttcaacgag gtgctggagg agtaccccat gatgcggcgc
gccttcgaga cggtggccat 3060cgaccgcctg gaccgcatcg gtgagcgggc
cgggggcgtg gccggggcgg gtgccctggc 3120gggggagggg cgtggccaag
gcatcaggag agtggcttgg acagtggcag ggggaagggc 3180gtggctgtgg
catcaggggc acggttgggg cagagacgtg gccaaggcat caggagtgtg
3240gccatggcag caggggcgtg gctggggcag gggcagcggc tggccgctcc
taggacccct 3300ttgggtctag aggctgattt tctgacctat tgtcctactt
cagccagagg cagcctgttt 3360cccaagggag ggaatgcaca gggtgtttgc
ggttgtgccg aatgctcggt gagcacctgc 3420tgtgtgctgg gggtgcaggg
gacagacccg ggggcccact cagactccca gggaggctta 3480tggactggtg
atgaaatcac acacgactgg gctgtgtgcc agcagggcag gtggggccgg
3540tgggcttccc tgagttggga atgcagagtg gagaccaggg taagggatgc
catgtggaaa 3600cggggaggaa gatgtgttcg tggagtggac acagcacatc
ccaaggccct gaggtggaaa 3660agaggcctag agtccagaga gccagggagg
cctggaggag gttggggaag aaggggaggc 3720cagacacaca gggcccagtg
ggcggcaggg agagtttaga ctaaatcagg agcatcaggg 3780agccatggag
ggttctaggt gggcggagga cctggtcaga ttgtatccgc caaggcgggc
3840cgtgtccagg agggagacgg tgacctggcc tctcaggggg gcagtctctg
gggcagggag 3900cggcagagcc ctgatgactg gatgtaggcg ccagagagat
ggcggctcat gctgctgttc 3960gtgggaatgg gaatgaagac catggctgaa
acgcaggaca ggtgcgacgg agtggtgtca 4020gggagctccc tggtgtacag
taggaagctc tccacaactt gctctataca gtgagtatgc 4080aacccgttcc
tgagtatcag gtgcttaggt tataacttct gtatacagca ggtgctcagc
4140acaggctgtg tacaggcagg tgttttcggt atgcctgtgg cacactggag
gcagtcatta 4200cataatcagc gtatacaggt ggtacacatg catacttggt
gcacagtgat acctgctcca 4260tgtacacagc aggcattaaa tacctgttta
ctgccaggcg cggtggctca cgcctgtagt 4320cccagcactt tcggaggcca
aggtgggtgg atcacgaggt caggagattg agaccatcct 4380ggctaacatg
gtgaaacccc gtctctacta aaaaaaaaat acaaaaaatt agccgggtgt
4440ggtggcgggc gcctgtagtc ccagctactc gggaggatga ggcaggagaa
tggtgtgaac 4500ccgggaggtg gaccttgcag tgggccgaga tcgcgccact
gcactccagc ccgggcgaca 4560gagcaagact ccgtctcaga aacaaagcaa
aacaaaagcc ctgctttctg tatgcaggtg 4620cttcatgcat gctggctgtg
catagcaggt gctcagcctg tatatggcag gtactcaata 4680tccatactat
aggccagaga tgctacatat gtgcttattg tatacagtag gtggtaaatg
4740catgcttgct ctacacggca agcactgtgt gcgcacccgc ggtgcagagt
aggtgctcgg 4800tgcccgctgt acgcagcagg cgctccctgt gcacacgcta
acgccccctc tcccgcaggc 4860aagaagaatt ccatcctcct gcacaaggtg
cagcatgacc tcaactcggg cgtattcaac 4920aaccaggaga acgccatcat
ccaggagatc gtcaagtacg accgcgagat ggtgcagcag 4980gccgagctgg
gtcagcgcgt gggcctcttc ccgccgccgc cgccgccgcc gcaggtcacc
5040tcggccatcg ccacgctgca gcaggcggcg gccatgagct tctgcccgca
ggtggcgcgg 5100ccgctcgtgg ggccgctggc gctcggctcg ccgcgcctcg
tgcgccgccc gcccccgggg 5160cccgcacctg ccgccgcctc acccgggccc
ccgccccccg ccagcccccc gggcgcgccc 5220gccagccccc gggcaccgcg
gacctcgccc tacggcggcc tgcccgccgc cccccttgct 5280gggcccgccc
tgcccgcgcg ccgcctgagc cgcgcgtcgc gcccactgtc cgcctcgcag
5340ccctcgctgc ctcacggcgc ccccggcccc gcggcctcca cacgcccggc
cagcagctcc 5400acaccgcgct tggggcccac gcccgctgcc cgggccgccg
cgcccagccc ggaccgcagg 5460gactcggcct cacccggcgc cgccggcggc
ctggaccccc aggactccgc gcgctcgcgc 5520ctctcgtcca acttgtgacc
ctcgccgacc gccccgcggg cccaggcggg ccgggggcgg 5580ggccgtcatc
cagaccaaag ccatgccatt gcgctgcccc ggccgccagt ccgcccagaa
5640gccatagacg agacgtaggt agccgtagtt ggacggacgg gcagggccgg
cggggcagcc 5700ccctccgcgc ccccggccgt cccccctcat cgccccgcgc
ccacccccat cgcccctgcc 5760cccggcggcg gcctcgcgtg cgagggggct
cccttcacct cggtgcctca gttcccccag 5820ctgtaagaca gggacggggc
ggcccagtgg ctgagaggag ccggctgtgg agccccgccc 5880gccccccacc
ctctaggtgg cccccgtccg aggaggatcg ttttctaagt gcaatacttg
5940gcccgccggc ttcccgctgc ccccatcgcg ctcacgcaat aaccggcccg
gcccccgtcc 6000gcgcgcgtcc cccggtgacc tcggggagca gcaccccgcc
tccctccagc actggcaccg 6060agaggcaggc ctggctgcgc agggcgcggg
ggggaggctg gggtcccgcc gccgtgatga 6120atgtactgac gagccgaggc
agcagtgccc ccaccgtggc cccccacgcc ccattaaccc 6180ccacaccccc
attccgcgca ataaacgaca gcattggcgc caagcctggc cgcgtgtgat
6240tgcccgagac ccgcagggcg tgcacccttc ctgaagacag tggctcctgg
gggtggcaaa 6300agagctttat ttacacactg acaaggctca cggggtgtca
gctgaagaag taggtggaac 6360gcttcacctg ctccaggtcg aaggcccctg
cggaggaagc agagcggacg gcgtgggtgg 6420cgggaaagcc ccgccctggc
ccgcagttcg agccaccctt gcgaggctgc ccacccgcct 6480acctggcttg
ggcaccgcct gcagtgtctc cttcagctgg ctggcctcca agatcttctg
6540gggcctgggg ttggaagcag ggtggggtga ggctgaggcc aggttttggg
gtgggggggg 6600aatccaggta gttggggtca gggagcgcct tactcagagc
agaaccgctt gaccaggaat 6660ctggacaggt cctgcaggat gggctcgctg
tgcaagcgga caaactgctc ccggcacacc 6720tgggcaggag tcagaggatc
cccaggggtg atcaggcagg ctctgggcac cacccctacc 6780caacgcccca
gtgtgggggc cccacccatg ggtggactga ggctcagact acgggggcac
6840ctggttcatg acggagacat cagctgcgtg agtccagtaa cagtcgtgca
cagagacgaa 6900ggtcaggccc ttcctgtggc agagcggagg actcctgaag
ggaggggagc tcacagggcc 6960acccagtgac cagcatcctg gccctgcgct
cagccccctc cacttgaggt ccagggaagc 7020ccaccctcct gcaggcctcg
ccccacccct cgccccgccc ctccccaaac atcctgggtt 7080aggtatcagt
acagggggag gaaatgttcc cagaagcctc ctcgccccac ccctgccgcc
7140ccccacgctg ctgtgggagc ctcagctccg agggcggcta cgaggtcccc
tcctgccagg 7200gccaccaccc cgcatcctga gcattcccag ctcccgtggc
cggtagattc tgctggaacg 7260acctccacgt gctccagatc taaccacaca
tcgcggtgcc aagaaatgcc cagcaggaag 7320gggcagcgcc catgctcggc
tctccctgtc gggccacagg aggggagctg ccaggaccac 7380ctacattcgg
ggcacacagc ctcagggcct ctacacaggc cccacagaca cagcagatcc
7440actctgccca gtccctgccc ccagctagac ccagccttgc cagctgtgcc
ctgctagcca 7500gaagacgccc ctgggaggcg agcggcaccc acgccgtccg
gagacgccca cctgtagcag 7560tgcagggcgg tgagcatcat gtgggaggag
tccagcgagt ggatgaagtt gggcgggaag 7620ccgttcttct gcttacgtgt
gttgggcttt ctgaggacgg aacaggtgcc ggtgggggcg 7680gcccagggac
acccctaact ggccgctgtc tccaccgtgg ctgctctcca gacccccggc
7740caggccccag cccgggcccc ccactcaccg gctgatgtct ccgttgtggg
tgtaggtgat 7800gctctgaatt ccacctccta tttgctaaaa aggggaaggg
gccggtgagt cccacccgag 7860gcccagcacg gtggtggtac attgaggtgg
tggtacactg gggtagtggc acgctaggat 7920ggtggcacac tggcgcggga
tggggtggca cactggggcg gtggtacact ggggtggtgg 7980tacgctgggg
cactggtaca ctgggacgct gttacactgg gatggtggca cactggggag
8040ggatggggtg gtacactgac cttgaccttg gagtccaggc gatagggctg
gatgacgggg 8100acgcccaggg gtgtgaccca ctccaccaca gagcccatgt
gggagatgag gcgggcactc 8160tcggtcagcc agtgctgtgg gacacaggcc
gtctcagggc agggggctca ggccggggat 8220cccgtccact tgcttaggga
gtcctggccg agcggggaca ggacaggacg tacctggatg 8280gcccgggtcc
ccgagaacat ctcctgtaga ctcttgaaga cctggcgtac gagatagtga
8340gaggcctccc acacgaactc ctgcagaggg cgggcagcag gtgcaggtcc
tcaggggctg 8400gcccgttcac gccctactcc cccctatttc agagccactg
aggcccaagg cctagggcct 8460agcagggggg caggggaatg gggcctggcg
cccacgcagt cagcaagaaa cgcccaagcc 8520ctaacaggca gccagtggtc
tgggggagca gccagggctc ctgctgggag gctgggtcgg 8580gggcacaccc
gtctgagttt taaatggcag tgaaaccaac gtgttcgcag cgcgacatgc
8640ctggcgcacc tggggaaagt cgctcagctc ccggaggcgc ttctcaatct
gcaggcgccc 8700gccatagcgc gtgaccccgt acaccaccgt catcaccgtc
tgcttcacca ccttgcgggt 8760gatgaaacct tccagcacct gtgccacccg
catgccccgc tgggcgtcct gcctacggaa 8820cacctccacc tgcacggcgg
gtgggccggg ggcgcgggtc agccccgcta gcagcccagg 8880ggccaccaag
cacccatgaa gcccccgccc cagccccacc acatcctcag gacaggccaa
8940ggtgagggca cctggggccg agactcaggg ctcacattgc ccccacgccg
agatgccccc 9000gggcagcagg gcacacccta cctgcgcggc cacgccgctg
tacacgtcct gcggcacatc 9060cgagggctcc aggttgacgg aggcggcgcc
cacgctgtcg cggcccagag cagcataatg 9120ctgcaggccg ttgcaagagc
cgtcctgagg aaggggcggc aaacgggaga tggaagctag 9180agaggcagag
acgtgtggga ccccaaacca ccccccaggt cgagccgttc ctagggccgt
9240gcacccccca gccaagtgca ccggagcccc cgcacgctcc cgggagagac
caggagccat 9300ggctcccgca cactctagga ccacctccag agaataccac
gagcgaaggt gaaatctcac 9360accctcaagt cgagccccag gcccagtgca
cactgcacgg cctcgggggc cagacccagc 9420tggctcacct gatggacggg
gaggtgggag acataggcgg cagggtcgga ggcgcgcaca 9480gcgttcgcca
cctccataca gcaggccagc gtctgccagg gttcctccgc gcccatccac
9540cactttcggc cctgcgggga cagcggatgg ggggcagtga ggcccgggcc
cgatccctga 9600gcccgctggg aggctgtgtt gcggggaggt gggaaatggg
gaggagacgc acacccgtga 9660tagtgaacac gggacgcatg tgggcgagag
acggggcggt ggctggatga gttctccata 9720gccacggatg gaggatggga
gctgcgggtg gaccgggctg aaacaagcgt gtccggagct 9780gccgggggag
gagggtggac agaggacctg ggggcgccgg gggaggaagc agctcggcgg
9840atgcagggga ggggggaacg tggggaacgc gggggccctg gggcagggga
gaagggagaa 9900gcaggacggg cagggggcgc gggggaggag agcgggcggg
ggacgtgggg gcgccagggg 9960agggggaggg gaggaggaag acgggcaggg
ggcgccaggg gagggggagg ggaggaggaa 10020gacgggcagg gggcgcgggg
gcgccggggg agggcgcggg ggcgccgggg gagggcgcgg 10080gggtgccggg
agggcgggga atgcgggggc cccgccccta ccgtcaaggg ttggtccgcg
10140gagtccagga tgtcatccat cacctcctcc gcaaaggcca ggcgcttccg
cagcggctcc 10200cgcttcttca accccgtgag attgaccagg tggatcttga
gccaatccag gccgtgcggg 10260ccgagcgggc ggccctgggc gaactccagc
agggcccgcg ccacgtcgct gcccaggtgg 10320ttgaagtgcg gcgggcaggg
gtaggtgcgg ccgcggaagt ccatgttgtg cggcagccag 10380aagacgcggt
cccgcaggtg ctgcgccagc gagaggcggt acagcgcctc cgcccgcagg
10440ctgtgcatct cccgggccac cttctggcag tgcgccagct cacggcgcag
ctcggccttg 10500cgggcgggcg cggcgctgtg cggcaggtgg gcctcgggcg
gctggggcgc ctcggagggc 10560ggggccggca cgcctagctg ggggcagccc
ttggcctgga agagctgcag caccaggtcc 10620agcacgcgcc cgttgacgcg
ccaggcgcag ttgcccagtt gggtgagggc gtccagtgcg 10680ccatgcagcg
cggtgggcgg gcaggtttcc agcagctcct ggtgctgcgt ggcgccttcc
10740accgtgcgca tcagcttggt ggggctgagc aggaaagcac cagagtgcgg
cgatgtccag 10800ggcagcgggg ggcaaagcat gggtacatcc accgcctcga
aggtcagcgt gggctccgcg 10860gccttctcca gcagctgcac gtaggccggg
tgcggcttca ggatgccgat ctggggtgcg 10920acaggcagac gggtcagggc
cccggtgctg gggctttcct gttcccaccc cttaaacttg 10980ggtgagaggg
gccggctccc cggccaacaa gaaaccagtg tggcctccca cgaacagaag
11040ccacctccag aaacggccgg acacctgcat ggacacccat ggtgtgtccc
gagtcctggg 11100aggtactgac ggctgcgctg agatcaaggc tccgcccaaa
ggcgccaacc ccatggggtc 11160cctggtcctc ccagcgggat gccccccagc
tcaggagggc actgcctggc acctgctgga 11220cgttgcggaa ggaatacacg
tggtagagca cggggacaag
ccgagaggaa cgatgcggct 11280tgtccaggct gcatggcatc tgcgtagcct
gcaccagcat ctccgccagc agcttgccca 11340gctccatctg cactggcagg
ggccagggct gctcccgcag ggcctcgggc gcccccagct 11400cctcccagta
ctgccgcggc aggcagggct cgggcacctg taggacaggg cggtcagggc
11460gctgggcacc ggggcccctg agctagatgc cccaccgccc gtgcctgacg
cccggtgggg 11520catctgtcag cccaagcata cagatgaaca gactgaagct
tgggtgcaaa cccggctgct 11580ccagggaggg agagcgccca cccaccactg
gccccagcca ggaggagagg gggtgcgagc 11640ctcacctcgg cgtcggaggc
cagcaagcag aggtacttcc tgtagtggtt ctgcagcgcc 11700tgcacctggc
cactgacccg ctgcctctgc accacgtgcc ggctgaaagt gcgcgcactc
11760agctcccggg ccagggtggt gaaggactca ccttgggcgg gcagcgcctg
caggacctgc 11820ggaaggcagc cgtgagtgcc tgcccgcccc gcccggggac
ccggccgcgc ggaggaagac 11880gcacctgcag gagcatccgc accacctcgc
gctcgtccag caggcacagg aaggggtaaa 11940gtgagaaccg gccctcgtac
acctcgcgct ctaggcggtt cttggtctcc cgcagcgccc 12000ggcacagtgc
tttctcccat tggtcccgca gggtcttcag ggtcttccgc tgcgggggat
12060gaacgggccc ggtgagcccc gtggcagctg gtgggaccca ggctcacagg
acgggggtca 12120ccgcagctcc ctgcagagac ctcatggccc tcaaggtccc
tgctgtgtgt tccgggtagc 12180tcctcacccc ggcctgccct ctgccggctt
cagcgtgcct gacgcagcca agagcaaaag 12240cccagctgca gtgtgcgcag
aagcacaggc caagacccaa cctcgggacc ccacaagttt 12300tccctgagcg
gcagccaggc tgagttccta ggccctgcat gaccagacca gggcatgagc
12360aattcaaccg catacacgga gctcagcccc tgcggcggac acgcgacccc
ggctcagccc 12420ctgcggcgga cacgggaccc cggctcagcc cgtgcggtgg
acacgcgacc ccggctcagc 12480ccctgcggcg gacacgggac cccggctcag
cccctaccgc gtgcttgacc tccttgcttg 12540gcaacgtggg cttctccacg
gacaccacgc acaccctgct ggccagctcc atgtggagct 12600gcttctcaaa
gaggcactgc agggtcttca agggcaggtg cagcttcggg taggacacac
12660gcccatcctg cagggatggg ggtagtgagg ttgggggctt gccagagggc
gacctgccct 12720cccaggaccc cgagacagca tgggtgcacg cgtttctgcg
tctcctgcaa gttgctggtg 12780gctatcgctg acgcggggaa aggcgggctg
cgggtaaagt cagtgccagc agtgcaaacc 12840aaaggccttg accctcctgg
cctcgacccc tctagaaggg acactgggca ccgtgcaggg 12900ggtggcaggg
gcggtgatgc tgggagctgg cagagcctgg ggagaccgtt cactgcaccc
12960ccagatgttg gctgttttct cctcaaactc agaactgtat gaatgtgacc
catccagaaa 13020tagatgaatt aaaaataaca actaaagcct agcgctttga
gaatcaaaga cgcacgtcca 13080cataaaagct tgtacacaaa cgttcacagc
tgcatgactc gcagtcgata agtagaaaca 13140gcccaacgtc ccataaacgg
acgaacagac gggcacggcg cggccatcca cgcaccggag 13200catgactcag
ccctgaccca ggtcgcctcc cggaggcacc atgaggacgt cacgctcagt
13260gggagatgcc aaacacaaaa ggtctcgcag tgtgtggtcc catttctatg
gaatgtccag 13320agcagactca tccacagatg gggaggggat ggggagtgac
ggggatgggg acgaggcttc 13380cttttagggt gatggaacat tctagaatta
gacaaccgtg actacactaa aatcgctgaa 13440ttacaccttt aagagggttt
tatggcaggt gaattacacc tcagtaacag acgagcccac 13500tgcgtgcacc
tggcagcccc actcaaacgc actgctctcc tgtcacccca ccctctctct
13560gcggcccccg accacctcgt ccccctgagc ccacaccctc agggccaaga
ccctcccagc 13620tctgggtcct cccatcttct cagaggagga agggaggaat
tcagggccca gcccaggtga 13680gccctgggca ccggggaggc ccattggtct
gagctgaggc tccaggaacc cccaaagggc 13740agctataagg actgaagtct
gccggggccc acgtgggctc accttggcat acacgtccct 13800gagcagcttg
gaggtgttga ccgggggcgg cagctgcggc gggaggctga aggtgggctt
13860caccttgtgc acggccttca gaacagtggc ccgatcctcc tcagacagca
gaacggcggt 13920gaagagtgcc tgcagcttca gcccctcctg gctcatctgt
tccagacacc tgtggtgcag 13980gcggcctgct cgagggacgg gccagcccca
cgctgggctt ccacagaccc caggggaacc 14040tcgtgaccac ctcctgctag
cctgcaggtc tcggtgtggc tgtcaggccc tctgggggtc 14100cccagccccc
agcccaggca ccgtcccaga tcttaaaacc ctgggaggga catggtgggg
14160ggtgggggcc ctcccgacac cacctacctt tcgatggtcc cggcgtcctg
gtcctgcctc 14220cccatgcact ggagggcagc cgcataggac agcaggtccg
gagtcaagcc ggcatccttc 14280accatgaata acacatatac cagctccttg
aaggcaccct gggagaccaa gccagggtga 14340gggtctgggg ggatggccca
acctccacat cctccctgct ccctggagac cccttctctg 14400tagccaccag
ctcagcaggg gacagggtca ccaggcagga gtggccagct gggcagaccg
14460atgcatcccc ctgaggttct gacacacaag ctccacctgc agaggcagcc
gcatggcccg 14520ccaggtggga ctgtgggagg ttcacgttcc tctgggaggc
agcttgttaa acctccagat 14580ttgtcaattg tgtggatctt ttcaaaggac
tgacttggct tgactgttct ctgctgtttc 14640tgccttccat ttcatcgatt
tgttttaatc tttgtaactt cctctcatct acttgcttta 14700ggtttagtga
cagcttcttc ttctagtttc ctaaggtgaa aggtgacgta tttggtctga
14760gatgtttcac tttttttccc cccaagatgg agtcttgctc tgttgcccag
gctggagtgc 14820agtggcacaa tctcagctgg gccgggttct ctgcctccca
ggttccagca cttctcctgc 14880ctcagcctcc tgagtagctg ggattacagg
cacacgccac cacaccagct aattttttgt 14940attcttagca gatacggggt
ttcaccatgc tggccaggct ggtctcgaac tcctgacatc 15000gtgatccgcc
agcctcagcc tcccaaagtg ctgggatgac aggtgtgcac caccgcgccc
15060ggccatcacc tttccgaata taggcatttt gtgactataa attaccctgc
gagcactgtg 15120tcagctgcat cccaggactt ctgacaggtg gtgttttcat
tttcattatc tccaagtgtt 15180ttcgaacttc atagtttact tcttctttgg
aaattttatt taattatttt tttagataga 15240gtctcgctct gtcgcccagg
ctggagtgca gtggcgcaat ctcagctcac tgtcaacctc 15300cgcctcccgg
gttcaaccga ttctcctgcc tcagcctcct gagtagctgg gactacaggc
15360acatgccacc acacccagct aattattttg tatttttagt agagatgggg
tttcgccctg 15420ttggccaggc tggtctccaa ctcctgacct caggggatcc
acccgcctcg gcctcccaaa 15480gtgctgggat tacaggtgtg agccaccacg
cccagccatg tatagcttaa atatcccctg 15540caattttttt ttttttcatt
taatttttgg ccaggcacag tggctcatgc ctgtaacccc 15600agcactttgg
gaggccaaga caggaggatc acaaggtcag gagtttaaga ccagcctggc
15660caacatagtg aaaccccatc tccactaaaa atacaaaaaa aaaaaaaaaa
aattagctgg 15720gcgtggtggc tcatgcctgt gctccctcca ctaaaaatac
aaaaaaaaaa aaaaattagc 15780tgggcgtggt ggcacatgcc tgtaatctca
gctactggga gcctggggca ggagaatcac 15840ttgaacgcag aaagcggaaa
ttgcggtaag ccgggatctc accactgcac tccagcctgg 15900gagacagaaa
ctttgctgtc gacagacttg gagactctgt cttaaaatat acacacacac
15960acatatatat atatatataa aataacatat atatataatt tttttcttgt
attcattttt 16020cctgacatcc ctgttctgag caatttctcc tttgacccag
tggctgctta agagtggcct 16080gtaactgtaa cagactattc caaagggaaa
aaaattccct tacatcctcc caccccatag 16140tcctgcagct gaagacatgc
tgtgacatga ggtggccaca caccagagac cagagacatg 16200agttttgggg
catttttttt tttttttttt tttgagacgg agtctcgctc tgtcgcccag
16260gctggagtgc agtggctcga tctcggctca ctgcaagctc tgcctcccag
gttcactcca 16320tcctcctgcc tcagcctccc aagtagctgg gactgcaggc
gcccgccacc acacccggct 16380aattttttgt atatttttag tagagacggg
gtttcactgt gttagccagg atggtctcat 16440ctcctgacct cgtgatccgc
ccgcctcagc ctcccaaagt gctgggatta caggcgtgag 16500ccactgtgcc
cggccggttt tggggcagtt tctaaacaac ctctgtatgg tagacctcac
16560tggccacaca tagtccttaa attgaaatat tcagttcttc cctttcacca
gcttcaagtg 16620ttcagtagca cacacagctg ttggcagatg cggaaaattc
ccaacatcat agaaagttct 16680actggatggt gctggttaga atacgtggcc
gggcgcggtc gctcacgcct gtaatcccag 16740cacttaggga ggctgaggcg
ggcggattac ctgaggtcag gagtttgaga ccagcccggc 16800caacatggca
aaagcccgtc tctactaaaa atacaaaaat tggccgggcg tggtggtgag
16860tccctgtaat cccagccact caggaggctg cggcagggag aattattgaa
cccaggaggc 16920ggaggctgta gtgagccgag atcatggcac tgcaccctag
cctgggcaac agacagagag 16980tctatctcaa aaaaaaaaaa aaaaaaaaga
tagaagcaat gccttagcct ggctaacatg 17040ctgaaacccc acctctacta
aaaataaaaa ttaaaacaat tatccggggg tggtggcaca 17100cgcctgtaat
cccagctgct cgggaggctg agctcgcagt ccagcgacat ccaggactgc
17160tggccacccc ggaacgctgg gagaggcagg aggggcccct gctagagcct
ctggagagac 17220ttcgggtctg cagacatctt gattccagac ttctgggctc
gtgctaagag tgcgtttctg 17280ctgtgcaagc cgccaggttt gggacacttt
cgtaggggcc gatcccaaaa gcgccctgtt 17340acagtgtggg ctctctgccc
agggaatcca gggggcttgt gaccttggag gggaaaatac 17400acgaccctca
tcctcagtcc tcccggagtc tggcgccccc tgcagcaagg aggaaccagg
17460cagcacgccg cctccacctc gcggtaagag cactgcggac ttcaccgcaa
gactggcccc 17520acctgatcct gaatttcgct gtttgatgcg ttaataaaga
agcacatcaa gttctctacc 17580acgaattggt cttaatattg cgatatctgt
attttaatat aatagtatcc catgtttacc 17640caaatattaa gagaagcttt
tactgttgtt tctcaaatta gggctgaagg atcatggggg 17700gggagaaagc
tgggaacgtt tgctgctttg aaagggtgtg taaacaacac cctccaaaac
17760aaccaagagt tccgaggaga aactttggcc ggatacggtg gctcacgcct
gtaatctcag 17820ctcctcggga ggctcagggg ggcagatcac gaggtcagga
gtttgagacc agcttggcca 17880acacggtgaa acccccgtct ctactcaaaa
tacaaaaatt aatcgggggt ggtggcgggc 17940acctgtaact ccagctactt
aggaggctga ggcaggataa tcacttgaac ctgggaggtg 18000gaggtggcca
tgagccgaga tcgcaccacc gcactccaac ctagtaacag ggagagtatg
18060tcccagaaaa caaataaata aacaaacaaa aagaaaacgg caagggaaat
tggaaaatac 18120tccagatgaa ccacaacgaa gatgggtggg atacatctaa
agctgtgctc agagggaatg 18180cggcgccagt gaacacccac atttcacaca
gaaggatctc agcacagcag cccgaccttc 18240cacctcagga aaccagaaaa
aggagcaaag tcaaccccaa caccaaagcc tcatcctgac 18300gagggctctg
caggctgccc cccgacgagg ccaaaagcac ccctgcccag acagattcac
18360gagccccgag aaagaacgga aggaaatgct caaggcatta gcagaatttc
tccctacttt 18420tttggtcatt ttcaaaattt gagagtcaca cgtgatttgt
atttgaaaag cctaaaagaa 18480ttattaaaat aaaaaacaaa ggacttgaac
ctgggggcta agagagaaaa gtccagtcta 18540aatgagggca agttcctgtc
tccaacgacc agggcaggtg gcccggctcc cggctgcact 18600cacctgccgc
gcccagccaa gcatcacggc gttgtacatg tccagcgtga gcagcttccg
18660cttctgccgc tggccgtggt ggacgaccag caggtggtgg gcgaggggca
gctggtcagt 18720gagcaggcag cacttgaaga aggccaggag cctctgctgc
tgacctgaga gctgggcctg 18780cgagtgctgc cccgacgggg cctgctccac
atcgaggctc agcttcccag gggcctcctg 18840cagcagccgg gccagctgct
cctcccaggg gctctcgggg gcctggcgcg tgcagtcctc 18900caggcacccg
gccatctgct tgctcaggag ccggggctcc acctgcaggc gcctggtcag
18960cgccttgaac tccccgctct ggaatggcat ctgcagcttc gccttcaacc
gctgcatacg 19020catctgctgg gtccgcttat ccttctccag tatctttgcc
cagcggccac agggcaccgg 19080ggtggcatcc ttggccccca tctggacctt
cctgggtggc tggaggctac catctccact 19140gccacattct gggagccgcg
ccacatccac cctgttcacc accacctccg acacgctctc 19200agcctgcagc
tgccgcaccc gcgcctggag cactgtgagg ggcagaaggc gaggacatga
19260gagggacccc ctccccattc gagcacccgt ctctctggac cctgagccag
gccaggaggt 19320gcaggtggct gagctcgctg ggacccaagg cgtgaattcc
tcatacttgc caacaacgtt 19380gtaaggtctg cccgctgctt tccagacaca
cgcaccccac cacctccgca cctccccacc 19440cgagcctcac agaactcagc
agccctaaca agctgccacc gaaacctgca gcaccacgtc 19500tccccggtca
ctggccgctc agaccctcca ggtgcacagg cccagaaccc ggggtctgtg
19560acaactccct ccgtccacct ctcagtacct cctctgggct tgcctccaga
atctatccag 19620gtggcccccg cctcccctgc ccctctcact gtctagctca
gggcctctgc acagactccc 19680aggaccctga accgcccact ccctggctca
accatggcct gcaagttcgc accccgcctc 19740agcaagaccc ccccagctgg
tggagctgcc acacacacac tcctaggctc ccagtgtcta 19800caccggtgga
cgctgagcca ctagctcgca gggaaaacgc ggctcctgct cgtgccgcct
19860caggttgcat ttttgccaac caatcaatgc ctaagtgttc tgtatctctt
taaagaagcc 19920ttgttggaaa tctattgctg gccgggcatg gcggctcacg
tcggtcatcc cagcactttg 19980ggaggccgag gcaggaagat cacctaaggt
caggagttcg agaccagcct ggccaacatg 20040gtgaaacccc gtctctatta
gaaatccaaa aaattagctg ggcgtggtgg catgtgtcta 20100tagtaccagc
tacttgggag gctgaggcag gagaattgct tgagcctggg aggcagaggt
20160tgcagtgact caagatagcg ccattgaact ccagcctggg caacagaaca
ataatccatc 20220taaaaaaaaa agactgttga aataagccgg gtacagggcc
gcgcacctgt ggtcccagct 20280actccggtgg ctgaggtgaa agaatcacct
aagcctagga gttcctggct gctgtgagcc 20340gtgatcaggc caccgtgctg
cagcctgaga gacagagcag gaccctgtct caaaaaaaaa 20400aagggggggg
gggacccagg tgtccagatg tggtggctca cgcctgtaat cccagcactt
20460taggaggccg aggcaggcgg atcacgaggt caggagatca agaccatcct
ggctaacacg 20520gtgaaacccc gtccctacta aaaatacgaa aaattaaccg
ggcgtggtgg tgcgcgcctg 20580tagttccagc tactcgggag gttgaggcag
gagaattgct tgaactcggg aggcggaggc 20640tgcagtgagc caagatcgca
ccattgcact ccagcctagc aacagattga gaatccgtct 20700caagaaaaaa
aaaattgctg aaataaaaag acaagcgtga tgtccgcctt cagagtgctc
20760caaaactcag gagatacttt taggattaac agttgagagc tttgttttgt
tttgttttgt 20820ttttgagatg gaatttccct cgttgcccag gctagagtgc
aatggcatga tctcggctca 20880ccgcaacctc caccttccgg gttcaagcga
ttctcctgtc tcagtctccc cgggttcaag 20940cgattttcct gcctcagcct
cctgagtagc tggcactgca ggcgttcacc accatgccca 21000gctaattttt
gtatttttag tagagacagt gtttcaccat gttggccagg ctggtcttga
21060actcatgacc tcttgatccg cccgcctcgg cctcccaaag tgctgggatt
acaggcgtga 21120gccaccgcac caggcctcgg acccttgacc tcttgatccg
cccaccttgg ccacccaaaa 21180gtgctgggag tacaggcgtg agccaccgca
ccaggcctcg aacccccgac ctcttgatcc 21240gcccacctcg gccacccaaa
agtgctggga ttacaggcgt gagccaccgc acctggccag 21300gttttttccc
tttataaagg ttctcccgcc tctcccttcc cggctgccta atggacgcag
21360acaggatgtg ggacagaagc accggcggga agcaagcaca gggaagctcc
cacctccctc 21420ccacaccacc agccaggcca ggacgagggc ctgccaccgc
tggagcctgg gctgtccctc 21480ccaagtttcg cagtcatcca gtctccatta
ggcgcctacc ccccagagcc aagccaggac 21540agctgagtca gttcagggtt
cacatcctgg ctctgcacat gtggccttgg cggcggggcc 21600gggggggggg
tctctccaga cataatcttg ggcctcacct atgtccctgg aaagtgggag
21660cacctggtgg ggttctgggg agggggaatt acgagagctc caggaaggag
cctgctcagc 21720aaggacaggg cccatgagcg gtgcaagaga tgtttcagca
acgccgtctg ggcgtgtcct 21780gggacccgag aggtggagac cgccctcagc
ctgtctcaga atctgagcct ttgccttttc 21840tcccggcagc agggagcgga
ctctcctctc ccgggccgcc gtgggggtcg cgctcaccct 21900ccagcagctc
cacgtggccc cagtccttcc tgcggtcttg gtcttgctcc tgggggctgg
21960cggacgagct cctcctgggg ccgcagacgc caccggcggt ccctgcggga
aagacgagag 22020cggctgagcg gggccgggcg tgtgggcggg ggcctccata
aaggcagaag ccgaagggtc 22080gaagggcaaa ggagccctaa acgcagcgga
aactctcgga gcacgggctt aagttggaaa 22140gaaactaaga cagcgaaggt
ggaagggccc cgccgcggcg aacacgggcg cggaaccgcc 22200gagagagggt
tcctcgcact cgaggtgcag caggtcaaag gttaagagcc ctaaacacca
22260cacctggggt caggaggctg cataagaaac cacgagtcaa aggtcagact
gcacggagga 22320gcctcagtcg aaaagcgggc aagggcgagt ggaaagcggg
gccgggtcgg tgggctgcgc 22380acgcccaggt gcaaagaggc aaaggtcaaa
gcgccaaagg ccccggccgc gcggggagga 22440gcccacgccg tggcccccgg
gctgcctggc cgtctccctt tgtgttacct tctttgccgg 22500ggagtcccgg
gcggccgcaa ggccgtaggg ctcgtttgag ccccgccgct ccgcggcccc
22560agcaaagtgc cgacattacg cacgccgctc caggccaccc caccggcccg
cgcctgcgca 22620tgcgcccgcg ccgcctgccg ggagttgtgg tttcatggtc
gacggaggct gcgaagggaa 22680accccagccg gaagtagact cccaggatgc
agcggaggcg cgaaggcatg cgccggtgga 22740cgctctgatt ggttcctcct
gctgttttta aagggagggg gcgggacaga gctgttgccg 22800tggcaactgg
gaggcactct caggctgttt tcccgaggac ctcaaatccg gacttttttt
22860ctgtttttct ttcttttttg gttttgtttt ggacgcgttg tggcccaggc
tggagtgcag 22920tggcgtgatc atagctcagt gcagcttcga actgctgggg
taaagagatc ctcgcccctc 22980ggcttcccaa agcgctggga ttgcagacgc
cgccaccgtg cccggctttt tttttttttt 23040tttcaaggca tactcatcta
ataacgagga cagcatctgc aatttagaga ttcctgtccg 23100caaccttcat
tgctccaacg acaacttttg ggtaagagtc attaggatgc cgtctatcat
23160ggaggaagct gaggctcaga gagggccacc aagttgctgg aagacacagc
acgtgcgacc 23220tcagggaggc tgcaaggaga gaaagcccca gtccgcgaga
ctcccagcct ccagcttcag 23280tttaccctcc aatccccaag ccctcagggg
caggagccga atggagcggc aggcttggat 23340tcacctgcta agtggggtga
ggtcaaggga atgaaataaa cctcggagcc tagagcctgc 23400cctggtctcc
gcgtgatcct gcctaggagg agcagggcgg gagctttaga atggaacctg
23460gaaggtgtgc ccacctgtgt cgttcagccg gggcagcagg ccagaggcgg
gagcgcctgc 23520tgtggggcag taggcttggg aagggtgaga ataggaatat
ctgggggtaa ctgtgttcca 23580ggctaatatc ccagttgcaa aggggagctg
gtttggtggc tcaggcctgt catcccagca 23640ctttgggagg ctgaggcggg
cggatcacct aaggtcagag ttcgagacca gcttggcaaa 23700tacgcaagca
tgcctggcaa catggcaaaa ccccgtctct agtaaaaata caaaaattat
23760ccgggggtgg tggcgggcac ctgtaatccc agctactcgg gaggctgagg
caggagaatc 23820gcttgaaccc gggaggcgga ggttgcagtg agccaagatc
tcgccactgc actccagcct 23880gggtgacaga gcgagaacct gtctcaaaaa
aaaaaaagtg caaagggagg tcagttcagt 23940gcctcaggcc tgtaatccca
gcactttggg aggctgcggc gggaggatcg cttgagccca 24000ggagttccag
acaagccttg ggcaaccgag atactgagac ccagtctcca ccaaaggaaa
24060aaaagaaatt agccaggcat ggtggtgcac acctgtggtc ccagatactc
gggaggctga 24120ggcaggagga ctgcttgagc ccaggaggtt tagactgcag
tgagctgaga tggcgccact 24180gtactccagc ctgggttgac agaacaggac
cctgtctcaa aacaaaacaa gtgcaaaggc 24240cctgaggcag gaacaagcgt
ggacagagga gcaatttgag cagagtgggg ctggggagag 24300ggagcaaaga
tgtagctggg gctcagttag ggggcctgac cacacggggg ctcgggggcc
24360tcagctcaag ctatcctcca tccccaaacc ctggcacttc agtttcccca
tcagcccaga 24420acgaggactc gacctcactc tggaagggcc tggcagcctc
cttacagcac attccagacg 24480ctgctgccga cgcctgcgtg agcgcactga
tgccaccggc tgggaatgtt ttcgacagac 24540ggcagcaccc tccctcacct
gcctcagtcc acctcagggt gccccagcgg gctgtgacct 24600cagacctcac
ccactactgg ggtcacctgc ctggccctga atcagccagg cctggtgtgc
24660caagacctac agacaccccc tgcacccctg caggctggca gagccagaaa
cttgggtgga 24720aaccgacttc tgaactattt caccattcct tatgcgttag
tcttttcttt tatttgatga 24780gatcccagca ctttgggagg ccgaggcggg
cggatcacgt gaggtcagga gtttgagacc 24840agcctggcca acatggtgaa
accccgtctc tactaaaaat acgaaaatta gccgggcatg 24900gtggcctgtg
cctgtaatcc cagctactca ggaggccaag ggaggaaaat cacttgaacc
24960tgagaggtgg aggttacagt gagccaagat cgcaccactg cactccagcc
ttgggcaatg 25020tagccaaacc ccatcactac aaataataca aaaaaatttt
gttggctgtg atggtgcctg 25080cctgtggccc catctacttg ggaggctgag
gtgggaagat gtagaattgc ttgagccagg 25140aggcagaggc tgcagtgagc
tgtgattgag ccactgcact ccagcctggg cgacagagcg 25200agaccctgtc
tcaaaaaaaa aagaacataa tctgggtttt ggaataagac agcagtttct
25260gaaacagctc attgcccaaa ttccagcctc gcaactctgt agccgccacc
accccccagc 25320cccaccattt attttaacta catctgtctc caccactcct
gtattaagta aatgcaatat 25380tggctggtca tggtggctca tgcctgtaat
tccagcactt tgggaggctg aggcaggcag 25440atcccctgag gtcaggagtt
cgagactggc ctggccaacg tggtgaaacc ctgtctccac 25500taaaaattca
aaaattagcc ggacgtggta gtgggtggtg cctgtaatcc cagctacttg
25560ggaggctgag gtaagagaaa tgcttgaatc caagagactg aggttgcagt
gagctgagat 25620ctcgccgctg cactccagcc tgaacgacag agcgagactc
cgtctcaaaa ataaattaat 25680aaatacaaca ttaattattt ttcttgctta
agttttacga agagacttaa tatcaccatc 25740aaaagtggga aaccatatat
ctggccgggc gtggtggctc ccgcctgtca tcccagcact 25800acgggaggcc
gaggcgggcg gatcccctga ggccgggagc tggagaccag cctggctaac
25860atggtgaaac cctcatctcc aataaaaata acaaaaatta gccgggcatg
gtgggtgcct 25920gtaatcccag ctattcagga ggctgaggca gaagaatcac
ttgaacccgg gaggcggagg 25980ttgcagggag ccgagatcac accactgccc
tccggcctgg gcgacagagc gagactctgt 26040ctaaaaacaa aacaaaacaa
aacccaacca agcaaacccc acagagtcga gaatcgctag 26100atggaagggg
atggcccagg tccctggagc ccctgtgaca aattaccaca aactcggtgc
26160cttaaagcaa cgttcatttt cttacatttc tggaaatgaa aagtccaaaa
tcaggactgc 26220ggggctgaag tcaaggtgtg tggaggcctc gctccctcca
gaggccctgg ggctccttcc 26280tgcctctccc agcttttgaa ggctccaggt
gtgcttggcc
tgcggccaca tcactcccgt 26340ctcggtctct gtggtcgcac tgcagcctcc
tcgtctgcct gtgtgaaatc tcctcctgtc 26400tccgtattgt gaccgcgttt
aggatgcccc aggacaatct tctccatatc gttcagatct 26460tcatggtgtc
aatatattga gactcttttt ccaaataagg caaatgtcac attctaggga
26520tcagggtggg gacttacctt tgggccaacc acagaggcta caaagaggaa
gacaccactc 26580aatacaaagc gtgcgccagc ccagccctga tcggtgtttg
ttgttgttgt ttttgtttga 26640gacagagtct cgctctgtcg cccaggctgg
agggcagtgg catgatctca gctcattgca 26700acctccgcct cctgggttgt
atagattctc ctgcctcagc ctcctgagta gctgggatta 26760caggcgtgaa
aaggagcaag gctctgcccc agccacagcg cggatgcacc ttgaggatgt
26820catgctcagt gaaagacgcc agacacagaa ggacacacag tgtgtgatcc
cctttatatg 26880aaatgtccac aacaggccca tccacagagg caggaagggg
atgtgtgggt gccgggggct 26940ggcagagggg atgagtgaca gctgatgggg
cttcttcttg cggtgatgga atcttctgga 27000actagacagt cgtggtggtt
gcacaactct acgaggtact aaaatcactg aactggctgg 27060gtgcagtggc
tcatgcctgt aatcccagca ctttgggagg cagaagcagg tagatcacga
27120ggtcaggagt ttgagaccag cctggccaac atggtaaaac tctgtctcta
ctaaaaatac 27180aaaaattagc tgggtgtggt ggcaggtgcc tgtaatccca
gctactcagg aggctgaggc 27240aggagaatcg cttgaaccag ggaggcagag
tttgcagtga gccgagatcg caccactgca 27300ctccagtctg ggtgacagag
ccagactccg tctcaaagaa ataataataa aataaaatca 27360ctgaactgta
cagtgtaagt gggtgaattg tgtggtatat gagtgatgtt tccgaggtgt
27420cattaaagaa actcagacgc ctggggtggg gccagtctca ccgctgtggg
tcccatcccc 27480atcatttctc acaaggccct cagatcaccc ttccgcggtg
gggggcggac actctaagaa 27540gggaagacct gggctcctgc tggcgagaag
gcggtggaca tttcttcagt gtctggtgcc 27600gcgccctctg cccagcgtgc
tccgtggagg gtctcattgt cttcctccag acgtctcttt 27660actggcccat
tttacagagg cggaaccgaa gcttggggtg ttggccacag ggctctagtg
27720tgggaagcca ggccaggctg gacctcagcc atggggaccc ctgtccctga
gactgtggca 27780cctgccacac cctctgtgtg acccgcctaa gccaggaaga
gagggtcagg agatgcctga 27840gccaccaaga aggcatccca gcgtccagcc
agaccggtta tccctccaga gggctccccg 27900gcaggacagg ctggtcgcca
tgtcttcagc ctggtgctat ttaaaggtgg gtgccacctg 27960gggctgtggc
cgcagggcca ggactgggct gctgggagct gtgtccccac agcggaggtc
28020gccgcccctc tcaggcctcg gtttccccag ttgtcaatgc ctccacttgg
ctgtgagtct 28080gtgagggtca ctgtgctcac cttttggggc ccagcgcatg
gggcaggcag aggaagggtg 28140ggggccagcc gccttgctgg gtggttcccc
gtggggcctg gggtatggct ctaagggagg 28200agcaagtgtg ggtgcgaatg
gggccgcccc attcctgccg cctccgacgt gccccgccag 28260ccggccaccg
acaggtctac gtggctatcc tccctcctgc ccacctacct gcccaaacac
28320acgtccccag tcgtcacctg cccacccacc cgcgcattcc cacacccttg
tgggcctggc 28380tttcgggaaa ctacaatttg cggggagaga agtcccacga
gggcatgccc cggagcctgg 28440ctggtcccac ggctgacgca cgcggcagga
cctcccgtgt ccatctctgt ccccaagcat 28500ctccgcctct gcccctctct
gtctctgtgt ctctctcgtc tctcccggtc atcttccttg 28560tgtctcttga
ctgccgccgt ctttctgtct ctgtctccct ccgggtctct gtctccctcc
28620aggtctctgc ggcccgcgtc tcacactccc gcccccgcaa cccgaggtcc
tagcccgccc 28680ggggactcgg ctgactcacg gacacgcccc gcgagacaaa
caacaaacgc gcggaggccg 28740agcgcggagt cccgcacggc cgcgcccctg
tgcacctggc ccccgccccc gagacgtccc 28800attggccggc gccctagcct
ggtcccgccc aagtggaccc cgcccccgcc ccgaggcacc 28860ccattggccg
gcgtccccgc cccagcgaac ccggccccgc ccccgaggcg ccccattggc
28920cccgccgcgc gaaggcagag ccgcggacgc ccgggagcga cgagcgcgca
gcgaaccggg 28980tgccgggtca tgcgccgccg cctgtggctg ggcctggcct
ggctgctgct ggcgcgggcg 29040ccggacgccg cgggaacccc gagcgcgtcg
cggggaccgc gcagctaccc gcacctggag 29100ggcgacgtgc gctggcggcg
cctcttctcc tccactcact tcttcctgcg cgtggatccc 29160ggcggccgcg
tgcagggcac ccgctggcgc cacggccagg acagtgagtg cggggcggcg
29220ggggcctggg gtggggaggc ggcgggtgac ggcaacgcgg ccgccgtctt
cacggtgacc 29280tgcgcccgcg ggggagtccc ggaggctcct ctgtgcagcc
tcggcctcag tttccgtggt 29340ctgtgagatg ggtgcagcct gcctggtggg
agggttgcac tgttaaagcg aaggctgcag 29400cggcggaccc ggctcagggg
cagagaagcg tccgtgtggt acaaccctgt gggtggggcc 29460acccatctgc
aggtgggaaa ctgaggctcc agaggggctg gggcaggccc agctgcatgg
29520cggaagcggc ggggggctga cctccggact cctgacatca cagaatccag
tcagggctgc 29580ctgagtcggg gccccctctg cttcttccca gacaccccat
ctggcaggtg aggacaagga 29640ggcacacaga agggatggga cctgcccagg
gtcacactga caggggtggc ggagctgggt 29700ccccacaggg cccaggacgt
cacggagcgg gcgtctctgt ccccagggtc tgccgagcac 29760actgaggtag
gccctcagtg tttgtggaat gtcaggagca agaggagagg ctgggcacag
29820caggggatgt gggtacctgg aggccagggg agtcggtgtc cccgccgggc
ggggggcact 29880gggaaggggg cccgggcccg ctggctgccg cctgaatcac
caccatcagg gcaggtaatc 29940accccctgtc cttcccaccg ctttcatctg
ggcgccaagg ccctcattag gccgcacgtg 30000acgagggcgg acaggggact
ggctgggccg gtccatccat ggcgggcatg gccaggcggg 30060gtggcctcgg
gccggggcag aggcctggct ccgctgcctg acctggaaca gtctctgcct
30120ctctccaagc ctcggtttcc ccagctggac ggtgatgggg gtgagggcta
gctgagggct 30180ctcctgccct tcgtgcattc gctggtcact aatcgggcac
cttgtgggtg ctgtgctccg 30240catgggggac ccagtggtga cagagacgcc
caccctcctg gggctcccag agcagaggcg 30300cgcagcagtt agacacgtga
acaagggcgc aggtgggtgc acagaacagt gaacggttgg 30360ccgggtgcag
tggctcacgt cggtaatccc agcactttgg gaggccgagg cgggcagatc
30420acgaggtcag gagatcgaga ccatcccggc taacacggtg aaaccccgtc
tctaccaaaa 30480atacaaaaat tagccgggtg tggtggcggg cgcctgtagt
cccagctact cgggaggctg 30540aggcaggaga atgacgtgaa gccgggaggt
ggagcttgca gtgagctgag atcgcgccac 30600tgccctccac cctgggcgac
agagcgagac tccgtctcaa aaaaaaaaaa aaaaaaagaa 30660cagtgaatga
cgtgaacaag ggtgcaggtg ggtgcgcaga acagtgaacg gcggtgttgg
30720gaggcacctt gccaggggag gggaggtgca gggcgaggaa ggggccaggg
gagatcgtga 30780cacagacgcc ccagaacaac cacctcaaag acgttcctgt
gtgtcctgga aggtcgggct 30840gggaggctgc cccgaggagc tttcactttg
acagggagct ggccgggcac gcagggaact 30900gtacacccag ctgacaaagc
ggcagacacc caggccgggg tgagcgagtg tgggtgagga 30960gtggcggctg
gccccagggt ccttgctgga caagacactt cagctcaggg tggggcaggg
31020ctcacccagg gctacccaca gacgatggcg tccaaatctg gctctgccac
tcccaggcct 31080caactggccc ctctgcaacg tgggctgctg agcgggcttg
gtaggacagc tggcatacag 31140tcggcgctca agcatgtctg tggtgtccca
taaaccaccg gtgtcccact ctaggccact 31200gccagcccgg cctccagtcc
agagtcccag tccggagtcc cagtgactgt gcgtgggccg 31260ggcagctgag
ctgtgagggc cgggctgggg gctccatatg gggtggtgtg agctgtgagg
31320gccgggctgg gggctccata tggggtggtg tgagctgtga gggccgggct
gggggctcca 31380tatggggtgg tgtgagctgt gagggccggg ctgggggctc
catatggggt ggtgtgagct 31440gtgagggccg ggctgggggg tccctggggt
ggtgtgagct gtgagggccg ggctgggggg 31500tctctggggt ggtgtgagct
gtgagggccg ggctgggggc tccatatggg gtggtgtgag 31560ctgtgagggc
cgggctgggg gctccatatg gggtggtgtg agctgtgagg gccgggctgg
31620ggggtccctg gggtggtgtg agctgtgagg gccgggctgg gggctccctg
gggtggtgtg 31680agctctgagg gccgggctgg ggggtctctg gggtggtgtg
agctgtgagg gccgggctgg 31740gggctccata tggggtggtg tgagctctga
gggccgggct gggggctccc tggggtgctg 31800ctggtcgctg gctcattgac
agttatcagt ggtctgggtg ggccctgccc cttctgactc 31860ccacatccca
ggaacccttt cccaaccttc ctcgtggtgt tgctgccccc ctgacgtccg
31920tccctctggg tgtgtgggag cccccccgcc atacacacac acagatgctg
ctcttgggct 31980gagctgcagg gacagcgctg acctggccct cccacggggt
cctcatcgat ctctgcactc 32040ccccagctcg tgggggccgt cctgcttccc
gttccctctg cctgctcctt gctcctccct 32100cacatgctgg ggggggctcc
tggtgtcagt cacggctctg ggggatcctg agtgtccgtc 32160gtggtcggga
ggggactcgt ggtcccgggg gtctcctggt atctgtcgtg gtcctgaggg
32220ccctgcacga agcacagcgg acagcagcgg tgctgggggt gagccagcaa
ggccctcccc 32280gacccccgcc tcccccaggc atcctggaga tccgctctgt
acacgtgggc gtcgtggtca 32340tcaaagcagt gtcctcaggc ttctacgtgg
ccatgaaccg ccggggccgc ctctacgggt 32400cggtgagtgc cgggcagggc
tgggcggcgc gggcagggtg gggagggtgg gccggcctca 32460cccccgcccg
cagcgactct acaccgtgga ctgcaggttc cgggagcgca tcgaagagaa
32520cggccacaac acctacgcct cacagcgctg gcgccgccgc ggccagccca
tgttcctggc 32580gctggacagg aggggggggc cccggccagg cggccggacg
cggcggtacc acctgtccgc 32640ccacttcctg cccgtcctgg tctcctgagg
ccctgagagg ccggcggctc cccaaggtgc 32700ctgggctggt ggcgaggggc
ccggccacgc ttgttcttcc ccctgcgggc tctgtaagcg 32760ctgagtgccc
accgtgtgcg ggcgctgtgg acacagccca ggagccctcc aggggggtcc
32820cagcctgagg gggtggtggc caccaagcag gttcaatcct gagttgggga
cctcgaggac 32880ccaacagggc gcctctcggg ctgaaggacg cagacgtcga
aaggtcgagg gggacgtccc 32940aggcagggcc cggcagaggc aggggctcgg
ggtggggagc acgttgggag tgggggcagg 33000agcggagggg aggggagggg
gccggggaga cggtgacaga cgccgcagaa caccagcctc 33060gaagccggtc
ccgtcccggg aatctgcaaa tacaacgcct tgcgaggaca aaggcacctg
33120caggtgggac ggagatggag gagcatccag ggtggggggt ccagggcccc
agtgtcctca 33180cagggtcctc acgacaggag gcgggacagt gagagccaga
gagagatggg gatgggccgc 33240gctgtggccg tgaaggggag gaagggccct
aagctgaggg acgtgggtgc ctccagatgc 33300tggggaaggc gggaacggtt
ccgcactgga gcccccggga gggaccggcc tgctcctgcc 33360ttgatatgag
cccagtggga cccagtttgg actctggcct ccagaaccgc cagaaaataa
33420acgtagtaag ccatcaactt tgtggtcttt tgttacagca gacgtcggaa
atatgcacac 33480ggtgtctgaa actgttctca tgacaaaata agcctcagat
cccccgggga agggcggagg 33540ccaacgcctc ggtgttcctc cgatcccccg
ggaagggcgg aggccgacgc ctcggtgttc 33600ctcggatccc ccgggaaggg
cagaggccga cgcctcggtg ctcctcagat cccccgggaa 33660gggcagaggc
tgagggcagg agccgtgctg ggtgcagggc aggcctgggg gcttcatgcc
33720gctgtcctgc gggacgcaga gagggctggc cgtcggtgtg ggggcgcccc
cacctgtgcc 33780cagcgccctc ctgacatcct gactccgctg ggacttctgc
ctacagccct gggagtcaaa 33840ctccagcctc tcagagaaaa ggtcagagcc
aagagcccca cagcctggag ccaggcagtg 33900acaccctggg cctgtctccc
cttctgtgtg tggggcgaca gcagcatcgc cctggtgaag 33960tccccgggga
cggccagggc tccatcccca gccgccgcct tccacataaa tacaggaaga
34020ctgggccgag gcacttgctg ggaggtgctg agcagcctga cacggaaaac
ccttctggga 34080agggagggtc gtgcccggcc cgagagcttc tgctcaccct
gcagacagaa gcgagcccca 34140ccccagggga caccaggcgg cctctgggga
catctttggc tggcatggag tgggtggagg 34200acagggctgc acccaggatg
tccccaggtt ggcagtgtga ggggagatcg gcccacgttg 34260gccagtcgga
gggcgtcgcc acttgagttg tcactgggag ctgcacaggt caccacagct
34320gaaataaaac ttgctggcac cccacgcagg aacgtaacat gtgcctcgaa
gaaacgggtc 34380agcaggccgg gcgcgggggc tcacgcctgt catcccagca
ctttgggagg ccgaggcggg 34440tggatcacga ggtcaggaga tcaaggccat
cttggtcaac atggtgaaac cccgtgtcta 34500ctaaaaatac aaaaaattag
ccgggcgtgg tggcgggcgc ctgtaattcc agctacttga 34560gaggctgagg
cggggaatcg cttgaatccg ggaggcggag gttgcagtga gctgagatcg
34620cgccactgca ctccagcctg ggcgacagag cgagactccg tctcaaaaaa
aaaaaaaaaa 34680aagaaacagg tcagcagttg tttctttgtt tctaaaacag
agcgtggaat gggcgtacag 34740ctccgcacat cccagggcag tgaaatcccg
gttcacacag agccctcagc agcttattcg 34800caagcccaaa cctggggacc
cccgttgtcc tcaggcagtg aggtgggggc cccccaacag 34860agaggagcgg
cctgggggca cagaaccagc ggctccccag gaaatcgcca gcagtgaaaa
34920taagacaacc ccaaactgtt gcaaactgtg cttccgctta cgaagcactc
ctgagcggca 34980gggcggatgg ggagagggcg gctgcaggcg cgaggggccc
ggggacgcag gggtgcgggc 35040cttaccaggg ccctgtcctg tcgtgcagca
ggctcctggg gcagggaaga caccaggggc 35100ggccacttct tactgctgtc
tgacctcgag caatgcggcc tcacagcccc caccagggtg 35160ccggtgtcct
ctgggcccag cgcccccgag gctcatgcct gggtggggcg aaccaatcgg
35220tcctgctcct ctggccactc cacgcgaggg aagtcccagc ctcacaggca
ggcgcacacc 35280ccggcagcat ctctgacaaa ggccctccag ttccgagtct
ccaggtcccg ccgctgcaag 35340cctcacctgc ccagccctcc tctccagctc
caactccaac tcccaagaac caccacggac 35400acacagaacc cgagccttgt
ctccctcaac gcctcctgac tcaaaactcc atcttccaac 35460aggaaaacgg
ctcggccggg ggactgtgac ccggagcagg cggcccagcc tgtcgcgcag
35520actcggggcc taaaacactt gttctctcag tccggagatc aaggacgatc
cgaggtaacc 35580tccctacctc ggtgtcctcc atgcaacctc gtcttagggc
accgggtacg ttacctcgtg 35640aggagccgag tccgcgggtc ctggggttga
gatgtggacg ccctcagggc tggcactctg 35700ccctggcggc cacagtcatg
gaagtcccaa cgcttctctc ggctccgcaa ccccagaggg 35760cggccacgag
gagggcccgc cacgcacgac cccagagggc ggccaccagg agggcccgcc
35820acgcgcgacc ccagagggcg gccaccagga gggcccgcca cggcgttgcg
gcagcagccc 35880agaaggtgcc ctgcgcacgg tccggacagg tgggatccga
gttacctggc caagggggct 35940gacgcagaca cgtcgcggga cacagtgaag
agtgtggtgc agagcggagg gcgggagtct 36000ttggagaaca ggtaggggcg
tggggcacgc gcctcccacg cgcaggagcc gtctaccgtg 36060gagggacacg
ggtggtcctg ctggaggctc ctctccgtta gctgtctcca tcgtctgatt
36120cttggatccc aggatggtgg gatcatcagc aactgagatg aacccactgc
cccggccccc 36180tgagcccgca ggtccccacg ccttgccagc tgtgcccgag
ctggctgcac cccgggccag 36240gcatccagca accttgagca gtggggtccg
gcttttcaga aggggccagg aacccgcgtg 36300gctgaggtgt gaccgaagcg
tggggcagag gcgctgggcc ctggcgcttt aacgctggtg 36360tttctggttt
taaatttcac gacccagtga cactgccacc ctgctacctc gccagcagcc
36420ctcctgggct taacttcggg agagcagttt tgctagccgg ccctgggtgc
caagccctgc 36480aggaggcgca gacccctgga gacaggaccg gactctgcag
agcccgacca gcctcccagc 36540ttggcctttt cctgacgcac gggcgcagaa
ggaaagccac agcaccggct tctctttgta 36600agtagtgtat tttaaatagc
tttcaagata cacatatttt ttcctttaaa aaagtctgtt 36660ggagcagttt
tgttcttgaa ttttgctggt catcctcatg gtcccgagcc cccctactcc
36720gggtcgtgga ggcggccgag ggggaggctg ggggcccacg tggcccgtcc
tggcggcacc 36780tgcagcactg ggggagccgc tgaaccccgt gcttcagcgc
tgggggagcc gctgggcccc 36840gtcttccgcc acaaaccatg catggccgcc
acgtgagctc aaacgtccgt ttatttcaaa 36900gcagtaataa tttaaaatta
taaaaatctt tccaccgctg aacgtttaga gggtgaggtt 36960agacagagga
cggggaggct ggggacgccc cagaggggac catgtggccc acgccttccc
37020aagccagggg gccggtgggc cgggcccggg tcctgccctg gaacaggcgg
gacctgcagc 37080gctgaccagc caagcgtggc gccgccgggg cacccagtct
gtgggtgccg tgtggcgctg 37140gctgagggtg ggtgggaaag gccccgtgct
ttcccgacgg ccgacgtggg ctcacgagtt 37200gcttgtggcg ttctcgttgc
tgggcgagct ggaggaggac gatgacgacg aggaggagaa 37260gctcacccca
gtgaggccag gggggttcgt ggccgtgttc tgtcccgtga ggctttttcg
37320gcagacgggg cagctgtcgt gctttgtggg gacagaggca gggacgggag
aaggggcagg 37380ttagaggcgg gagggccgcg gtcggggtgg gggggcgggt
gggcggggca ctcacctgct 37440ccagccaggg cacgatgcag ccgtcgtgga
acaggtggtt gcagggcagc tgccgcacac 37500gctcacccag cgcgtagtcg
tccttgcaca cagggcactc gagcccggag cctgcgggag 37560tgtgcagctg
cggtcacagc gggcgtgggg ggcctgccga gccttcaagg gcaggctact
37620ccacagcctc agccggaggc cgcccctgag cccagcgagg ggagaaaagc
cgtgtgtgtg 37680tcccccgggc tgccagaggg gacctggaca gaaccctctc
ctcccagccc accttcaggg 37740aaatgctcga ggccgggtgc ggtggctcac
gcctgtcatc ccagcacttt gggaggccga 37800ggcaggagga tcacctgagg
tcaggagttc gagacctgcc tgaccaacat ggtgaaaccc 37860tgtctctact
gaaaatacaa gtatgagcca ggcgtggcgg cgggtgcctg taattcccac
37920tactcgggag gctgagctct catacctacg tgctcctcag tgacggggac
ggtggggagg 37980gcctggattt tctctttatc tgccggtggg gggcctgtgt
tttcaaactg attgaggagc 38040tgaaagacaa gaggcgagag tgccgggagc
tcctcggggg cccggcccgg ggctctgaaa 38100cgcgaggctg caggacctgc
aaaagcaccg aggccgcgtt tgtcctgggc cctgggcccc 38160ttggagcccg
cccggggtcg gagatc 3818639720DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number Z69589 39cgccggcgct
tgacctgact ttcatgaatc gaaaaggaaa tcctctatga acgcactgca 60tcgcatcggc
gccggaacgc tactggccgt gttgctcgct tttggcctga ccggctgcgg
120ggagaaggag gaggttcagc agtcgctcga gccggtggct tttcacgact
ctgacgagtg 180tcacgtgtgc ggcatgatca tcactgactt ccccggcccc
aagggccagg cggtcgaaaa 240gcggggagtg aagaaatttt gttccaccgc
cgaaatgctt ggttggtggc tgcagccgga 300aaaccgtctg ctcgatgcca
agctctacgt ccacgacatg gggcgcagcg tttgggaaaa 360gccggatgac
ggtcatctga tcgacgcaac cagcgcctac tatgtggtcg gtacgtcact
420caaaggcgcc atgggcgcgt cgcttgcaag ctttgccgag gagcaggacg
ccaaggcgct 480tgccggcatg cacggcggtc gtgtgctgcg cttcgaggaa
atcgatcagg cgctgctgca 540ggaggctgca agcatgcagc acggcggcat
gcacgaccat gcgccaaacg gtgcacataa 600cgcacacgca ggccactgag
cagcagtggt ctgaacagca cacacaagaa atcgaggtaa 660gcacaatgat
gggtatcagc gtctggcaac tcctgatcat tcttctgatc gtcgtcatgc
72040127DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number Z22279 40gcggccgcnc ggcgctggct gctgtgcgga
ggccacggcg ggccgcgagc cgcctcgtcc 60tcgccctcct gccctgggtg cggccccccg
ggtcccggcg ncccactcgc cccggcgtnc 120ccgcgct
127416858DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number X17524 41actcgccaag tgatcgaccg gcccctgagg
gccgcgacgc agagggcgcc ccgtgcactg 60gcacaggcgg ccttgtgcgt tagactctga
tattcgtgcg ccctctcgtt ggcaggacca 120tccatcctgt gtgccggggg
ccgcgcacac cgatcccgga tccgcctcgg ccctgccctg 180cgcgcccctc
cgttctcgac ctccccgacg ctgtctgaac acgcgtcgcc gggggacgac
240ggcgggcggc ccgcctcggg ggaggggtaa gcgtcccggg atgcccgttc
aaccgttccg 300caaggctcgc ccatcgtggg ggagaaccgg cgcgacgcta
ggagagacaa gtgatccagc 360aggagtcgcg gctcaaggtc gccgacaaca
ccggtgcgaa ggaaatcctg accatccgtg 420tgctcggcgg ttccggacgc
cgctacgcag gcatcggcga caccatcgtc gccaccgtga 480aggacgccat
ccccggcggc aacgtcaaga aggcgcacgt cgtcaaggcc gtggtggtcc
540gcacccgcaa gcagtcccgc cgtcccgacg gctcgtacat caagttcgac
gagaacgcgg 600cggtcatcct gaagaccgac ggcgagcccc gtggcacgcg
catcttcggc cccgtgggtc 660gcgagctgcg tgacaagaag ttcatgaaga
tcgtgtcgct cgccccggag gtgatctgac 720ctcatggcca agatcaagaa
ggacgacctc gtgcaggtca tcagtggcaa ggacaagggc 780aagcagggca
aggtcctgcg cgtgttcccg acggatgagc gcgtgctcgt cgagggcgtg
840aaccgcgtga ccaagcacct gcgcgccggc caggacaaca acggttccac
cgagggcggc 900ctgcaggtcg tcgaggcccc gatccacatc tcgaacgtgg
ccgtggtgga cccggagacc 960aagaagccga cccgtgtggg ctaccgcttc
gagaccgtcg agaaggacgg cgtgacgaag 1020accgtgaagg tccgcttcgc
caaggcctcg gggaaggagc tgtgatgacc gaggtgcagc 1080agaccgagaa
ggtcaccccg cgtctgaaga ccaagtaccg cgaggagatc cgcggacgcc
1140tgcaggagca gttccagtac gggaacgtca tgcaggtgcc gggcctcgtg
aaggtcgtcg 1200tcaacatggg cgtcggcgag gccgccaagg actccaagat
catcgacgac gccgtcaccg 1260acctcaccgc catcaccggc cagaagccga
tgatcaccaa ggcccgcaag tccatcgcgc 1320agttcaagct gcgtgagggc
atgcccatcg gcacgcacgc caccctccgt ggcgatcgca 1380tgtgggagtt
cctggaccgc ctggtcacgc tgccgctgcc gcgcatccgt gacttccgcg
1440gcctgtccga ccgccagttc gacggcaacg gcaactacac cttcggcctg
tccgagcaga 1500ccgtgttcca cgagatcgat caggacaaga tcgaccgcgt
gcgcggcatg gacatcaccg 1560tggtgacgac cgccaagaac gacgacgagg
gccgcgcgct gctcaaggcg ctgggcttcc 1620cgttcaagac cgaccagtaa
gacctccacg ccacaggtcc tccaccggtg aaccggtggc 1680ggaaaccacg
gcgagaaagg gcgtgaagca catgaccatg accgatcccg tcgcagacat
1740gctgacccgt ctgcgcaacg caaactcggc ctaccacgac accgtgtcca
tgccgtcctc 1800gaagctgaag actcgcgtcg ccgagatcct caaggccgag
ggctacatcc aggactggcg 1860cgaggaggag gccgaggtcg gcaagaagct
gaccatcgac ctgaagttcg gcccgcagcg 1920tgagcgtgcg atcgccggcc
tgcgccgcat ctccaagccg ggcctgcgcg tgtacgcgaa 1980gtccacgaac
ctgccccacg
tgctgggcgg cctcggcatc gccatcctgt ccacctcctc 2040tggtctcctc
acgaaccagc aggccgccaa gaaggctggc gtgggcggag aagtcctcgc
2100ctacgtctgg tgacgggcaa gacggaagaa aggctgaact gacatgtctc
gaatcggacg 2160tctcccgatc accatccccg ccggcgtcga tgtgaccatc
gacggcgacc gcgtctccgt 2220gaagggcccc aagggcccca agggtcagct
cgagcactcg ctgcccacgc ccatcacggc 2280caccctcgag gaggggcagg
tcaccgtggc ccgccccgac gacgagcgtg agtcccgctc 2340cctgcacggt
ctgacccgta ccctcatcag caacatggtc gagggcgtga ccaacggctt
2400ctccaagcag ctcgaggtcg tcggcaccgg ctaccgcgtg caggccaagg
gccaggacct 2460cgagttcgac ctgggctact cccaccccgt cccggtgaag
gtgtcccagg gcatcacctt 2520cacggtggag ggtaacaggg tcaccgtcgc
cggtatcgac aagcagcagc aggtcggcga 2580gaccgccgcc aacatccgca
agctgcgccg ccccgacccg tacaagggca agggcgtcta 2640cgcgggcgag
cagatccgcc gcaaggccgg aaagaagtga tgtctactct gaaggtgaag
2700ggcaagggca agttcaacgc ccgcacccgc cgccacctcc gggtgcgcaa
gcggatctcc 2760ggcaccacgt ccgtcccccg cctcgtcgtc aaccgctctg
cacggcacat gttcgtgcag 2820gtcgtggacg acacgcagag ccgcacgatc
gcgtacgcct ccaccatgga ggccgacgtg 2880cgtgcgctcg agggtgacaa
gacggccaag gccaagcgcg tgggcgagct cgtcgccgag 2940cgtgccaagg
cggccggcat cgaggccgcg gtcttcgacc gggcgggcaa caagtaccac
3000gggcgcgtcg cggccgtggc cgacggtgcg cgagagggtg ggctgcagct
gtgaccgaga 3060acatcaacca gaaggacact caggtgaccg agagcaccga
gaccaccgtc tccgagaccg 3120ggtcgggctc gcgagccaga ccaccgagcg
cgccaccggt ggccgcggcg gtcgcgacgg 3180cggccgcggt ggccggacgg
cgatcgtcgt ggcggccgtc ggacgaccga accgtcgtgg 3240cgcccaggac
gacgaggaag gaccagttcc tcgagcgcgt cgtgggcatc aaccgcgtct
3300ccaaggtcgg ccgccgcttc tccttcaccg ccctcgtggt ggtgggtgac
ggcgacggca 3360ccgtcggcgt cggctacggc aaggcgaagg aggtccccgc
tgcgatccag aaggccgtgg 3420aggaggccaa gaagtccttc ttccgcgtcc
cccgcgtcgg ctccaccatc ccgcacctgg 3480tgcagggtga ggacgccgcc
ggcgtcgtgc tgctccgccc ggcctccccg ggtaccgcgg 3540tgatcgccgg
cggtccggtg cgcgccgtgc tcgagtgcgc cggcatccac gacgtgctct
3600ccaagtccat gggctccgtg aacgcgatca acatcgtgcg cggcacggtg
gagggcctca 3660agaagctgaa gagcccccag gccgtcgccg cccgccgcgg
caaggccctg gacgagatcg 3720ccccccatgc gatgctgcgc accatggaga
acgatcgcgc ccagaagagc gcgaaggcag 3780gtgcgtgacg cgtgtttgag
tccactcgca agaacatcca gccctcggac gccaccctgg 3840tcatcaccca
gacccgcggc gtcacgggct ccaagcagaa ccatcgggac accctgcgct
3900cgctgggcct gaagcggatc ggccaccagg tcacccgcaa ggccgacgcg
gtgacggtcg 3960gcatggtcaa caccgtgccg cacctggtgt ccgtggagga
ggtcaacaat ggctgacaac 4020gacgccatca aggtccacga cctgcgtccg
gcccccggtg ccaagaccgc caagacccgc 4080gtgggtcgcg gtgaggcgtc
gaagggcaag accgccggtc gcggcaccaa gggcaccaag 4140gcccgttacc
aggtccgtgc gggcttcgag ggcggtcagc tgcccctgca gatgcgtctg
4200ccgaagctcc gcggcttcaa gaacccgttc cgcacggagt accaggtcgt
gaacctggac 4260aagctctccg cgcacttccc cgagggcggt gaggtcaccg
tggacgcgct cgtctccaag 4320ggcctcgtcc gtcgtggcca gcccgtgaag
gtgctgggca cgggggagat caccgcggcc 4380gtgcaggtga aggcgaacgc
cttctctgcg tccgccgtgg agaagatcca ggccgccggc 4440gggtccaccg
agaccctctg acacgccgac ccatcgaccg agggccctgg ccggagcagc
4500cgctcgggcc aggccctggt ccgtccgtgt agactcgcac agccgccccg
gtgtggccgc 4560cgtctcgtgc ccccgccccg cggaacggcg cacgccccac
aggaccagcc gcaggaggac 4620tcgtgctcaa ggccatcgcc cggatcgtcc
ggacgcctga cctgttgcgg aagatcgcct 4680tcacgctcgg gctcatcgcc
gtctatcgga tgggcgactt cgtgccggcc accggcgtgg 4740actacccggc
ggtgcagcag tgcctggcag cgggcaacgc ccagggcggc ctgtactcct
4800tcgtgaacat gttctcgggc ggggcgctcc tgcaggtgtc tgtcttcgcg
ctgggcatca 4860tgccgtacat cacggcgtcg atcatcgtgc agctgctgcg
cgtggtgatc ccgcgcttcg 4920agcagctcca ccaggagcgc cgcaggggcc
aggcgacgct gacgcagtac acccgctacc 4980tgaccctcgc cctcgccctg
ctgcaggcga ccacgatggc ctcgctggcc cgcaccgggg 5040ccctgctcgg
atgcagcctg ccgctgctgc gcgacggctc catcctcacg gtgctgctcg
5100tggtcatcgc cctgaccacc ggctgtctca tcgtcatgtg gttcggggag
cggatcaccg 5160agaacggcgt gggcaacggc atgtccctgc tcatcttcac
ctccatcgcg gcaggcttcc 5220cggccggtct cggccaggtg gtccagacgc
agggctggcg cgtgttcgcg atcgtcatgg 5280ggatcggcct gctcaccatg
ctggccatcg tcttcgtgga ggagtcgcag cgccggatcc 5340cggtccagta
cgccaagcgg cagatcggct cacggaccgt gggcgggtcg agcacctaca
5400tcccggtcaa ggtgaacatg gccaacgtca tcccggtcat cttcgcctcc
tccgtgctga 5460tgctcccggg catcctcatc cagttcaaca cgccgcagga
cggcagtgcg ccggccccgt 5520ggatcacgtg gctgagccgg tacttcggct
ccggtgacca cccggtgtac atggccctgt 5580acttcctgct catcatcggc
ttcacgtact tctacgtgtc catcacgttc aacccggtgg 5640agatctcgga
caacatgaag cgctacggcg gcttcatccc ggcgtccgcg ccggccggcc
5700ccaccgagcg ttacctgcag tacgtcatca gccgcatcac gttcgtggtg
ggggccctct 5760acctcggtat cgtggccatg atcccgctga tcgccttcgc
ggtgatcggc accagccaga 5820acttcccgct cggcggcacg tccatcctca
tcatggtggg cgtcggcctc cagaccgtga 5880agcaggtcag cgcacagatg
gagcagcgcc actacgaggg cctgctgcgc tgagccccga 5940cccgatcccg
caacgccgtc cgtatcgaca gtgaggaaca cacgatgacc cgcatgctgc
6000tcatgggccc tcccggttcc ggcaagggca cccaggccac ccggatcgcc
gacaagctgg 6060ggatcgtccc gatctccacc ggtgacatct tccgccacaa
cgtgaagtcg atgacgccgc 6120tcggcgtcga ggccaagagg tacatcgaca
acggcgactt cgtccccgat gaggtcacga 6180accgcatggt cgccgaccgc
atcgcccagg ccgacgcgga gcacggcttc ctgctggacg 6240gctacccgcg
cacgaagggc caggtcgagg cgctggacgc catgctcgcc gaggccggcc
6300agtcgctgtc cgccgtcgtc gagctggagg tgcccgacga ggagctcgtg
gagcgcctgc 6360tcaagcgtgc cgagatcgag ggccgcgcgg acgacaccca
ggaggtcatc gagcaccgcc 6420tggacctgta ccaccgcgag accgagtccg
tcatccagga gtacgtggag cgcggcatcg 6480tcgcccgcgt ggacggcacc
ggccagatcg acgacgtcac cgagcgcctg ctgcaggccg 6540tgtactccgt
gcgctccgcc acgggctccc tgcccgtgat ccagccgggc gcggagtcct
6600gaccccgtga tcggccgccg ctcgctcgag ctcaagaccg ccccccagct
gctggccatg 6660cagcgcgcgg gggtggtcct gtccgaggca ctggacgccg
cgctggccgg cgcgccgggc 6720ttcaccaccg cggagctgga cgccgtgttc
gcggtggtgc tggccgaacg cggtgcgacc 6780tccaacttcc tgggctacta
cgacttcccg gcctcgatct gcacctcggt caacgaggag 6840gtcgtgcacg gcatcccc
685842578DNAHomo sapiensMisc_feature(5)N is any nucleic acid
42ttctngtcta tggcagagat ggncaggttg ncgttgagca ggtactgncc atcagccgtc
60ttcagcgcca ggtagttccc atcgttctgc acacccgggt ggctccgctg cttcacgtca
120atattagtgg caccagctgg gatggtgaca atgtcattgt agccataatt
ggtgggggtg 180agggacccgg agaccttcct gcaggagttg nctttgcccc
cacacacccc gcatttgtcc 240agcttccgag gcgagtccac cacatggtca
cagccggcct tgacacactg gncacggaca 300cagatggnca gtgtttctgg
cccacacagg gtgccatcaa tcaccttggn ctcgaacact 360ttggaactcg
ctcctccccc gggntcggga ggaacaactt gcaggggtcc cgggggggac
420aacccagcat tcttggggga cccactgcag gaggattccc cgtccatgtc
aagtgtnatt 480ggtgggcatt attcttctca caattgntgc tccctgaagg
ttttcccgnc aaggggggat 540tcccccccng ntggaatnat tggtacttgg gtctccga
57843305DNAHomo sapiensMisc_feature(128)N is any nucleic acid
43catttaagtt tgctagtcct ttgcaaacag actgacgctg agtgtcctgt ctgagtcaat
60aagtgcactt ttacctttta acctatgccc tctacttgaa cccgagcaag gtccagtcca
120ctggacangt tgatgatagg gtctgncgcc ccataccctc tcctcttccc
ccttaggaat 180ttgtgcagta ctggaggggt tgcggcaatg ggaggcctgg
gtgggccgtg ctgccttgat 240atggccaagg gacccagtca ccacagtgga
gacccttgtc tgcacctcag taccgcatgt 300ccagg 30544333DNAHomo
sapiensMisc_feature(82)N is any nucleic acid 44ggcacaggtg
actttagcat gcagagcagc aaagagagag caaccaccaa catcatccag 60ccgctgctcc
acgcacagtg gntgctgggg gactggtctg agtgctctag cactgcgggg
120ccggctggca gaggcgaact gtagagtgca gggacccctc cggtgcaggc
ctctgccacc 180tgcaacaagg ctctggaaac ccgaggatgc caagccctgg
cagaaccagc tgtgccccct 240gtgatttcag ggggncaggg gccattttgt
gctcngggac atgcggtaat ggaggttgnc 300agacaaggtc ttncattgtg
gtgnatgggt tcc 33345102DNAUnknownDescription of Unknown Organism
Sequence from Applicant's cDNA clone HCE3Z95R 45gcagcagcag
cgcagcgcag agagagcagc agcagcagca gcagcagcag cagagcagat 60cntnctggna
nnaaaaaatc gcggcagcag ctgctctagc ag 10246123DNAUnknownDescription
of Unknown Organism Sequence from Applicant's cDNA clone HTLEQ90R
46caggcaagnc ggcacgtagg agcagcagca gcagcagcag cagcagtaac nnagtcnacg
60agggggngcc cgggacccaa ggcgcccgaa cagagaggcg gagcacaatc cactggtcgg
120cgn 12347109DNAUnknownDescription of Unknown Organism Sequence
from Applicant's cDNA clone HMWEF45R 47ggcacgcagg agcagcagca
gcagcagcag cagcagcagc agagagagag cagcagagag 60agagagcagc agagcagagc
agagcanagt agagnagagc anagcnnac 10948293DNAHomo
sapiensMisc_feature(86)N is any nucleic acid 48ggcacgaggg
ggaaactgct ccgcgcgcgc cggggaggag gaaccgcccg gtcctttagg 60gtccgggccc
ggccgggcat ggattnaatg cctgagcccg ggtcccgctg tcttctgctt
120cttcccttgc tgctgctgct gctgctgctg ctgccggccc cggagntggg
cccgagccag 180gccgnagctg aggagaacga cttgggttng cctncccana
aaatgggaag gganttgggg 240ttaatcgaag tcattgggac cattttaaaa
ggggcttcct ggattatagn ctt 29349506DNAHomo sapiensMisc_feature(283)N
is any nucleic acid 49aattcggcac gagcacccgg ccactgcagt cttctgccct
gctggacagc agcagcagca 60gcagcagcag cagcagcagc agcagcaaca gtaacagcag
cagttcgtcc ggacccaacc 120cttctacctc ctttgagccc atcaaggcag
accccacagg tgttttggaa ctccccaaag 180agctgtcaga aatctttgat
cccacacgag agtgcatgag ctcggagctg ctggaggagt 240tgatgtcctc
agaagtgttt gcccctctgc tttcgtcttt ctncaccccc gggagaccac
300gattatatct acaacctgga cgagagtgaa ggtgtttgtg anctcttttg
atgtgnctgt 360tntnaacntt tgactgacag ggacatgcct tttttggttg
ggacccagat tttttgactt 420gggggtttnc ttgggacttt tcaaccgacc
ctanagagtt nagagcaaan aggttggttt 480ttcggcttcc ttaacgaaag ttttgg
50650419DNAHomo sapiensMisc_feature(137)N is any nucleic acid
50tttaagcacc aaaacttgtg ttttaatgat gttggatgga aatctttcct aaatgtgtca
60tgcatgctct tgtctccctt aatggagaga gtgtgacact gcttagcact tggatggctt
120ggggtggtgg ttatgancag cagtctgtca cagctcagcg aggtgaagcc
tgtgggcgtt 180ttgctctgtg ctgaatggct cagtggccct acaaagcgga
ntcagctctt ggtggctttc 240tgttgtggtg ggctgctgnt gctgctgctg
ctgctgctgc tgctgccctt gcctctaaaa 300gaactcactt cctcttcctc
ctgctgncac ctgtcttttg gcttgtggga ttggagtcat 360ggggcccaga
tggagccttg ctccntgant tatgataggc ccctcggtct cttttntnc
41951495DNASaccharomyces cerevisiaeMisc_feature(177)N is any
nucleic acid 51aattcggcac gagcaaagtt ctgcgctcca ttgtgggcat
caaacgacac gtcaaagccc 60tccatctggg ggacacagtg gactctgatc agttcaagcg
ggaggaggat ttctactaca 120cagaggtgca gctgaaggag gaatctgctg
ctgctgctgc tgctgctgcc gcagacnccc 180agtccctggg actcccacct
ccgagccagc tcccaccccc agcatgactg gcctgcctct 240gtctgctctt
ccaccacctc ttgcacaaag cccagtcctc cggcccagaa catcctgggc
300ccggagttcc ttccttgcct tnaggggntt ttcagcaagt tnagttcctt
gggtcctttt 360tgggaaantt naggnagttn aaggantacc aggttnttgc
catnctttcc agatccaagt 420ttnacnaaaa attttnaaca gtntaaattg
ggtttnttgn ccctttnngg nggntgtttt 480ttttttcggg tccgg
4955281DNAUnknownDescription of Unknown Organism Sequence from
Applicant's cDNA clone HMCAO46R 52ggcacgcagg agcagagcag cagcagcaga
gagagcagca gcagcagcag cagcagcaga 60gagananata natanatata t
8153305DNAHomo sapiensMisc_feature(11)N is any nucleic acid
53aggcacttga nttgaaaatg gaaaacccta ctgctggtgg tgctgcggtg atgaggccta
60tnatgcagcc ccagggtttt nttaatgctc aaatggtcgc ccaacgcagc agagagctgc
120taagtcatca cttccgacaa cagagggtgg ctataatgat gcagcagcag
cagcagcagc 180aacagcagca gcagcagcag cagcagcagc aacagcaaca
gcaacagcaa cagcagcaac 240agcagcaaac ccaggncttc agcccacctc
ctaatgtgac tgcttcccnc agcatggatg 300ggctt 30554307DNAHepatitis C
virusMisc_feature(212)N is any nucleic acid 54tggggtgtga agctccggtg
ctggtgcggc gggggactgc ggggccagcc tcagtttaaa 60ccccctcagc agtctttctg
tcgttgccct ccacactgcg agactctgga gggcgatctg 120gaggtctgga
agataaccga ttcctgggag atttgggggt agtctccaat ctgtccctgg
180ctcatcttgt gacccgaagc cggcggcctt gncaggagta ttctagaatg
agtgcacata 240aaaatacctt caaacggtag cagcagcagc agcagcagca
gcagcaagca gcagcagcag 300cagcagc 3075588DNAUnknownMisc_feature(6)N
is any nucleic acid 55ggacanngac tactctctct ctctctctct ctctctctgc
tgctgctgct gtgctgctgc 60tgctgctgct gctgccgntg tgngcana
8856346DNAUnknownMisc_feature(278)N is any nucleic acid
56ggcacagccc aactggtgat gctgctgctg ctgctgctgc tgccgccgcc gcctctattg
60ctgatactct agtggggctg gaagggtggt tcctattcgc accatcgcca accagagaca
120gagggaaaaa aaaaaccggc agccactgct gaatgttggg ttcggaggct
gcatccgact 180cggtcacaag gaaaatggat tcagtttgca tctctccctc
ctttaaacag cttctccggg 240tctcagcatg ggcttccagg gcagcgattg
aggagacntt accaaggngc accacacant 300agatgctgag acntcgtgac
tccaggataa gaaacattaa cngggg 34657496DNAUnknownDescription of
Unknown Organism Sequence from Applicant's cDNA clone HLHTP36R
57gaattcggca naggtgcaca gatgtggtgg atggggaggg ccgcacggga cagaagttct
60ccctgtgtat tctgacgnct gagaaaggag catttcatcc gggcggagac caaggagatc
120gtcaatgggt ggctggagat gctcatggtc tatccccgga ccaacaagca
gaatcagaag 180aagaaacgga aagtngnagc cccccacacc acaggagcct
gggactgcca agttgggctg 240ttaccagcag cagcagcagc agcagcagca
gcagcagcat ccccantgct ntnggaaagt 300tcccaccacc aagtnccaca
atttgggnna aaaccaaggt tgtngnagac gngntttngg 360gatttnggca
ttgtgggttg cttgcatgga aggacattng gttgtnggtn ccttggangn
420tacaattacc atttncggtt gtnaaggtta aanntccgnc attcagaagg
ntnnaaggtg 480ntttgaagtc catttg 49658268DNADrosophila
sp.Misc_feature(16)N is any nucleic acid 58aacacttatc cttganagct
ctgtttggga agcaggacaa agctacatgt naggaaactn 60tggagcctcc gcagactctc
caccagcagc agcagcagca gcagcagcag caagagaagc 120ttccaattag
gcagggggtt gtacgctccc tgtcctatga ggaacccaga agacactcac
180cccccattga gaagcagctc tntccagcca ttcagaaact catggtcagg
agcgcagacc 240tccacccatt gtcagagctg cctgaaaa 26859471DNAHomo
sapiensMisc_feature(249)N is any nucleic acid 59tcgacccacg
cgtccgctga ggaacagacg ttccctggcg gccctggcgc cttcaaaccc 60agacatgctg
ctgctgctgc tgctgctgcc cctgctctgg gggacaaagg ggatggaggg
120agacagacaa tatggggatg gttacttgct gcaagtgcag gagctggtga
cggtgcagga 180gggcctgtgt gtccatgtgc cctgctcctt ctcctacccc
caggatggct ggactgactc 240tgacccagnt catggctact ggttccgggc
aggagacaga ccataccaag acgctccagt 300ggccacaaac aacccagaca
gagaagtgca ggcagagacc cagggccgat tccaactcct 360tggggacatt
tggagcaacg actgcnccct gagcatcaga gacgccagga agagggataa
420ggggtcatat ttctttcggc tagagagang aagcatgaaa tggagttaca a
47160379DNAUnknownDescription of Unknown Organism Sequence from
Applicant's cDNA clone HBNBG53R 60anttcggcan aggnaaggga gagggtgacc
ngcatcccaa ctagatttca gtggagtgaa 60gttcaggagg catggagctg acaaccatga
ggcctcggca gccaccgcca ccaccgccgc 120cgccaccacc gtagncagca
gcagcagcag cagcagcagc aagagttaac tctgacttag 180ggaatagaga
cagccagaga gaaatgtgat caatgaagga gacatctgga gtgtgcgtgc
240ttcttcagag gggacgggtg atgggcagat ttggaaaaag caccgcagat
tgggaacctt 300atcttttctt tttcntaaaa ttgttgttat gnaaatttgg
gtttttccng taacttntta 360aaaacttaaa agtnggttt
37961255DNAUnknownDescription of Unknown Organism Sequence from
Applicant's cDNA clone HMSCH94R 61aattccgaca atggaaagca ctcttagcct
tgcagtggtc tacattttta aggaaccaat 60atttcagcat tctttattac ccggcacgct
gtgtcctttg tcagagttca agtttatggt 120nactgccagg gtcagacagt
ccatttgctg ctgctgctgc tgctgctgct ttctcgaact 180ggnatggcat
tagggaagct gctgtctgag tgttagggaa tgtcttggct aagtaaagcc
240aatgttcttt cctnn 255625289DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number AB005287 62cgagctctcc
cagccgcagc ctccgaatcc acggcctcca ccccgcgcct ctccagcgct 60ctatcccgtc
gctgcgccct tgtcgccggc cccggccgct gcatccgcgt ccgcacaggc
120tccttgctgg gcacaaatag ctccaccatg gggctggcct ggggactcgg
tgtcctgctc 180ctgttgcatg cctgcggctc caaccgcatt ccagagtctg
ggggagacaa cagtgtgttt 240gacatctttg aactcaccgg agctgcccgc
aagcggtctg ggcgccgact ggtgaagggc 300cctgaccctt ctagcccagc
tttccgcatc gaggatgcca acctgatccc ccctgtgcct 360gacaagaagt
tccaagacct agtggatgct gtgcgggcgg agaaaggttt cctcctcctg
420gcctccctga ggcaaatgaa gaagacccgg ggtaccctgc tggctgtgga
gcggaaagac 480cactctggcc aggtcttcag cgtgatctcc aatggcaagg
cgggcaccct ggacctgagc 540ctgaccgtgc aggggaagca gcatgtggtg
tcggtggaag aagcactcct ggcgactggc 600cagtggaaga gcatcaccct
gtttgtgcag gaggacaggg cccagctgta catcgactgt 660gagaagatgg
agaatgcgga gctggatgtc cccatccaga gcatcttcac cagggacctg
720gccagcatcg ccaggctccg cattgccaaa ggaggtgtca acgacaattt
ccagggggtg 780ctgcagaatg taaggtttgt ctttggaacc acaccagaag
acatcctcag gaacaaaggc 840tgctccagct ctaccagtgt ctttgtcacc
cttgacaaca acgtggtgaa tgggtccagc 900cctgccatcc gcaccgacta
cattggccac aagacaaagg acctgcaagc catctgtggc 960atctcatgtg
acgagctgtc cagcatggtc ctggagctca ggggtctacg caccatcgtg
1020accacgctgc aggacagtat ccgcaaagtg accgaagaga acaaagagct
ggccaacgag 1080ctgaggaggc ccccactctg ctaccacaac ggagtgcagt
acaggactgg cgacgagtgg 1140acggtggaca gctgcactga gtgtcgctgc
cagaactcag ttaccatctg caaaaaagtg 1200tcctgtccca tcatgccctg
ctccaatgcc acagttccgg atggagaatg ctgcccacgg 1260tgctggccca
gcgactctgc agacgatggc tggtccccgt ggtctgagtg gacctcttgc
1320tctgtgacct gtggcaatgg aatccagcag cgtggccgct cctgcgacag
cctcaacaac 1380agatgcgagg gctcctctgt gcagacgcgg acctgccaca
tccaggagtg tgacaagaga 1440tttaaacagg atggcggctg gagccactgg
tccccatggt catcttgctc cgtaacatgt 1500ggagacggtg tgatcacaag
gatccggctc tgcaactccc ccagccccca gatgaatggg 1560aagccatgtg
agggcaaagc ccgggagacc aaagcctgcc agaaagactc ctgccccatc
1620aatggaggct ggggaccttg gtcaccatgg gacatctgtt ctgtcacctg
tggaggaggg 1680gtacagaaac gtagccggct ctgcaacaac cccaaacccc
agtttggagg caaggactgc 1740gttggtgatg tgacagaaaa ccagatctgc
aacaagcagg actgtcccat tgacggatgc 1800ctgtccaatc cctgctttgc
tggtgtccag tgtaccagct accctgatgg cagctggaag 1860tgtggtgcct
gtcccccagg ctatagtgga gatggagtcg agtgcaaaga cgttgatgag
1920tgcaaagaag tccctgatgc ctgcttcaac cacaatggag agcacaggtg
tgagaacaca 1980gaccccggct acaactgcct gccctgccca ccgcgcttca
ctggctcgca gccctttggc 2040cggggcgtgg aacatgccac cgccaacaag
caggtatgca agccccgaaa cccctgcaca 2100gacgggacac acgactgcaa
caagaacgcc aagtgcaact acctgggcca ctacagcgac 2160cccatgtacc
gctgcgagtg caagcctggc tacgccggca acggcatcat ctgcggggag
2220gacacagacc tggacggctg gcccaatgag gacctgctgt gcgtggccaa
cgcaacttac 2280cactgcagaa aggataattg ccccaacctt cccaactcag
ggcaggaaga ctatgacaag 2340gatggaatcg gcgatgcctg cgatgatgac
gatgacaatg ataagattcc agatgacagg 2400gacaactgtc cattccatta
caacccagcc cagtacgact atgacagaga tgacgtggga 2460gaccgctgtg
acaactgccc ctacaaccac aacccagacc aggctgacac agataacaat
2520ggggaaggag acgcctgtgc agctgacatt gatggggaca gtatcctcaa
tgaacgggac 2580aactgccagt atgtctacaa tgtggaccag aaagacactg
acatggacgg ggttggtgat 2640cagtgtgaca actgccccct ggaacacaat
ccagaccagc tcgactctga ctcggaccgc 2700attggagaca cctgtgacaa
caatcaggat attgatgaag acggccacca gaacaatctg 2760gacaactgtc
cctacgtgcc caacgccaac caggctgacc atgacaagga tggcaaaggc
2820gatgcctgtg accatgatga cgacaatgat ggcattcctg atgaccggga
caactgcagg 2880ctggtgccca atcctgacca gaaggactct gatggtgatg
gtcgaggtga tgcttgcaaa 2940gatgattttg accaggacaa ggtgccagac
attgatgaca tctgtcccga aaatgttgat 3000atcagtgaga ctgatttccg
ccgattccag atgattcctc tagatcccaa agggacatcc 3060cagaatgacc
ctaactgggt tgtacgccat cagggtaaag aactcgtcca gactgtcaac
3120tgtgaccctg gacttgctgt aggttatgac gaatttaacg ccgtggactt
cagtggcacc 3180ttcttcatca acaccgagag ggatgacgac tatgccggct
ttgtgtttgg ctaccagtcc 3240agcagccgct tctatgttgt gatgtggaag
caagtcactc agtcctactg ggacaccaac 3300cccacgaggg ctcaggggta
ctctggactt tccgtgaagg ttgtaaactc caccacgggg 3360cctggcgagc
acctgcggaa tgccctgtgg cacacaggaa acacctctgg ccaggtgcgc
3420acactgtggc atgaccctcg tcacattggc tggaaagatt tcactgccta
cagatggcat 3480ctgagccaca ggccaaagac aggtttcatc agagtggtaa
tgtatgaagg gaagaaaatc 3540atggctgact caggacccat ctatgacaaa
acctatgctg gtgggaggct aggcttgttc 3600gtcttctctc aagaaatggt
gttcttctcc gacctgaaat atgaatgcag agactcctaa 3660tcatcaaact
gttgatcaaa agactgatca taaaccaatg ctggtattgc accttctgga
3720accatgggct tagaaaaccc ccaggatcgc gcctcgctgc ctgcctttgc
tctctgcttg 3780catgagtgtg gactcctaga acatgtgact tgcctcaaga
aaatgcaatt ttccaaatca 3840gaccctgcat tcagcctctg actgagaaga
atcttccaag gagacaaaca atgactttgg 3900ttggcttttg caaaagcaaa
agcatccaca tgctttggtt ggaaggtgcc tgtcccactc 3960tgcttttgtc
agagcagaat gcgactgtga ggccagctct gagcagtgga ctccaaaatg
4020ttttcaggca tgtgagagaa gggaggactc actagaattg acaaacaaaa
ccagccctga 4080cctactccct ctggaatggg ggcgggtggg ggggccaaag
cccaaagggg aggatgcata 4140cccaagagat gattgtatga agaaaatatg
gaggaactgt tacatttttg gtactaaatc 4200attttcaggg gattgaaaga
ctattgctgg atttcatgat gctgaccggt gttagctgat 4260taacccacat
aaataggcac ttaaatagga gcagggaagg aaggaaaaga ctggcttctg
4320gacttcctcc cagatttcca ccccttaaca catcacctgt agtgaccaga
acagggagtc 4380ggagttaaac cgacacaagg cagggccagc tgctgcagct
tggttctatt gaaattgtca 4440gttgtattcc agatgtagct tctgcagatg
tagcagcaaa ataagaatac ccaccatctc 4500agcgagcacc aggctgtctc
ccaagggacg gcagccatgc ttgtattttt atggttagaa 4560aggcacaaaa
ttatcaacta agacattcct tctttctctt tttttcctga acatcatgga
4620gttttccagt tgtctctttt ggactgtagt ttttagtgtt ttaaacaaac
actttacaat 4680gtaaactatt tattttttac ttattctggg ggatctgtct
gaaagactat tcatggaaca 4740ggaagaagcg taaggactat ccatatcatc
tttgctacaa gtcattatga ctgtaagatt 4800gtaaatacag attatttatt
aactctgttc tacctggaat ctagtttcat atggaaagtg 4860tttgagagca
ggtagttgag atcgatcagc aaatctttca caggaatggc acaaggaaac
4920cagcatagca agctgctctt caccttgtgc ttagactgga tgatttggaa
ttcttttttc 4980cttttttttc ccaagtggaa ttacttggtt gtccatttgc
aagtgttttt agtttgcaaa 5040gaaagccaag aggccattaa tactgtctta
tcccatccct tgtgcctatt tccagggaga 5100tgaaaagcat ctacatttat
tatttttgcc tttttccaaa agaaaaaaat gacaaaggtg 5160aaacttgtat
acaaatatta cctcatttgt tgtgtgactg agtaaagaat tttgggatca
5220aacagaaaga gtttaagtgt ctaacaaact taaagctact gtagtaccta
aaaaaaaaaa 5280aaaaaaaaa 5289632053DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number X87619 63gaattccggc
ggccgctgag agcccaccct ggcgagctct cccagccgca gcctccgaat 60ccacggcctc
caccccgcgc ctctccagcg ctctatcccg tcgctgcgcc cttgtcgccg
120gccccggcgc tgcatccgcg tccgcacagg ctccttgact gggcacaaat
agctccacca 180tggggctggc ctggggactc ggtgtcctgc tcctgttgca
tgcctgcggc tccaaccgca 240ttccagagtc tgggggagac aacagtgtgt
ttgacatctt tgaactcacc ggagctgccc 300gcaacggtac tgggcgccga
ctggtgaagg gccctgaccc ttctagccca gctttccgca 360tcgaggatgc
caacctgatc ccccctgtgc ctgacaagaa gttccaagac ctagtggatg
420ctgtgcgggc ggagaaaggt ttcctcctcc tggcctccct gaggcaaatg
aagaagaccc 480ggggtaccct gctggctgtg gagcggaaag accactctgg
ccaggtcttc agcgtgatct 540ccaatggcaa ggcgggcacc ctggacctga
gcctgaccgt gcaggggaag cagcatgtgg 600tgtcggtgga agaagcactc
ctggcgactg gccagtggaa gagcatcacc ctgtttgtgc 660aggaggacag
ggcccagctg tacatcgact gtgagaagat ggagaatgcg gagctggatg
720tccccatcca gagcatcttc accagggacc tggccagcat cgccaggctc
cgcattgcca 780aaggaggtgt caacgacaat ttccaggggg tcctgcagaa
tgtaaggttt gtctttggaa 840ccacaccaga agacatcctc aggaacaaag
gctgctccag ctctaccagt gtctttgtca 900cccttgacaa caacgtggtg
aatgggtcca gccctgccat ccgcaccgac tacattggcc 960acaagacaaa
ggacctgcaa gccatctgtg gcatctcatg tgacgagctg tccagcatgg
1020tcctggagct caggggtcta cgcaccatcg tgaccacgct gcaggacagt
atccgcaaag 1080tgaccgaaga gaacaaagag ctggccaacg agctgaggag
gcccccactc tgctaccaca 1140acggagtgca gtacaggact ggcgacgagt
ggacggtgga cagctgcact gagtgtcgct 1200gccagaactc agttaccatc
tgcaaaaaag tgtcctgtcc catcatgccc tgctccaatg 1260ccacagttcc
ggatggagaa tgctgcccac ggtgctggcc cagcgactct gcagacgacg
1320gctggtcccc gtggtctgag tggacctctt gctctgtgac ctgtggcaat
ggaatccagc 1380agctggccgc tcctgcgaca gcctcaacaa cagatgcgag
ggctcctctg tgcagacgcg 1440gacctgccac atccaggagt gtgacaagag
atttaaacag gatggcggct ggagccactg 1500gtccccatgg tcatcttgct
ccgtaacatg tggagacggt gtgatcacaa ggatccggct 1560ctgcaactcc
cccagccccc agatgaatgg gaagccatgt gagggcaaag cccgggagac
1620caaagcctgc cagaaagact cctgccccat caatggaggc tggggacctt
ggtcaccatg 1680ggacatctgt tctgtcacct gtggaggagg ggtacagaaa
cgtagccggc tctgcaacaa 1740ccccacaccc cagtttggag gcaaggactg
cattggtgat gtgacagaaa accagatctg 1800caacaagcag gactgtccca
ttgacggatg cctgtccaat ccctgctttg ctggtgtcca 1860gtgtaccagc
taccctgatg gcagctggaa gtgtggtgcc tgtcccccag gctatagtgg
1920agatggagtc gagtgcaaag acgttgatga gtgcaaagaa gtccctgatg
cctgcttcaa 1980ccacaatgga gagcacaggt gtgagaacac agaccccggc
tacaactgcc tgccctgccc 2040accgcccgga att
2053644339DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number M87276 64agccactgcc tggagtcagc cagcctcatc
ggacttctgc aggcaatcgc gaagctgcta 60tccagttctg ccacggtctc tcccggcgca
ccggcagtct cagcgtcttc accggactca 120gcgtccttgt ccttcacttc
acctttgcca cctctccggg ttactgagcc ccggtgcaca 180caggctccgt
gttgggcaca aaggctccac catggagctc ctgcggggac taggtgtcct
240gttcctgttg catatgtgtg gaagcaaccg cattccagag tctgggggag
ataacggtgt 300gtttgacatc tttgaactca ttggaggtgc acgaaggggc
cccggtcgcc gactggtgaa 360gggccaagat ctatccagcc ccgccttccg
cattgagaat gccaacctga tccccgctgt 420gccggatgac aagttccaag
acctactgga cgctgtgtgg gccgacaaag gcttcatctt 480cttggcttcc
ttgaggcaga tgaagaagac ccggggcaca ctcctggctg tggaacggaa
540agacaacact ggccagatct tcagtgtggt ctccaacggc aaagctggca
ccctggacct 600gagcctgagc ctgccaggga agcaacaagt ggtgtcagtg
gaggaagctc tcctggccac 660tggccagtgg aagagcatca cgctgtttgt
tcaagaggac cgggctcaac tctacataga 720ctgtgataag atggagagcg
cggagctgga tgtacccatc cagagcatct tcaccaggga 780tctggccagc
gttgccaggc tccgagttgc aaagggagat gtcaatgaca attttcaggg
840ggtgctgcag aatgtgaggt ttgtctttgg aaccacccca gaagacattc
tcaggaacaa 900aggctgctcc agctctacca acgtccttct tacccttgac
aacaacgtgg tgaacggttc 960cagccctgct atccgcacca actacatcgg
ccacaaaaca aaggacctcc aagctatctg 1020tggcctctcc tgtgatgaac
tatccagcat ggtcctggaa ctgaagggcc tgcgcaccat 1080cgtgaccact
ctgcaggaca gcatccgaaa agtgacggaa gagaacagag agctggtcag
1140tgagctgaag cggcctcccc tctgctttca caatggagtc cagtacaaga
acaacgagga 1200gtggactgta gacagttgca cagagtgtca ctgccagaac
tcggttacca tctgcaaaaa 1260ggtgtcctgt cccatcatgc cctgctccaa
cgccacagtt cctgatggtg aatgctgccc 1320acggtgctgg cccagcgact
ctgctgacga tggctggtct ccctggtctg agtggacctc 1380ctgctctgcc
acatgtggca atggaattca gcaacgtggt cgttcctgtg acagcctcaa
1440caacagatgc gagggctctt cggtacagac gaggacctgc cacattcagg
agtgtgacaa 1500aagatttaaa caggatggtg gctggagtca ctggtctcca
tggtcgtcct gttctgtgac 1560ctgtggtgac ggtgtgatca caaggatccg
tctctgcaac tcccccagcc cccagatgaa 1620cgggaagccc tgtgaaggtg
aagcccggga gaccaaagcc tgcaagaaag acgcctgccc 1680aattaatgga
ggctggggtc cctggtcacc atgggacatc tgctctgtca cctgtggagg
1740aggagtgcag agacgcagcc gactctgtaa caaccccaca ccccagtttg
gaggcaaaga 1800ctgtgttggc gatgtgacag aaaatcaagt ttgcaacaag
caggactgcc caattgatgg 1860atgcctgtcc aatccctgct ttgctggtgc
caagtgtact agctaccctg atggtagctg 1920gaaatgtggt gcgtgtcctc
ctggctacag tggaaatggc atccagtgca aagacgtcga 1980tgagtgcaaa
gaagtgcctg atgcttgctt caatcacaac ggagaacatc ggtgcaagaa
2040cacagatcct ggctacaact gcctgccctg cccaccacga ttcactggct
cacagccctt 2100cggccgaggt gtcgaacatg ccatggccaa caaacaggtg
tgcaaaccgc gaaacccctg 2160cacggacggg acgcatgact gcaacaagaa
cgctaagtgc aactacctgg gtcactacag 2220cgaccccatg taccgctgtg
agtgcaagcc cggctatgca ggcaatggca tcatctgcgg 2280agaggacaca
gacctggacg gctggcctaa tgaaaacctg gtgtgtgtgg ccaacgcaac
2340ctaccactgc aaaaaggaca actgccccaa ccttcccaac tcggggcagg
aagactatga 2400caaggacggg attggcgatg cctgcgatga tgacgatgac
aacgacaaga tccctgatga 2460cagggacaac tgtccattcc attacaaccc
agcccagtat gactatgaca gagatgatgt 2520gggagaccgc tgtgacaact
gcccctacaa ccacaaccct gaccaagcag acacagacaa 2580aaacggggag
ggcgatgcct gtgctgtgga catcgatgga gatggaatcc tcaatgaacg
2640agacaactgc cagtacgttt acaacgtgga ccagagggac acggacatgg
atggggttgg 2700agatcagtgt gacaactgcc ccctggaaca caatccagac
cagctggact ctgactcaga 2760cctcataggg gacacttgtg acaacaatca
ggacatcgat gaggatggcc atcagaacaa 2820cctggacaac tgtccctatg
tgcctaacgc caaccaggcc gaccatgata aagatggcaa 2880aggagatgcc
tgtgaccatg acgatgacaa tgacggcatc cctgatgaca gagacaactg
2940caggctggtg cccaatcctg accagaagga ctctgatggt gatggccgag
gtgacgcctg 3000caaagacgac tttgaccatg acaatgtgcc agatattgat
gacatctgtc ctgagaattt 3060tgacatcagt gaaaccgatt tccgacgatt
ccagatgatt cctctagatc ccaaaggaac 3120ctcccaaaat gaccctaact
gggttgtccg ccatcagggc aaagaactcg tccagactgt 3180aaactgtgac
cctggacttg ctgtaggtta tgatgagttt aatgctgtgg acttcagcgg
3240taccttcttc atcaacaccg agagagatga tgactacgct ggcttggtat
tcggctacca 3300gtccagcagc cgcttctacg ttgtgatgtg gaaacaagtc
acccagtcct actgggacac 3360caaccccaca agggctcagg gatactcagg
cctgtctgta aaggttgtga actccaccac 3420cggccctggc gagcacctgc
ggaatgcact gtggcacaca ggaaacaccc ctggccaggt 3480gcgcaccctg
tggcatgacc ctcgccacat cggctggaaa gatttcactg cgtacagatg
3540gcgtctcagc cacaggccaa agaccggtta tatcagagtg gtgatgtatg
aaggaaagaa 3600aatcatggct gactcgggac ccatctatga caaaacctac
gccggcggta gactaggcct 3660gttcgtcttc tctcaggaaa tggtgttctt
ctcagacatg aaatacgagt gtcgagattc 3720ctaatcatca gctgccaatc
ataaccagcg ctggcaatgc accttctaaa aacaagggct 3780agagaaaccc
cccacccctg ccgggatcgc ctttcctcgc cttccttgcc tctcttcttg
3840catagtgtgg acttgtaaag cctgagacct gcctcaagaa aatgcagttt
tcgaacccag 3900agtcagcact cggcctttaa cgaatgagaa tgcatcttcc
aagaccatga agagttcctt 3960gggtttgctt ttgggaaagc caaagcgcct
atttacttcc cactaggaag gtgcccgctc 4020cactctgcct tactcacaga
gccagaactt cttcgaggcc acctctgagc agcacacaca 4080gaagcatttt
caggcatgtc aaagaaagga aaaatgactc actagaactc accgccaaac
4140aacctctgac ataggtcctg agatgtgggg aggcaggagc caaagctcta
gggagggcat 4200gtacccaaga gatgactgta tgaagaaaat gtggaggagc
tgttcggtac taaatcattt 4260tcaggggaca gacagacttg ctgcatttcc
gcatgctgct ggtgagagct gattgaccca 4320atcttccaca caggcactt
433965186DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number M62458 65gcacagttaa tggaggctgg ggtccctggt
caccatggga catctgctct gtcacctgtg 60gaggaggagt gcagagacgc agccgactct
gtaacaaccc cacaccccag tttggaggca 120aagactgtgt tggcgatgtg
acagaaaatc aagtttgcaa caagcaggac tgcccaattg 180gtaagc
186665774DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number AB002364 66gtcactttgg ttgatagcag ccgctctggt
agaggttagg acttcagctg atggacaagc 60tggtaatgaa gaaatggtgc aaatagattt
accaataaag agatatagag agtatgagct 120ggtgactcca gtcagcacaa
atctagaagg acgctatctc tcccatactc tttctgcgag 180tcacaaaaag
aggtcagcga gggacgtgtc ttccaaccct gagcagttgt tctttaacat
240cacggcattt ggaaaagatt ttcatctgcg actaaagccc aacactcaac
tagtagctcc 300tggggctgtt gtggagtggc atgagacatc tctggtgcct
gggaatataa ccgatcccat 360taacaaccat caaccaggaa gtgctacgta
tagaatccgg aaaacagagc ctttgcagac 420taactgtgct tatgttggtg
acatcgtgga cattccagga acctctgttg ccatcagcaa 480ctgtgatggt
ctggctggaa tgataaaaag tgataatgaa gagtatttca ttgaaccctt
540ggaaagaggt aaacagatgg aggaagaaaa aggaaggatt catgttgtct
acaagagatc 600agctgtagaa caggctccca tagacatgtc caaagacttc
cactacagag agtcggacct 660ggaaggcctt gatgatctag gtactgttta
tggcaacatc caccagcagc tgaatgaaac 720aatgagacgc cgcagacacg
cgggagaaaa cgattacaat atcgaggtac tgctgggagt 780ggatgactct
gtggtccgtt tccatggcaa agagcacgtc caaaactacc tcctgaccct
840aatgaacatt gtgaatgaaa tttaccatga tgagtccctc ggagtgcata
taaatgtggt 900cctggtgcgc atgataatgc tgggatatgc aaagtccatc
agcctcatag aaaggggaaa 960cccatccaga agcttggaga atgtgtgtcg
ctgggcgtcc caacagcaaa gatctgatct 1020caaccactct gaacaccatg
accatgcaat ttttttaacc aggcaagact ttggacctgc 1080tggaatgcaa
ggatatgctc cagtcaccgg catgtgtcat ccagtgagaa gttgtaccct
1140gaatcatgag gatggttttt catctgcttt tgtagtagcc catgaaacgg
gccatgtgtt 1200gggaatggag catgatggac aaggcaacag gtgtggtgat
gagactgcta tgggaagtgt 1260catggctccc ttggtacaag cagcattcca
tcgttaccac tggtcccgat gcagtggtca 1320agaactgaaa agatatatcc
attcctatga ctgtctcctt gatgaccctt ttgatcatga 1380ttggcctaaa
ctcccagaac ttcctggaat caattattct atggatgagc aatgtcgttt
1440tgattttggt gttggctata aaatgtgcac cgcgttccga acctttgacc
catgtaaaca 1500gctgtggtgt agccatcctg ataatcccta cttttgtaag
actaaaaagg gacctccact 1560tgatgggact gaatgtgctg ctggaaaatg
gtgctataag ggtcattgca tgtggaagaa 1620tgctaatcag caaaaacaag
atggcaattg ggggtcatgg actaaatttg gctcctgttc 1680tcggacatgt
ggaactggtg ttcgtttcag aacacgccag tgcaataatc ccatgcccat
1740caatggtggt caggattgtc ctggtgttaa ttttgagtac cagctttgta
acacagaaga 1800atgccaaaaa cactttgagg acttcagagc acagcagtgt
cagcagcgaa actcccactt 1860tgaataccag aataccaaac accactggtt
gccatatgaa catcctgacc ccaagaaaag 1920atgccacctt tactgtcagt
ccaaggagac tggagatgtt gcttacatga aacaactggt 1980gcatgatgga
acgcactgtt cttacaaaga tccatatagc atatgtgtgc gaggagagtg
2040tgtgaaagtg ggctgtgata aagaaattgg ttctaataag gttgaggata
agtgtggtgt 2100ctgtggagga gataattccc actgccgaac cgtgaagggg
acatttacca gaactcccag 2160gaagcttggg taccttaaga tgtttgatat
accccctggg gctagacatg tgttaatcca 2220agaagacgag gcttctcctc
atattcttgc tattaagaac caggctacag gccattatat 2280tttaaatggc
aaaggggagg aagccaagtc gcggaccttc atagatcttg gtgtggagtg
2340ggattataac attgaagatg acattgaaag tcttcacacc gatggacctt
tacatgatcc 2400tgttattgtt ttgattatac ctcaagaaaa tgatacccgc
tctagcctga catataagta 2460catcatccat gaagactctg tacctacaat
caacagcaac aatgtcatcc aggaagaatt 2520agatactttt gagtgggctt
tgaagagctg gtctcaggtt tccaaaccct gtggtggagg 2580tttccagtac
actaaatatg gatgccgtag gaaaagtgat aataaaatgg tccatcgcag
2640cttctgtgag gccaacaaaa agccgaaacc tattagacga atgtgcaata
ttcaagagtg 2700tacacatcca ctctgggtag cagaagaatg ggaacactgc
accaaaacct gtggaagttc 2760tggctatcag cttcgcactg tacgctgcct
tcagccactc cttgatggca ccaaccgctc 2820tgtgcacagc aaatactgca
tgggtgaccg tcccgagagc cgccggccct gtaacagagt 2880gccctgccct
gcacagtgga aaacaggacc ctggagtgag tgttcagtga cctgcggtga
2940aggaacggag gtgaggcagg tcctctgcag ggctggggac cactgtgatg
gtgaaaagcc 3000tgagtcggtc agagcctgtc aactgcctcc ttgtaatgat
gaaccatgtt tgggagacaa 3060gtccatattc tgtcaaatgg aagtgttggc
acgatactgc tccataccag gttataacaa 3120gttatgttgt gagtcctgca
gcaagcgcag tagcaccctg ccaccaccat accttctaga 3180agctgctgaa
actcatgatg atgtcatctc taaccctagt gacctcccta gatctctagt
3240gatgcctaca tctttggttc cttatcattc agagacccct gcaaagaaga
tgtctttgag 3300tagcatctct tcagtgggag gtccaaatgc atatgctgct
ttcaggccaa acagtaaacc 3360tgatggtgct aatttacgcc agaggagtgc
tcagcaagca ggaagtaaga ctgtgagact 3420ggtcaccgta ccatcctccc
cacccaccaa gagggtccac ctcagttcag cttcacaaat 3480ggctgctgct
tccttctttg cagccagtga ttcaataggt gcttcttctc aggcaagaac
3540ctcaaagaaa gatggaaaga tcattgacaa cagacgtccg acaagatcat
ccaccttaga 3600aagatgagaa agtgaaccaa aaaggctaga aaccagagga
aaacctggac aacctctctc 3660ttcccatggt gcatatgctt gtttaaagtg
gaaatctcta tagatcgtca gctcatttta 3720tctgtaattg gaagaacaga
aagtgctggc tcactttcta gttgctttca tcctcctttt 3780gttctgcatt
gactcattta ccagaattca ttggaagaaa tcaccaaaga ttattacaaa
3840agaaaaatat gttgctaaga ttgtgttggt cgctctctga agcagaaaag
ggactggaac 3900caattgtgca tatcagctga ctttttgttt gttttagaaa
agttacagta aaaattaaaa 3960agagatacca atggtttaca ctttaacaag
aaattttgga tatggaacaa agaattctta 4020gacttgtatt cctatttatc
tatattagaa atattgtatg agcaaatttg cagctgttgt 4080gtaaatactg
tatattgcaa aaatcagtat tattttaaga gatgtgttct caaatgattg
4140tttactatat tacatttctg gatgttctag gtgcctgtcg ttgagtattg
ccttgtttga 4200cattctatag gttaattttc aaagcagagt attacaaaag
agaagttaga attacagcta 4260ctgacaatat aaagggtttt gttgaatcaa
caatgtgata cgtaaattat agaaaaagaa 4320aagaaacaca aaagctatag
atatacagat atcagcttac
ctattgcctt ctatacttat 4380aatttaaagg attggtgtct tagtacactt
gtggtcacag ggatcaacga atagtaaata 4440atgaactcgt gcaagacaaa
actgaaaccc tctttccagg acctcagtag gcaccgttga 4500ggtgtccttt
gtttttgtgt gtgtgtgttc ttttttaatt ttcgcattgt tgacagatac
4560aaacagttat actcaatgta ctgtaataat cgcaaaggaa aaagttttgg
gataacttat 4620ttgtatgttg gtagctgaga aaaatatcat cagtctagaa
ttgatatttg agtatagtag 4680agctttgggg ctttgaaggc aggttcaaga
aagcatatgt cgatggttga gatatttatt 4740ttccatatgg ttcatgttca
aatgttcaca accacaatgc atctgactgc aataatgtgc 4800taataattta
tgtcagtagt caccttgctc acagcaaagc cagaaatgct ctctccaggg
4860agtagatgta aagtacttgt acatagaatt cagaactgaa gatatttatt
aaaagttgat 4920ttttttttct tgatagtatt tttatgtact aaatatttac
actaatatca attacatatt 4980ttggtaaact agagagacat aattagagat
gcatgctttg ttctgtgcat agagaccttt 5040aagcaaacta ctacagccaa
ctcaaaagct aaaactgaac aaatttgatg ttatgcaaac 5100atcttgcatt
tttagtagtt gatattaagt tgatgacttg tttcccttca aggaaacatt
5160aaattgtatg gactcagcta gctgttcaat gaaattgtga attagaaaca
tttttaaaag 5220tttttgaaag agataagtgc atcatgaatt acatgtacat
gagaggagat agtgatatca 5280gcataatgat tttgaggtca gtacctgagc
tgtctaaaaa tatattatac aaactaaaat 5340gtagatgaat taacctctca
aagcacagaa tgtgcaagaa cttttgcatt ttaatcgttg 5400taaactaaca
gcttaaacta ttgactctat acctctaaag aattgctgct actttgtgca
5460agaactttga aggtcaaatt aggcaaattc cagatagtaa aacaatccct
aagccttaag 5520tctttttttt ttcctaaaaa ttcccataga ataaaattct
ctctagttta cttgtgtgtg 5580catacatctc atccacaggg gaagataaag
atggtcacac aaacagtttc cataaagatg 5640tacatattca ttatacttct
gacctttggg ctttcttttc tactaagcta aaaattcctt 5700tttatcaaag
tgtacactac tgatgctgtt tgttgtactg agagcacgta ccaataaaaa
5760tgttaacaaa atat 5774675535DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number AB005297 67ggactttaga
agccgttgct gccctctctg tcacctgaag cggggccctc tcccatccca 60cccttgcccc
gcctccctgc ccccaccggg ccggccctgc ccgccgccgg accctggcat
120gtcaagacct ggtccgcgcc tgcctgccca gcccgcggaa ccccggcggc
cccgcgagct 180aggatgaggg gccaggccgc cgccccgggc cccgtctgga
tcctcgcccc gctgctactg 240ctgctgctgc tgctgggacg ccgcgcgcgg
gcggccgccg gagcagacgc ggggcccggg 300cccgagccgt gcgccacgct
ggtgcaggga aagttcttcg gctacttctc cgcggccgcc 360gtgttcccgg
ccaacgcctc gcgctgctcc tggacgctac gcaacccgga cccgcggcgc
420tacactctct acatgaaggt ggccaaggcg cccgtgccct gcagcggccc
cggccgcgtg 480cgcacctacc agttcgactc cttcctcgag tccacgcgca
cctacctggg cgtggagagc 540ttcgacgagg tgctgcggct ctgcgacccc
tccgcacccc tggccttcct gcaggccagc 600aagcagttcc tgcagatgcg
gcgccagcag ccgccccagc acgacgggct ccggccccgg 660gccgggccgc
cgggccccac cgacgacttc tccgtggagt acctggtggt ggggaaccgc
720aaccccagcc gtgccgcctg ccagatgctg tgccgctggc tggacgcgtg
tctggccggt 780agtcgcagct cgcacccctg cgggatcatg cagaccccct
gcgcctgcct gggcggcgag 840gcgggcggcc ctgccgcggg acccctggcc
ccccgcgggg atgtctgctt gagagatgcg 900gtggctggtg gccctgaaaa
ctgcctcacc agcctgaccc aggaccgggg cgggcacggc 960gccacaggcg
gctggaagct gtggtccctg tggggcgaat gcacgcggga ctgcggggga
1020ggcctccaga cgcggacgcg cacctgcctg cccgcgccgg gcgtggaggg
cggcggctgc 1080gagggggtgc tggaggaggg tcgccagtgc aaccgcgagg
cctgcggccc cgctgggcgc 1140accagctccc ggagccagtc cctgcggtcc
acagatgccc ggcggcgcga ggagctgggg 1200gacgagctgc agcagtttgg
gttcccagcc ccccagaccg gtgacccagc agccgaggag 1260tggtccccgt
ggagcgtgtg ctccagcacc tgcggcgagg gctggcagac ccgcacgcgc
1320ttctgcgtgt cctcctccta cagcacgcag tgcagcggac ccctgcgcga
gcagcggctg 1380tgcaacaact ctgccgtgtg cccagtgcat ggtgcctggg
atgagtggtc gccctggagc 1440ctctgctcca gcacctgtgg ccgtggcttt
cgggatcgca cgcgcacctg caggcccccc 1500cagtttgggg gcaacccctg
tgagggccct gagaagcaaa ccaagttctg caacattgcc 1560ctgtgccctg
gccgggcagt ggatggaaac tggaatgagt ggtcgagctg gagcgcctgc
1620tccgccagct gctcccaggg ccgacagcag cgcacgcgtg aatgcaacgg
gccttcctac 1680gggggtgcgg agtgccaggg ccactgggtg gagacccgag
actgcttcct gcagcagtgc 1740ccagtggatg gcaagtggca ggcctgggcg
tcatggggca gttgcagcgt cacgtgtggg 1800gctggcagcc agcgacggga
gcgtgtctgc tctgggccct tcttcggggg agcagcctgc 1860cagggccccc
aggatgagta ccggcagtgc ggcacccagc ggtgtcccga gccccatgag
1920atctgtgatg aggacaactt tggtgctgtg atctggaagg agaccccagc
gggagaggtg 1980gctgctgtcc ggtgtccccg caacgccaca ggactcatcc
tgcgacggtg tgagctggac 2040gaggaaggca tcgcctactg ggagcccccc
acctacatcc gctgtgtttc cattgactac 2100agaaacatcc agatgatgac
ccgggagcac ctggccaagg ctcagcgagg gctgcctggg 2160gagggggtct
cggaggtcat ccagacactg gtggagatct ctcaggacgg gaccagctac
2220agtggggacc tgctgtccac catcgatgtc ctgaggaaca tgacagagat
tttccggaga 2280gcgtactaca gccccacccc tggggacgta cagaactttg
tccagatcct tagcaacctg 2340ttggcagagg agaatcggga caagtgggag
gaggcccagc tggcgggccc caacgccaag 2400gagctgttcc ggctggtgga
ggactttgtg gacgtcatcg gcttccgcat gaaggacctg 2460agggatgcat
accaggtgac agacaacctg gttctcagca tccataagct cccagccagc
2520ggagccactg acatcagctt ccccatgaag ggctggcggg ccacgggtga
ctgggccaag 2580gtgccagagg acagggtcac tgtgtccaag agtgtcttct
ccacggggct gacagaggcc 2640gatgaagcat ccgtgtttgt ggtgggcacc
gtgctctaca ggaacctggg cagcttcctg 2700gccctgcaga ggaacacgac
cgtcctgaat tctaaggtga tctccgtgac tgtgaaaccc 2760ccgcctcgct
ccctgcgcac acccttggag atcgagtttg cccacatgta taatggcacc
2820accaaccaga cctgtatcct gtgggatgag acggatgtac cctcctcctc
cgcccccccg 2880cagctcgggc cctggtcgtg gcgcggctgc cgcacggtgc
ccctcgacgc cctccggacg 2940cgctgcctct gtgaccggct ctccaccttc
gccatcttag cccagctcag cgccgacgcg 3000aacatggaga aggcgactct
gccgtcggtg acgctcatcg tgggctgtgg cgtgtcctct 3060ctcaccctgc
tcatgctggt catcatctac gtgtccgtgt ggaggtacat tcgctcagag
3120cgttctgtca tcctcatcaa cttctgcctg tccatcatct cctccaatgc
cctcatcctc 3180atcgggcaga cccagacccg caacaaggtg atgtgcacgc
tggtggccgc cttcctgcac 3240ttcttcttcc tgtcctcctt ctgctgggtg
ctcaccgagg cctggcagtc ctacatggcc 3300gtgacgggcc acctccggaa
ccgcctcatc cgcaagcgct tcctctgcct gggctggggg 3360ctccctgcac
tggttgtggc catttctgtg ggattcacca aggccaaagg gtacagcacc
3420atgaactact gctggctctc cctggagggg ggactgctct atgccttcgt
gggacctgcc 3480gctgccgttg tgctggtgaa catggtcatt gggatcctgg
tgttcaacaa gctcgtgtcc 3540aaagacggca tcacggacaa gaagctgaag
gagcgggcag gggcctccct gtggagctcc 3600tgcgtggtgc tgccgctgct
ggcgctgacc tggatgtcgg ctgtgctcgc cgtcaccgac 3660cgccgctccg
ccctcttcca gatcctcttc gctgtcttcg actcgctgga gggcttcgtc
3720atcgtcatgg tgcactgtat cctccgtaga gaggtccagg acgctgtgaa
atgccgtgtg 3780gttgaccggc aggaggaggg caacggggac tcagggggct
ccttccagaa cggccacgcc 3840cagctcatga ccgacttcga gaaggacgtg
gatctggcct gtagatcagt gctgaacaag 3900gacatcgcgg cctgccgcac
tgccaccatc acgggcacac tgaagcggcc gtctctgccc 3960gaggaggaga
agctgaagct ggcccatgcc aaggggccgc ccaccaattt caacagcctg
4020ccggccaacg tgtccaagct gcacctgcac ggctcacccc gctatcccgg
cgggcccctg 4080cccgacttcc ccaaccactc actgaccctc aagagggaca
aggcgcccaa gtcctccttc 4140gtcggtgacg gggacatctt caagaagctg
gactcggagc tgagccgggc ccaggagaag 4200gctctggaca cgagctacgt
gatcctgccc acggccacgg ccacgctgcg gcccaagccc 4260aaggaggagc
ccaagtacag catccacatt gaccagatgc cgcagacccg cctcatccac
4320ctcagcacgg cccccgaggc cagcctcccc gcccgcagcc cgccctcccg
ccagcccccc 4380agcggcgggc cccccgaggc accccctgcc cagcccccac
cgcctccgcc cccaccgcca 4440ccacctcccc agcagcccct gcccccaccg
cccaatctgg agccggcacc ccccagcctg 4500ggggatcccg gggagcctgc
cgcccatccg ggacccagca cggggcccag caccaagaac 4560gagaatgtcg
ccaccttgtc tgtgagctcc ctggagcggc ggaagtcgcg gtatgcagaa
4620ctggactttg agaagatcat gcacacccgg aagcggcacc aagacatgtt
ccaggacctg 4680aaccggaagc tgcagcacgc agcggagaag gacaaggagg
tgctggggcc ggacagcaag 4740ccggaaaagc agcagacgcc caacaagagg
ccctgggaga gcctccggaa agcccacggg 4800acgcccacgt gggtgaagaa
ggagctggag ccgctgcagc cgtcgccgct ggagcttcgc 4860agcgtggagt
gggagaggtc gggcgccacg atcccgctgg tgggccagga catcatcgac
4920ctccagaccg aggtctgagc gggtgggcgg cggccacgca ctgggccacg
gaggagggat 4980gctgctccgc ccgctcctgc cgcagacggg cacagacacg
ctcgcgggca gcgggccagg 5040cccgcacccc ggcctcaggg cgctcagacg
gcggccaggc acagggcccg cagtgctggg 5100accagagcca gatgcaggac
aggaggcggc ccggccagcg ggcacagggc accagaggcc 5160gaaggtgcct
cagactccgc cctcctcggg ccgaggccca gcgggcagat gggcggacgg
5220ctgtggaccg tggacaggcc cagcgcggcc agcgtcccag ggtacccgcc
tgagctcctg 5280ctgcggagga gctgcctgct tggcccggcc ggcctggcac
cgttttttaa acacccccat 5340ccctcgggaa gcagccagct ccccacacct
tccagggccc taggcccctc ctagacccag 5400gtggagggca cagccctccg
accctcatgg cccccagggg caggactgag tcccctccag 5460gaagaagcag
gggggaatct attttttctc tccttttctt ttcttcaata aaaagaatta
5520aaaacccaaa aaaaa 553568398DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number X69161 68cggggcaacc
cgctggagtg gacgggccag gtgacggtgc gcaagaagcg caagccctac 60tccaagttcc
agacgctcga gctcgagaag gagttcctct tcaacgcgta cgtcagcaag
120cagaagcgct gggagctggc gcgcaacctc aacctcaccg agcgccaggt
caagatctgg 180ttccagaacc ggcgcatgaa gaacaagaag aacagccagc
gccaggcggc cagcagcagc 240agcagcaaca gcagcagcag cagcagcagc
aacagcagca agcggccgcc ggcggggcgt 300cggccgccgc caacggccac
cagggccacc aagcgcacca ccacgcgccc cccaacggcg 360ccgtcgcagc
cctcaagcac caccagtgac ccgtagcg 398698670DNAUnknownDescription of
Unknown Organism GenBank Clone Accession Number X16619 69cccgggtgcg
gtgtcgtgtg tggggctggg cgccatgttc ctggacatgc tgagggccaa 60gcgcgacacg
gcgcccgacc gccgccagct ggacgaccgg atgatggggg cggacccggg
120ggacatagcg gccaaggtga gggcagggtt ttgcgtgcgt gcttgattgt
gcgtgtgcgt 180gcgtgcgtgc gtgcgtgcgg tgttgcgtgt gtatttgaac
tgtgttttgt gtatgtactt 240aggggtaaga gtgcatacac atgcatgcga
ccggtggcct tacaaatcaa caacacgtac 300gcctgcatgt atccaggtgg
cagcgtggcg acgagcacgt ggcttcgagg gcccaggcac 360ggcgggcccc
agcggcagcg ccgccagtgg cagcggcgcc agcggctcgg caccgcaggc
420gcgctcgccc cgacctcagc caccgcggcc gcgctcacct tcacgcgggt
gaaccccggc 480gaggagccgc ccgtgtacgc gtgcgagcaa acaggtgcgt
aagcgacgtg tgggcagcgc 540gaagaggcgt gggggcgaga gagcaaaggg
actagggaaa cgcacagcca aatacggtat 600gcgggcaacg aggcgatggc
cctggaaatc gcagggccct tttgaaatcg tgtaaggcgc 660aattgctggg
cgactaccgt agtctactga tgcattgcac tacttgtatt actgtatcct
720actgcagtag tgccgttgcc agccgcgctg ctgccctttg gctcccttcc
caatccaaat 780ggcccatgcc tcgcgcactc cgagcaccca gagcacccag
aagccgttgc gtgcgctccg 840ccgccgccct ctcccccgcc ttcacttctt
aattaatcgt gaatgtaatc cccccccccc 900ccgcttcctc aggctgggtg
cacgtgtgcg cgacgcctgc acggagggtg tggtggatgc 960ccgcagcgaa
ctgctggtgt gcccggtgag tcgacgagga ggaggtgcaa gggggatacc
1020agcgcgtgtt tctcagggcc tgtgtgggac accgaaacgt ggtaaaagag
acccgcccgc 1080gaactgtgta tgtggagtag cgtggcgtgt gcggccggac
cgacaaggca gcttgtggac 1140tgccccacgt tgcagagtca gctgacaacg
acacgtgcgc cttcctgtca ttgcccgtgc 1200gcacgcacgt cctccgcact
cccaacaaat tgacagcgac acgtgcgcct tcctataagc 1260ctatgcccgc
acacgctccc gcgccctcag gtgtcgggcc agaccacaga ccggttggtc
1320cacgagtgcg aggaggatga ggcgggcggc tgcggcggcg ccggcggggc
gccgcggcga 1380ggaggacggc ctgggactgg gcatcacagg tgggtggcag
gctggcaggg actcacgcat 1440gggccttgta cgtgactgcg gttctgcatg
gctagtggct cacgcgctgc gcacgttcac 1500gtacggcttg tgggcatgca
gtgccttgac gtgaggctgc gctgccttgc tgctgccgcc 1560ttgccccgct
ccctgcacac actgcagccg gcttcgggcg ctacttcacc gcgggctacg
1620agtgcgagaa cgcgcagcag ctcaacaggc tgctggggta caaggcgctg
tgagagcgcg 1680ccgcaggggg agtgtgttca tattgtggtt gtttgggccg
tgggcgcggg ctgcatgtgc 1740gtattgcacg cgtacagcat tggtgactgg
tcaggtgtaa gcggccggca gtgcgccgcg 1800aggcgctgca gcgagttgtg
gggcatgcgt catgcgcaga cggcccctgg acgacaaggc 1860gttgagttgg
cgtttggagg tgtgggacga cgtggggttt gtgccgtcaa agcacagaac
1920agaaggcgtg accgttttac gagctcgtat gatgtagcat ggattgaata
atgacatgtg 1980atttttgtta caagcgacga atgcgtgggg ttttggatgg
caggggtttc agtcgcccga 2040ttgcgcatgc acacgtgacc aaatttatgc
tcaacgacgt gaccattgct ttatacatac 2100ttgtgtatcg gttggcactt
ataacaattg gctcgtcaaa ttgacgcgag gctgcacttc 2160gatcctgaaa
gccccagttc aacaagtcgg atagccaaat ggccccgctc gctctccagc
2220atcaaggggc ctctaagtgc ctcgcggcaa cccagcgcaa gtgtgctcgc
gttgcggtga 2280gctggactcg tgcacttgtc gacgccgtcg gcaccgcaat
cgaaagacgc gtgcgtcgag 2340caattgtgga agccgctgac gaattgtccg
catgtgacat tgcaggctcg cgtccccgct 2400cgtctcagcg tcatggccca
ggtgcggacg ttgggactgc acttgcacga atgtgatggg 2460gccgcaccga
gtctgcgcgg acgtctcgct gacgtttcgc gttgaatgca tctcgcaata
2520ggcagctgct gcgcctgctg acaacactaa gaagctgtgg ggcggtcgct
tcacgggcaa 2580gacggacccg ctcatggaga agttcaacga gtcgctgccc
tttgacaagc gcctgtgggc 2640tgaggacatc aaggtgcggc acagggaggg
gggcgagtgg tggggtgggg ctggggggga 2700cgcgggtttg gtggccaggg
cagggaggga agacgtgcgg ggctaggcaa gaggctgcga 2760gggcccaggg
taacaccaga ccgtgccgtg tcgcgtgccc ggcttgctgc ccaccttgcc
2820cggccatccc caccgccctc cccaccagca atgacacgta cacattcaca
cactccccca 2880cacccacata cccacacacc cacgcattcc ccaacagggc
agccaggcgt acgccaaggc 2940tcttgccaag gccggcattc tggcacatga
cgaggccgtg accattgtgg aggggctggc 3000caaggtgcgc acacccggca
gcagggcggg tgggtgggtg ggtggggtgg gggggcagag 3060agaggcgcgg
gctgagaggg ggctgagagg ggggtcagcg aggcgcaggc tcagggggag
3120gcgtctgagg ggggctgaga tggtggtggg ggagctgcgg gtgctggggc
tgctgcggtg 3180gcgggcgggc gggcgggcgg gcgacgtgta cgtgagtagc
cgctgaccgg gcgctgggcc 3240tttgcgcacg ccacagccca catgacaccg
ccgcaaggcc cgccgcgccc cacccacgtt 3300cacacactcc ccacacccac
gcgtgcgcgc gcctccttcc cctcaataca cgcgcctcct 3360tcccctggcc
cccgcctgct ccccccatcc ggccgccccg cctgcaggtg gctgaggagt
3420ggaaggcggg tgcctttgtg atcaaggcgg gtgacgagga catccacacg
gccaacgagc 3480ggcgcctcac ggagctggtg ggggcggtgg gcggcaagct
gcacaccggc cgctcgcgca 3540acgaccaggt gagggtgggt gggtgggggt
ggggtgggtg ggtgggtggg tgggtgggtg 3600ggtgggtggg tgggtgggtg
ggtgggtggg ggtttgagat accggtacca ggccaaacta 3660aaccgaaccc
aagggggtgg cgtaggggcg tgggaggggg ggagtgcgga agccgggagg
3720caggagtaag ggcgggagga gggggccgga ggagaagcag ggacgaagtc
gatgacaggc 3780gcagtcggtg gcggcggtgg cgggtgtgcc gttgtgcagt
ggctgtggag gccatgtgca 3840gggcggcggc ggggccgggc cgggggtggg
agacttgtcc agaccccgtg gccctcttcc 3900agccccgtcc gccactgccg
ccaccaccac cgccgccgcc gtagccacca cccctcacgt 3960cgaggcactt
cacagatgcg aagcaaccac accgttctcc acatgaacag ctaccctccc
4020aaacccaact ttcccttccc gccttaccta accatgaccc gctacccccc
ccccctttat 4080ttcttaacta accatgaatg cccccccccg gctgtacctg
gctacgactt cacttcgtaa 4140acttaatgtg tgtaaccccc cttacacaca
cacacacacc cctccccgcc cctccaaagg 4200ttgccaccga ctaccggctg
tggctggtgg gtcaggtgga ggtgatgcgg tccgaggtgg 4260gcgagctgat
gcgcgtggcg gcggaccgct ccgaggcaga ggtggaggtg ctcatgccgg
4320gtgagggggc agggaggggg ggagggggag ggggaggtgc tcatgccggt
gagggtaggg 4380aggggagggg cagaggaggg agggggagga gggggcggct
gagtgcggga gaggcaggga 4440tgagggcgat agaaagttgc gtattgtcgg
taaactcaaa ggactagacg aagagaacaa 4500acctaaacaa gggagctgga
gcgaggccaa atctgaacgt gacatcgccc gcctcctccc 4560gctgcctgct
cccccacctc ctcccccatc tcgccccccc ccccacacac acacaggctt
4620cacgcacctt cagaatgcca tgactgtgcg ctggagccac tggctgatga
gccacgccgc 4680ggcctggcag cgcgacgaca tgcggctgcg ggacctgctg
ccgcgggtgg ccacactgcc 4740gctgggctcg ggtgggtgag ggaggggagg
ggaggggagg gggggagggg gagggagagg 4800aggggagaag ggggggggag
acgaggaggg tggaagggtg ggggcggggc ggtggaggct 4860agagggtggg
gctgggtggg tggacggagt gcactggtag aggagggata gggtacattg
4920agacgggagg agggatgcag gggcgaaggt ggggaggagg ggaggggagg
aggcgtggag 4980ctggagtggg ccgacgagtg tgcggacggg gcaggcggca
acggggatta aacggcgggg 5040ggccggggcg tgtgcacgac aggggcttgc
gcgtctgcga ttgtgggggc acacagggac 5100aggagcacga cgtgggacac
gcatagatac gccgcattga caacacacac acacacacac 5160acacacacac
acacacacac acacacacaa acacaaacac acacaaacac aaacacacac
5220acgccccccc ccctacacac acgccccctc cccaggcgcc ctggccggca
acccctttct 5280ggtggaccgc cagttcatcg ccaaggagtt gggtttcggc
ggcggcgtgt gccccaactc 5340catggacgcg gtgaggggag gaggaggggg
aggagggcgg gggggggcag gaggggggag 5400gaggaggggg ggagggggtt
aactttgaag cgtaaggaaa cagtcgggag gaggggggga 5460aggagggggc
ctggaggagg gggggaggag gagggtggct ggagggggct gggggaggag
5520gagggggagg attgggaggg ggctggggga gggtgcccgc agctggggga
ggtggggagg 5580gagggggttg ctgctggtgt aaagggcctg taggcactga
gagcactgtg gggagccggg 5640gtactgcctg gggccccgcg ctgcagaggt
gtcgcgcagt gtggcggcgc atcccccgca 5700tccccacacg cgggccgctg
ccgctgcccg ccacaccctt gccactttgt gtgctttcct 5760aggatataca
cacacacaca cacacacaca cacacacaca cacacacaaa cacaaacaca
5820cacgggcgcg ggctttcgtt tcgtttttta acacaaacac acactccccc
tgtgctcctc 5880aacacactcc atctttctca cacaaacaca cacgcacaca
cacatgcgca ggtgtctgac 5940cgcgactttg tgatcgagac ggtgtttgcg
gccagcctgc tgtgcgtgca cctgtcgcgc 6000tgggcggagg acctcatcat
ctacagctcc ggccccttcg gctacgtgca gtgcagcgac 6060gcctacgcca
ccggctcctc gctcatgccg cagaagaaga accccgacgc cctggagctc
6120atcaggtgcg ggagggatgg ggtgggggtg ggggggttac attcatggtt
agttaagaag 6180tgaaggcgta gggggtggat ggggtgggtt acattcatga
acatttaaga agtgaaggcg 6240tagccaggaa cagtagtaga gcagacgcgt
tgtagtgtgt gggtttgggt gggagggatg 6300gttgggtaaa gcggtacagg
atgtactgag gactgcagac cgaaggagcg ggggaggggg 6360agcaggcagg
cggggcgagg ggcgtggggg cgggggttac tggcaccgtg ccgggtaagc
6420aacacgtgac acggagatgc accacacaaa gagggacgtg gggagtggca
ggcgggggcc 6480agggctgaga ggcgcgtgtg gaggggtgcg gggttgggcg
gggggctgtt tcatgatacc 6540gctgcctcca cctcctccac cgcctcctgc
cacctccacc tcccccactg cccctccccg 6600cctcctcctg ctgcaggggc
aagggcggtc gtgtgcaggg caacctgatg ggcgtcatgg 6660cggtgctcaa
gggcacgccc accacataca acaaggactt ccaggcgaga gagcgagagc
6720gagggaggga gggagagcga gggagaggga gggagaggga gggagaggga
gacagaggga 6780cagggacagg gacagggaca gggacaggga cagggacagg
ggcaggggca ggggcagggg 6840caggggcagg ggcaggggag gccccccggg
ggcggcgggc ccggggcatg aggtcagaca 6900taggggcgct gcactgaggc
cgcgaggcgg gcgggaggca gggggcgggg ggcggggggc 6960gggagcggac
atgcgccgca aacacagacg ggttgagaaa gcacaacgac tggaacgcag
7020tgggcttact gacaattcat cattgtgcgc atatgtgtgt atgtgtatgt
gtgtgtttgt 7080ttgtgcagga gtgttgggag ctgctgtttg acacggtgga
cacggtgcac gacgtggtgc 7140gcatcgccac cggcgtgctg tccaccctgc
ggatcaagcc cgaccgcatg aaggccggtg 7200agcgtagccg agcagggctg
gagcagcagc cgggcagcag tagcagcagg gcaggggagc 7260agcgggagcg
ggagcagcag gaggggtggt tgggaagcgg tgggggtagg gtgggagcgg
7320aggaagggaa ggaggagcag
gagcaggagg aagaggagga ggaagggcgg tggggggtgg 7380ggggtcgtgt
ccttggccgc atgggcggag gcggggaggc ggggaggagg cggggaagca
7440gagcctgcac ccacgctccg cgggtcccta ccgtcttgcg cctaaccccg
tgcgcctagc 7500ctcttgcgcc caccccctta gtgcatcctg tacccctctt
tccaaacatc cttgcaactc 7560cctgacctcc tcgccaaacc tcccccgccc
ccaggcctgt ccgccgacat gctcgccacg 7620gacttggccg agtacctggt
gcgcaagggc gtgccgttcc gggagacaca ccaccacagg 7680tgcggccggg
cgggagggcg tgagggcgtg ggtggggcat gcccggggtt gtgagagcta
7740tcgaacgttg tgccgcgcct gtttcacaat gtcgggccac agggtatgca
gtttcctctc 7800catatgtata acaaactgac caccaatcat gcacgctcac
acgctctccc acacacacgc 7860gcaccacgcc accacagcgg cgccgccgtg
aagatggccg aggaccgcgg ctgcacgctg 7920ttcgacctca ccgtggacga
cctcaagacc atccacccgc tcttcaccga cgacgtggcg 7980gcggtgagcg
gcggcgcgga gcagcagcag cagcagcagc agcagcagca gcagcagcag
8040tagcctgggg gggagcgtgt gggaggaacg gcgggggagg ggaggcgggg
ggtgtcgttt 8100gcagccgagc gcacgtggtg ctttgcccca ttccatgcca
gcagggtgac acacctgacc 8160atgctggtgt gctgctaggt ggttcacacc
tacgtgtgaa tttgtgctgg cgtgcgcaca 8220ccttactgtg gccatgtgaa
cggcatcctc atgtcctcgt gattgcgccc ggcacattgc 8280ccacaacccc
gcaccaccca gctcctcaat ccagtgcaag gaaaggaaat gcacgcccgc
8340cgcaccaaca acacgacgca tgtgtttgcc acgtgcgcgc acacacgcgc
aggtgtggga 8400cttcaaccgc agcgccgaga tgcgcgacac ggagggcggc
accagcaagc gctcggtgct 8460ggagcaggtg cagaagatgc gcacctacct
ggcggcggag ggacagcact gagcgggtcg 8520ggggaggggg ggcgggtgtg
tatgtgtgtg tgtgtgcgtg tgtaagtctc ggtggagggg 8580tggtcctcta
tatggcggcg gggccacagg gggacgggtg tgacagagtt acggccggag
8640ccagcggagt cccgggatgg attaaggatc 867070745DNAUnknownDescription
of Unknown Organism GenBank Clone Accession Number I36448
70atgagatggc gacgcgcccc gcgccgctcc gggcgtcccg gcccccgggc ccagcgcccc
60ggctccgccg cccgctcgtc gccgccgctg ccgctgctgc cactactgct gctgctgggg
120accgcggccc tggcgccggg ggcggcggcc ggcaacgagg cggctcccgc
gggggcctcg 180gtgtgctact cgtccccgcc cagcgtggga tcggtgcagg
agctagctca gcgcgccgcg 240gtggtgatcg agggaaaggt gcacccgcag
cggcggcagc agggggcact cgacaggaag 300gcggcggcgg cggcgggcga
ggcaggggcg tggggcggcg atcgcgagcc gccagccgcg 360ggcccacggg
cgctggggcc gcccgccgag gagccgctgc tcgccgccaa cgggaccgtg
420ccctcttggc ccaccgcccc ggtgcccagc gccggcgagc ccggggagga
ggcgccctat 480ctggtgaagg tgcaccaggt gtgggcggtg aaagccgggg
gcttgaagaa ggactcgctg 540ctcaccgtgc gcctggggac ctggggccac
cccgccttcc cctcctgcgg gaggctcaag 600gaggacagca ggtacatctt
cttcatggag cccgacgcca acagcaccag ccgcgcgccg 660gccgccttcc
gagcctcttt cccccctctg gagacgggcc ggaacctcaa gaaggaggtc
720agccgggtgc tgtgcaagcg gtgcg 745711986DNAUnknownDescription of
Unknown Organism GenBank Clone Accession Number L12260 71gaattccttt
tttttttttt ttttttcttt ttttttttgc ccttatacct cttcgccttt 60ctgtggttcc
atccacttct tccccctcct cctcccataa acaactctcc tacccctgca
120cccccaataa ataaataaaa ggaggagggc aaggggggag gaggaggagt
ggtgctgcga 180ggggaaggaa aagggaggca gcgcgagaag agccgggcag
agtccgaacc gacagccaga 240agcccgcacg cacctcgcac catgagatgg
cgacgcgccc cgcgccgctc cgggcgtccc 300ggcccccggg cccagcgccc
cggctccgcc gcccgctcgt cgccgccgct gccgctgctg 360ccactactgc
tgctgctggg gaccgcggcc ctggcgccgg gggcggcggc cggcaacgag
420gcggctcccg cgggggcctc ggtgtgctac tcgtccccgc ccagcgtggg
atcggtgcag 480gagctagctc agcgcgccgc ggtggtgatc gagggaaagg
tgcacccgca gcggcggcag 540cagggggcac tcgacaggaa ggcggcggcg
gcggcgggcg aggcaggggc gtggggcggc 600gatcgcgagc cgccagccgc
gggcccacgg gcgctggggc cgcccgccga ggagccgctg 660ctcgccgcca
acgggaccgt gccctcttgg cccaccgccc cggtgcccag cgccggcgag
720cccggggagg aggcgcccta tctggtgaag gtgcaccagg tgtgggcggt
gaaagccggg 780ggcttgaaga aggactcgct gctcaccgtg cgcctgggga
cctggggcca ccccgccttc 840ccctcctgcg ggaggctcaa ggaggacagc
aggtacatct tcttcatgga gcccgacgcc 900aacagcacca gccgcgcgcc
ggccgccttc cgagcctctt tcccccctct ggagacgggc 960cggaacctca
agaaggaggt cagccgggtg ctgtgcaagc ggtgcgcctt gcctccccaa
1020ttgaaagaga tgaaaagcca ggaatcggct gcaggttcca aactagtcct
tcggtgtgaa 1080accagttctg aatactcctc tctcagattc aagtggttca
agaatgggaa tgaattgaat 1140cgaaaaaaca aaccacaaaa tatcaagata
caaaaaaagc cagggaagtc agaacttcgc 1200attaacaaag catcactggc
tgattctgga gagtatatgt gcaaagtgat cagcaaatta 1260ggaaatgaca
gtgcctctgc caatatcacc atcgtggaat caaacgctac atctacatcc
1320accactggga caagccatct tgtaaaatgt gcggagaagg agaaaacttt
ctgtgtgaat 1380ggaggggagt gcttcatggt gaaagacctt tcaaacccct
cgagatactt gtgcaagtgc 1440ccaaatgagt ttactggtga tcgctgccaa
aactacgtaa tggccagctt ctacagtacg 1500tccactccct ttctgtctct
gcctgaatag gagcatgctc agttggtgct gctttcttgt 1560tgctgcatct
cccctcagat tccacctaga gctagatgtg tcttaccaga tctaatattg
1620actgcctctg cctgtcgcat gagaacatta acaaaagcaa ttgtattact
tcctctgttc 1680gcgactagtt ggctctgaga tactaatagg tgtgtgaggc
tccggatgtt tctggaattg 1740atattgaatg atgtgataca aattgatagt
caatatcaag cagtgaaata tgataataaa 1800ggcatttcaa agtctcactt
ttattgataa aataaaaatc attctactga acagtccatc 1860ttctttatac
aatgaccaca tcctgaaaag ggtgttgcta agctgtaacc gatatgcact
1920tgaaatgatg gtaagttaat tttgattcag aatgtgttat ttgtcacaaa
taaacataat 1980aaaagg 1986722003DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number I36352 72ggaattcctt
tttttttttt tttttttctt nntttttttt tgcccttata cctcttcgcc 60tttctgtggt
tccatccact tcttccccct cctcctccca taaacaactc tcctacccct
120gcacccccaa taaataaata aaaggaggag ggcaaggggg gaggaggagg
agtggtgctg 180cgaggggaag gaaaagggag gcagcgcgag aagagccggg
cagagtccga accgacagcc 240agaagcccgc acgcacctcg caccatgaga
tggcgacgcg ccccgcgccg ctccgggcgt 300cccggccccc gggcccagcg
ccccggctcc gccgcccgct cgtcgccgcc gctgccgctg 360ctgccactac
tgctgctgct ggggaccgcg gccctggcgc cgggggcggc ggccggcaac
420gaggcggctc ccgcgggggc ctcggtgtgc tactcgtccc cgcccagcgt
gggatcggtg 480caggagctag ctcagcgcgc cgcggtggtg atcgagggaa
aggtgcaccc gcagcggcgg 540cagcaggggg cactcgacag gaaggcggcg
gcggcggcgg gcgaggcagg ggcgtggggc 600ggcgatcgcg agccgccagc
cgcgggccca cgggcgctgg ggccgcccgc cgaggagccg 660ctgctcgccg
ccaacgggac cgtgccctct tggcccaccg ccccggtgcc cagcgccggc
720gagcccgggg aggaggcgcc ctatctggtg aaggtgcacc aggtgtgggc
ggtgaaagcc 780gggggcttga agaaggactc gctgctcacc gtgcgcctgg
ggacctgggg ccaccccgcc 840ttcccctcct gcgggaggct caaggaggac
agcaggtaca tcttcttcat ggagcccgac 900gccaacagca ccagccgcgc
gccggccgcc ttccgagcct ctttcccccc tctggagacg 960ggccggaacc
tcaagaagga ggtcagccgg gtgctgtgca agcggtgcgc cttgcctccc
1020caattgaaag agatgaaaag ccaggaatcg gctgcaggtt ccaaactagt
ccttcggtgt 1080gaaaccagtt ctgaatactc ctctctcaga ttcaagtggt
tcaagaatgg gaatgaattg 1140aatcgaaaaa acaaaccaca aaatatcaag
atacaaaaaa agccagggaa gtcagaactt 1200cgcattaaca aagcatcact
ggctgattct ggagagtata tgtgcaaagt gatcagcaaa 1260ttaggaaatg
acagtgcctc tgccaatatc accatcgtgg aatcaaacgc tacatctaca
1320tccaccactg ggacaagcca tcttgtaaaa tgtgcggaga aggagaaaac
tttctgtgtg 1380aatggagggg agtgcttcat ggtgaaagac ctttcaaacc
cctcgagata cttgtgcaag 1440tgcccaaatg agtttactgg tgatcgctgc
caaaactacg taatggccag cttctacagt 1500acgtccactc cctttctgtc
tctgcctgaa taggagcatg ctcagttggt gctgctttct 1560tgttgctgca
tctcccctca gattccacct agagctagat gtgtcttacc agatctaata
1620ttgactgcct ctgcctgtcg catgagaaca ttaacaaaag caattgtatt
acttcctctg 1680ttcgcgacta gttggctctg agatactaat aggtgtgtga
ggctccggat gtttctggaa 1740ttgatattga atgatgtgat acaaattgat
agtcaatatc aagcagtgaa atatgataat 1800aaaggcattt caaagtctca
cttttattga taaaataaaa atcattctac tgaacagtcc 1860atcttcttta
tacaatgacc acatcctgaa aagggtgttg ctaagctgta accgatatgc
1920acttgaaatg atggtaagtt aattttgatt cagaatgtgt tatttgtcac
aaataaacat 1980aataaaagga aaaaaaaaaa aaa
200373957DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number X15898 73tctcgcccca actttttccc ccgcgctccg
cagcagcagc agcagcagca gcagcagcag 60caaaatggca gacctcttca gcggactcgt
gggcggcgtc gtcggcgctg ttgctgcagc 120agatttgcct gcggagggcg
agagggcccc ccgccccgcc cccggcactg cctggacttg 180ctgctgcagc
aaactgcaag aaggggcccg cgagctggag ggttttgtgc agcagctgag
240ttttgttgca gggaagctgg cctgctgcct gcgggtgggg gcggagcagc
tggcgcgctg 300cgctgcggag gggcggctgc ccagcagcag cagcagcagc
agctgctgcg cgctgctgca 360gctcgagaag caggacctcg agcagagcct
cgaggccggc aagcagggcg cggagtgcct 420cttgaggagc agcaaactgg
ccctcgaggc cctcctcgag ggggcccgcg ttgcagcaac 480gcggggtttg
ctgctggtcg agagcagcaa agacacggtg ctgcgcagca ttccccacac
540ccaggagaag ctggcccagg cctacagttc tttcctgcgg ggctaccagg
gggcagcagc 600ggggaggtct ctgggctacg gggcccctgc tgctgcttac
ggccagcagc agcagcccag 660cagctacggg gcgccccccg cctccagcca
gcagccctcc ggcttcttct ggtagccctg 720cagcagcagc agcagcagca
gcagcagcag cagcgcgggc ggcagccgcg gcggggccgg 780ggcgccgctg
cagcaacagc agcagccgnn ncggctagcg ccgcggagca ctcgcaggga
840actccacagg cagcgggaga gcagcaggga cgagaagcag gtcatgtagc
gcaggcagca 900gcgccagctg cagcagcagc agcagcagca gcagcagcag
cagcagctcc tgcaccg 95774957DNAUnknownDescription of Unknown
Organism GenBank Clone Accession Number I07789 74tctcgcccca
actttttccc ccgcgctccg cagcagcagc agcagcagca gcagcagcag 60caaaatggca
gacctcttca gcggactcgt gggcggcgtc gtcggcgctg ttgctgcagc
120agatttgcct gcggagggcg agagggcccc ccgccccgcc cccggcactg
cctggacttg 180ctgctgcagc aaactgcaag aaggggcccg cgagctggag
ggttttgtgc agcagctgag 240ttttgttgca gggaagctgg cctgctgcct
gcgggtgggg gcggagcagc tggcgcgctg 300cgctgcggag gggcggctgc
ccagcagcag cagcagcagc agctgctgcg cgctgctgca 360gctcgagaag
caggacctcg agcagagcct cgaggccggc aagcagggcg cggagtgcct
420cttgaggagc agcaaactgg ccctcgaggc cctcctcgag ggggcccgcg
ttgcagcaac 480gcggggtttg ctgctggtcg agagcagcaa agacacggtg
ctgcgcagca ttccccacac 540ccaggagaag ctggcccagg cctacagttc
tttcctccgg ggctaccagg gggcagcagc 600ggggaggtct ctgggctacg
gggcccctgc tgctgcttac ggccagcagc agcagcccag 660cagctacggg
gcgccccccg cctccagcca gcagccctcc ggcttcttct ggtagccctg
720cagcagcagc agcagcagca gcagcagcag cagcgcgggc ggcagccgcg
gcggggccgg 780ggcgccgctg cagcaacagc agcagccgnn ncggctagcg
ccgcggagca ctcgcaggga 840actccacagg cagcgggaga gcagcaggga
cgagaagcag gtcatgtagc gcaggcagca 900gcgccagctg cagcagcagc
agcagcagca gcagcagcag cagcagctcc tgcaccg
957751089DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number I08144 75gaattccctc caactcttcg cgactctctc
tctctcgccc caactttttc ccccgcgccc 60cgcagcagca gcagcagcag cagcagcaaa
atggcagacc tcttcagcgg actcgtgggc 120ggcgtcgtcg gcgctgttgc
tgcagcagat ttgcctgcgg agggcgagag ggccccccgc 180cccgcccccg
gcactgcctg gacttgctgc tgcagcaaac tgcaagaagg ggcccgcgag
240ctggagggtt ttctgcagca gctgagtttt gttgcaggga agctggcctg
ctgcctgcgg 300gtgggggcgg agcagctggc gcgctgcgct gcggaggggc
ggctgcccag cagcagcagc 360agcagcagct gctgcnngct gctgcagctc
gagaagcagg acctcgagca gagcctcgag 420gccggcaagc agggcgcgga
gtgcctcttg aggagcagca aactggccct cgaggccctc 480ctcgaggggg
cccgcgttgc agcaacgcgg ggtttgctgc tggtcgagag cagcaaagac
540acggtgctgc gcagcattcc ccacacccag gagaagctgg ctcaggccta
cagttctttc 600ctgcggggct accagggggc agcagcgggg aggtctctgg
gctacggggc ccctgctgct 660gcttacggcc agcagcagca gcccagcagc
tacggggcgc cccccgcctc cagccagcag 720ccctccggct tcttctggta
gccctgcagc agcagcagca gcagcagcag cagcagcagc 780ggcggcggca
gccgcggcgg ggccggggcg ccgctgcagc aacagcagca gccgcggcgg
840ctagcgnnnn gagcactcgc agggaactcc acaggcagcg ggagagcagc
agggacgaga 900agcaggtcta tgtagcgcag gcagcagcgc cagctgcagc
agcagcagca gcagcagcag 960cagcagcagc agctcctgca ccgcagcgtt
gtgtcattta ttacgttggc agctctgagg 1020cctcggcgca gccaacgcgc
ctcaggtatc tttcagactc ttttctctaa ggtcttccag 1080acggaattc
1089761985DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number U31814 76cgccgagctt tcggcacctc tgccgggtgg
taccgagcct tcccggcgcc ccctcctctc 60ctcccaccgg cctgcccttc cccgcgggac
tatcgccccc acgtttccct cagccctttt 120ctctcccggc cgagccgcgg
cggcagcagc agcagcagca gcagcaggag gaggagcccg 180gtggcggcgg
tggccgggga gcccatggcg tacagtcaag gaggcggcaa aaaaaaagtc
240tgctactact acgacggtga tattggaaat tattattatg gacagggtca
tcccatgaag 300cctcatagaa tccgcatgac ccataacttg ctgttaaatt
atggcttata cagaaaaatg 360gaaatatata ggccccataa agccactgcc
gaagaaatga caaaatatca cagtgatgag 420tatatcaaat ttctacggtc
aataagacca gataacatgt ctgagtatag taagcagatg 480catatattta
atgttggaga agattgtcca gcgtttgatg gactctttga gttttgtcag
540ctctcaactg gcggttcagt tgctggagct gtgaagttaa accgacaaca
gactgatatg 600gctgttaatt gggctggagg attacatcat gctaagaaat
acgaagcatc aggattctgt 660tacgttaatg atattgtgct tgccatcctt
gaattactaa agtatcatca gagagtctta 720tatattgata tagatattca
tcatggtgat ggtgttgaag aagcttttta tacaacagat 780cgtgtaatga
cggtatcatt ccataaatat ggggaatact ttcctggcac aggagacttg
840agggatattg gtgctggaaa aggcaaatac tatgctgtca attttccaat
gtgtgatggt 900atagatgatg agtcatatgg gcagatattt aagcctatta
tctcaaaggt gatggagatg 960tatcaaccta gtgctgtggt attacagtgt
ggtgcagact cattatctgg tgatagactg 1020ggttgtttca atctaacagt
caaaggtcat gctaaatgtg tagaagttgt aaaaactttt 1080aacttaccat
tactgatgct tggaggaggt ggctacacaa tccgtaatgt tgctcgatgt
1140tggacatatg agactgcagt tgcccttgat tgtgagattc ccaatgagtt
gccatataat 1200gattactttg agtattttgg accagacttc aaactgcata
ttagtccttc aaacatgaca 1260aaccagaaca ctccagaata tatggaaaag
ataaaacagc gtttgtttga aaatttgcgc 1320atgttacctc atgcacctgg
tgtccagatg caagctattc cagaagatgc tgttcatgaa 1380gacagtggag
atgaagatgg agaagatcca gacaagagaa tttctattcg agcatcagac
1440aagcggatag cttgtgatga agaattctca gattctgagg atgaaggaga
aggaggtcga 1500agaaatgtgg ctgatcataa gaaaggagca aagaaagcta
gaattgaaga agataagaaa 1560gaaacagagg acaaaaaaac agacgttaag
gaagaagata aatccaagga caacagtggt 1620gaaaaaacag ataccaaagg
aaccaaatca gaacagctca gcaacccctg aatttgacag 1680tctcaccaat
ttcagaaaat cattaaaaag aaaatattga aaggaaaatg ttttcttttt
1740gaagacttct ggcttcattt tatactactt tggcatggac tgtatttatt
ttcaaatggg 1800actttttcgt ttttgttttt ctgggcaagt tttattgtga
gattttctaa ttatgaagca 1860aaatttcttt tctccaccat gctttatgtg
atagtattta aaattgatgt gagttattat 1920gtcaaaaaaa ctgatctatt
aaagaagtaa ttggcctttc tgagctgaaa aaaaaaaaaa 1980aaaag
198577476DNAUnknownDescription of Unknown Organism GenBank Clone
Accession Number AF001444 77ccaccctcct ccccctcccc cggccacttc
gctaacttgg tggctgttgt gatgcgtatt 60cctgtagatc cgagcaccag ccggcgcttc
agccccccct ccagcagcct gcagcccggc 120aaaatgagcg acgtgagccc
ggtggtggct gcgcaacagc agcagcaaca gcagcagcag 180caacagcagc
agcagcagca gcaacagcag cagcagcagc aggaggcggc ggcggcggct
240gcggcggcag cggcggctgc ggcggcggca gctgcagtgc cccggttgcg
gccgccccac 300gacaaccgca ccatggtgga gatcatcgcc gaccacccgg
ccgaactcgt ccgcaccgac 360agccccaact tcctgtgctc ggtgctgccc
tcgcactggc gctgcaacaa gaccctgccc 420gtggccttca aggtaagagg
ctaccccgcc ccccgccccc ggccgggagc ggcgga 4767824DNAArtificial
SequenceDescription of Artificial SequenceDNA Primer 78gcattttgga
tccgcctttt catg 247922DNAArtificial SequenceDescription of
Artificial SequenceDNA Primer 79gttgtgtgct gcagattgtt cc
228021DNAArtificial SequenceDescription of Artificial SequenceDNA
Primer 80gaaaaatggg gatccgaggt g 218120DNAArtificial
SequenceDescription of Artificial SequenceDNA Primer 81gcaggagaat
tccgtccatg 20825PRTHomo sapiensMisc_feature(3)Xaa is any amino acid
82Trp Ser Xaa Trp Ser1 5836PRTHomo sapiens 83Cys Ser Val Thr Cys
Gly1 5845PRTHomo sapiensMisc_feature(4)Xaa is any amino acid 84Gly
Cys Gln Xaa Arg1 585733DNAHomo sapiens 85gggatccgga gcccaaatct
tctgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60aattcgaggg tgcaccgtca
gtcttcctct tccccccaaa acccaaggac accctcatga 120tctcccggac
tcctgaggtc acatgcgtgg tggtggacgt aagccacgaa gaccctgagg
180tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg 240aggagcagta caacagcacg taccgtgtgg tcagcgtcct
caccgtcctg caccaggact 300ggctgaatgg caaggagtac aagtgcaagg
tctccaacaa agccctccca acccccatcg 360agaaaaccat ctccaaagcc
aaagggcagc cccgagaacc acaggtgtac accctgcccc 420catcccggga
tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
480atccaagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac
aactacaaga 540ccacgcctcc cgtgctggac tccgacggct ccttcttcct
ctacagcaag ctcaccgtgg 600acaagagcag gtggcagcag gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc 660acaaccacta cacgcagaag
agcctctccc tgtctccggg taaatgagtg cgacggccgc 720gactctagag gat
7338686DNAArtificial SequenceDescription of Artificial SequenceDNA
Primer 86gcgcctcgag atttccccga aatctagatt tccccgaaat gatttccccg
aaatgatttc 60cccgaaatat ctgccatctc aattag 868727DNAArtificial
SequenceDescription of Artificial SequenceDNA Primer 87gcggcaagct
ttttgcaaag cctaggc 2788271DNAArtificial SequenceDescription of
Artificial SequencePCR Fragment 88ctcgagattt ccccgaaatc tagatttccc
cgaaatgatt tccccgaaat gatttccccg 60aaatatctgc catctcaatt agtcagcaac
catagtcccg cccctaactc cgcccatccc 120gcccctaact ccgcccagtt
ccgcccattc tccgccccat ggctgactaa ttttttttat 180ttatgcagag
gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
240ttttggaggc ctaggctttt gcaaaaagct t 2718932DNAHomo sapiens
89gcgctcgagg gatgacagcg atagaacccc gg 329031DNAHomo sapiens
90gcgaagcttc gcgactcccc ggatccgcct c 319112DNAHomo sapiens
91ggggactttc cc 129273DNAHomo sapiens 92gcggcctcga ggggactttc
ccggggactt tccggggact ttccgggact ttccatcctg 60ccatctcaat tag
739327DNAArtificial SequenceDescription of Artificial SequencePCR
Fragment 93gcggcaagct ttttgcaaag cctaggc 2794652DNAHomo
sapiensMisc_feature(524)N is any nucleic acid 94ggcataagat
cacactttag ttcagagaca catttgcata aatacttgaa atggatccac 60ccctgcaggt
ggcagcctga gaacatggcg ctgcaggggg accagggcag cgtctggttc
120aggtggacga acagcggtgc catcacgtgg tgcttgccca tgggcccgaa
gagccgtgtg 180cagggcttgg agtcgtcgtg gggcatgctg aggacgtgcc
ctagttcatg ggccagggtg 240tgggccgcct ggagcccctc atcctcgatc
acggagcagc ttttgttggg gtcacaaatg 300gtcccgatgt ctgccacacc
cagggtgtca cacagcccct cctgcccaca gaagttctgt 360ctggtgagca
ggatggccgt gtcgtagtgc tctgggtggc ggtcgctggg ctggttgaaa
420cgccgctgcc agttgcagaa gttacgcagt gtaagccccc cattgtcgga
cacctctggg 480ccccattttt catcttctac gatcagcact tttaccacca
tcangttgat ggaattcttg 540atgctggggt gcttgtagaa tcgggcttgc
cacgaaaatt aacctcagga tgtggttctg 600caggtcggcc cgtaaagggc
gccatggacg catcggccac caacagcgtt tc 65295716DNAHomo
sapiensMisc_feature(578)N is any nucleic acid 95taagtttgct
agtcctttgc aaacagactg acgctgagtg tcctgtctga gtcaataagt 60gcacttttac
cttttaacct atgccctcta cttgaacccg agcaaggtcc agtccactgg
120acagttgatg atagggtctg ccgccccata ccctctcctc ttccccctta
ggaatttgtg 180cagtactgga ggggttgcgg caatgggagg cctgggtggg
ccgtgctgcc ttgatatggc 240caagggaccc agtcaccaca gtggagaccc
ttgtctgcac ctcagtaccg catgtccagg 300agcacaagac tggcccctgc
ccccctgaat cacagggggc acagctggct ttcgcagggc 360ttggcatcct
cgggtttcag agccttgttg caggtggcag aggcctggcc ggaggggtcc
420ctgcactcta cagttcgcct ctgccagccg gccccgcagg tgctagagca
ctcagaccag 480tcccccagca cccactgtgc gtggagcagc ggctggatga
tgttggtggt tgctctctct 540ttgctgctct gcatgctaaa agtcacgtca
ttaggaanca aagaaggtgt atttgacttt 600ttggggggaa gaacctcgcc
caggactgtc aggagctgca ctgtcagaag gctctgcnaa 660ggcccngaag
ctctgcangc gctccagggt ggcgatggag ccgtgtactt caggat 71696543DNAHomo
sapiens 96ggcataagat cacactttag ttcagagaca catttgcata aatacttgaa
atggatccac 60ccctgcaggt ggcagcctga gaacatggcg ctgcaggggg accagggcag
cgtctggttc 120aggtggacga acagcggtgc catcacgtgg tgcttgccca
tggcctcgaa gagccgtgtg 180cagggcttgg agtcgtcgtg gggcatgctg
aggacgtgcc ctagttcatg ggccagggtg 240tgggccgctg gagccctcat
cctcgatcac ggagcagctt ttgttggggt cacaaatggt 300cccgatgtct
gccacaccca gggtgtcaca cagcccctcc tgcccacaga agttctgtct
360ggtgagcagg atggccgtgt cgtagtgctc tgggtggcgg tcgctgggct
ggttgaaacg 420ccgctgccag ttgcagaagt tacgcagtgt aaggccccca
ttgtcggaca gctctggggc 480ccatttttca tcttctacga tcagcacttt
taaccacatc aggttgatgg aattcttgat 540gcc 54397377DNAMus musculus
97gcaaagtgcc accacccttc ggatccaaaa ctagaagcaa gaggtttgtg tccgaggctc
60gcttcgtgga aacacttctg gtggctgatg cgtccatggc tgccttctat gggaccgacc
120tgcagaacca catcctcacg gtgatgtcaa tggcagcccg aatctacaag
cacccgagca 180tcaagaactc cgtcaacctt gtggtggtga aagtgctaat
agtggaagag gaaggatggg 240gcccggaggt gtcggacaac ggggggctca
cactgcgcaa cttctgcagc tggcaacggc 300gtttcaacaa gcccagtgac
cgccacccgg agcactatga cactgccatc ttgttcacca 360gacagaactt ctgtggg
37798432DNARattus norvegicusMisc_feature(42)N is any nucleic acid
98ctaaagtaca gtggttccat ggccaccctg gagcggctgc anagcttcca agccctccct
60gagcctctta cagtacagct cctgactgtg tctggtgagg tcttccctcc aaaagtcaaa
120tataccttct tcgtccccaa tgacacggac ttcaacgtgc agagtagcaa
agaaagagca 180agcaccaaca tcattcagtc cttgccctat gcanagtggg
tgctggggga ctggtctgaa 240tgtccaagca catgtggagg tggctggcag
cggcggactg tggaatgcag ggacccctca 300ggtcaggcct ctgacacctg
tgatgaggct ctgaaacctg aggatgccaa gccctgtgga 360agccagccat
gtctcctctg atccccttgg tggacatgtc taaggcttat ggatttgggc
420tactggcgtt tt 43299354DNAMus musculus 99caaagtgcac cacccttcgg
atccaaaact agaagcaaga ggtttgtgtc cgaggctcgc 60ttcgtggaaa cacttctggt
ggctgatgcg tccatggctg ccttctatgg gaccgacctg 120cagaaccaca
tcctcacggt gatgtcaatg gcagccacga atctacaagc acccgagcat
180caggaactcc gtcaaccttg tggtggtgaa agtgctaata gtggaagagg
aaggatgggg 240cccggagtgt cggacaacgg ggggctcaca ctgcgcaact
tctgcagctg gcaacggcgt 300ttcaacaagc ccagtgaccg ccacccggag
cactatgaca ctgccatctt gttc 354100389DNAHomo
sapiensMisc_feature(136)N is any nucleic acid 100ttgtgcccag
aagacactgg ccctggggcc tgggttgagt tcaaaaccaa aagaaagaag 60aaaagtgctg
taaattcggg atttctccac cggatgctcc tgcttccgca tgggtgtcac
120ctccatgccg ttcctncctc tttctaggga aaagcttcag ggagcagcag
tgtgagaagt 180ataatgccta caattacact gacatggacg ggaatctcct
gcagtgggtc cccaagtatg 240ctggggtgtc cccccgggac cgcctggcaa
gttgttctgc cgagcccggg ggaggagcga 300gttcaaagtg ttcgaggcca
aggtgagaat caccctgggg gacttcagat ccagagatgg 360ggggagggaa
ggtcggcctg ttccccaca 389101305DNAHomo sapiensMisc_feature(128)N is
any nucleic acid 101catttaagtt tgctagtcct ttgcaaacag actgacgctg
agtgtcctgt ctgagtcaat 60aagtgcactt ttacctttta acctatgccc tctacttgaa
cccgagcaag gtccagtcca 120ctggacangt tgatgatagg gtctgncgcc
ccataccctc tcctcttccc ccttaggaat 180ttgtgcagta ctggaggggt
tgcggcaatg ggaggcctgg gtgggccgtg ctgccttgat 240atggccaagg
gacccagtca ccacagtgga gacccttgtc tgcacctcag taccgcatgt 300ccagg
305102152DNAHomo sapiensMisc_feature(105)N is any nucleic acid
102atcgtagaag atgaaaaatg gggcccagag gtgtccgaca atggggggct
tacactgcgt 60aacttctgca actggcagcg gcgtttcaac cagcccagcg accgncaccc
agagcactac 120gncacggcca tcctnctcac cagacagaac tt 152103632DNAHomo
sapiens 103tttaataata ataatgcccg gggctttatt atgctgtatc actgctcaga
ggttaataat 60cctcactaac tatcctatca aatttgcaac tggcagttta ctctgatgat
tcaactcctt 120ttctatctac ccccataatc ccaccttact gatacacctc
actggttact ggcaagatac 180gctggatccc tccagccttc ttgctttccc
tgcaccagcc cttcctcact ttgccttgcc 240ctcaaagcta acaccactta
aaccacttaa ctgcattctg ccattgtgca aaagtctatg 300aaatgtttag
gtttctttaa aggatcacag ctctcatgag ataacacccc tccatcatgg
360gacagacact tcaagcttct ttttttgtaa cccttcccac aagtcttaga
acatgatgac 420cactccccca gctgccactg ggggcaggga tggtctgcac
aaggtctggt gctggctggc 480ttcacttcct ttgcacactc ggaagcaggc
tgtccattaa tgtctcggca ttctaccagt 540cttctctgcc aacccaattc
acatgactta gaacattcgc cccactcttc aatgacccat 600gctgaaaaag
tggggatagc attgaaagaa tc 632104519DNAHomo sapiens 104tttttttcta
aacttgtaat agatgtaaca aaagaaataa taataataat gcccggggct 60ttattatgct
atatcactgc tcagaggtta ataatcctca ctaactatcc tatcaaattt
120gcaactggca gtttactctg atgattcaac tccttttcta tctaccccca
taatcccacc 180ttactgatac acctcactgg ttactggcaa gatacgctgg
atccctccag ccttcttgct 240ttccctgcac cagcccttcc tcactttgcc
ttgccctcaa agctaacacc acttaaacca 300cttaactgca ttctgccatt
gtgcaaaagt ctatgaaatg tttaggtttc tttaaaggat 360cacagctctc
atgagataac acccctccat catgggacag acacttcaag cttctttttt
420tgtaaccctt cccacaggtc ttagaacatg atgaccactc ccccagctgc
cactgggggc 480agggatgtct gcacaagggc tggtgctggc tgcccggac
519105475DNAHomo sapiens 105gagtcatgat gcgatcacaa ccagctttta
cacactgtcc ttgcacacag acagaggtgg 60aatctgggct acatggagta ccatctacaa
ccttgggctg caaaacgaag aagtagccaa 120tgcctttggc ttggcagatg
agcttgcacc tgtcctttgg tgagacgcca gcgtacttgg 180gaatccattc
caccgcaggc ccactcccaa aggaagcttt tgaaaactcg ttgtgtgctt
240cacattgttc ctctctaaag gtttttccat tattgtctgg acagtcctca
aggttacagg 300atctgtagcg cactcgtttg ccttcacagt acttccctcc
attctttggg actgggttgt 360cacattccct catcgtgtac tggactcctc
caccgcacgt tctcgaacag tctccccaag 420gcccccacat tccccagctt
ccatgaaaag gcgtatcaaa atgctttctg tcggt 475106455DNAHomo sapiens
106aataataata atgcccgggg ctttattatg ctgtatcact gctcagaggt
taataatcct 60cactaactat cctatcaaat ttgcaactgg cagtttactc tgatgattca
actccttttc 120tatctacccc cataatccca ccttactgat acacctcact
ggttactggc aagatacgct 180ggatccctcc agccttcttg ctttccctgc
accagccctt cctcactttg ccttgccctc 240aaagctaaca ccacttaaac
cacttaactg cattctgcca ttgtgcaaaa gtctatgaaa 300tgtttaggtt
tctttaaagg atcacagctc tcatgagata acacccctcc atcatgggac
360agacacttca agcttctttt tttgtaaccc ttcccacagg tcttagaaca
tgatgaccac 420tcccccagct gccactgggg gcagggatgg tctgg
455107515DNAHomo sapiens 107aacccttccc acaggtctta gaacatgatg
accactcccc cagctgccac tgcggggcag 60ggatggtctg cacaaggtct ggtgctggct
ggcttcactt cctttgcaca ctcggaagca 120ggctgtccat taatgtctcg
gcattcttcc agtcttctct gccaacccaa ttcacatgac 180ttagaacatt
cgccccactc ttcaatgacc catgctgaaa aagtggggat agcattgaaa
240gattccttct tcttctttac gaagtaggtg tatttaattt taggtcgaag
ggcattgcca 300cagtaagaac ctggatggtc aagggctctt tggagcaggc
taaagctgcg aattctttcc 360aatgccgcag aggagccgct gtacctcaag
acaacacctt tgtacataat gtcttgctct 420aaggtggaca aagtgtagtc
accataaaga atatatgtgc catcagcagc ttttgatggc 480aggaagctgt
cattgttctt ggatccctct gttcc 515108359DNAHomo sapiens 108acttcgtaaa
gaagaagaag gaatctttca atgctatccc cactttttca gcatgggtca 60ttgaagagtg
gggcgaatgt tctaagtcat gtgaattggg ttggcagaga agactggtag
120aatgccgaga cattaatgga cagcctgctt ccgagtgtgc aaaggaagtg
aagccagcca 180gcaccagacc ttgtgcagac catccctgcc cccagtggca
gctgggggaa gtggtcatca 240tgttctaaga cctgcgggaa gggttacaaa
aaaagaagct ttgaagtgtc ttgtcccatg 300atggaggggt gttatctcat
tgagagctgt gatcctttaa agaaacctaa acatttcat 359109320DNAHomo sapiens
109cagagaacat tcgccccact cttcaatgac ccatgctgaa aaagtgggga
tagcattgaa 60agattccttc ttcttcttta cgaagtaggt gtatttaatt ttaggtcgaa
gggcattgcc 120cacagtaaga acctggatgg tcaagggctc tttgagaggg
ctaaagctgc gaattctttc 180caatgccgca gaggagccgc tgtacctcaa
gacaacacct ttgtacataa tgtcttgctc 240taaggtggac aaagtgtagt
caccattaag aatatatgtg ccatcagcag ctttgatggc 300aagaaagctg
cccttgttcc 320110316DNAHomo sapiens 110aatgccgaga cattaatgga
cagcctgctt ccgagtgtgc aaaggaagtg aagccagcca 60gcaccagacc ttgtgcagac
catccctgcc cccagtggca gctgggggag tggtcatcat 120gttctaagac
ctgtgggaag ggttacaaaa aaagaagctt gaagtgtctg tcccatgatg
180gaggggtgtt atctcatgag agctgtgatc ctttaaagaa acctaaacat
ttcatagact 240tttgcacaat ggcagaatgc agttaagtgg tttaagtggt
gttagctttg agggcaaggc 300aaagtgagga agggct 316111318DNAHomo
sapiensMisc_feature(4)N is any nucleic acid 111agantnccga
gacattaatg gacagcctgc ttccgagtgt gcaaaggaag tgaagccagc 60cagcaccaga
ccttgtgcag accatccctg cccccagtgg cagctggggg agtggtcatc
120atgttctaag acctgtggga agggttacaa aaaaagaagc ttgaagtgtc
tgtcccatga 180tggaggggtg ttatctcatg agagctgtga tcctttaaag
aaacctaaac atttcataga 240cttttgcaca atggcagaat ncagttaagt
ggtttaagtg gtgttagctt tgagggcaag 300gcaaagtgag gaagggct
318112314DNAHomo sapiens 112ttttttttct aaacttgtaa tagatgtaac
aaaagaaata ataataataa tgcccggggc 60tttattatgc tatatcactg ctcagaggtt
aataatcctc actaactatc ctatcaaatt 120tgcaactggc agtttactct
gatgattcaa ctccttttct atctaccccc ataatcccac 180cttactgata
cacctcactg gttactggca agatacgctg gatccctcca gccttcttgc
240tttccctgca ccagcccttc ctcactttgc cttgccctca aagctaacac
cacttaaacc 300acttaactgc attc 314113316DNAHomo sapiens
113aaggaatcct tcaatgctat ccccactgtt tcagcatggg tcattgaaga
gtggggcgaa 60tgttctaagt catgtgaatt gggttggcag aaaagacttg tagaatgccg
agacattaat 120ggacagcctg cgtccgagtg tgcaaaggaa gtgaagccag
ccagcaccag accttgtgca 180gaccatccct gcccccagtg gcagctgggg
ggagtggtca tcatgttcta agacctgtgg 240gaaggggtac aaaaaaagag
gcgtgaagtg tctgtcccat gatggagggg tttatctcat 300gagaactgtg atcctt
316114265DNAHomo sapiensMisc_feature(10)N is any nucleic acid
114agcagtttan ncctntcaaa gagcccttga ccatccaggt tcttactgtg
ggcaatgccc 60ttcgacctaa aattaaatac acctacttcg taaagangaa gaaggaatct
ttcaatgcta 120tccccacttt ttcagcatgg gtcattgaag agtggggcga
atgttctaag tcatgtgaat 180tgggttggca gagaagactg gtagaatgcc
gagacattaa tggacagcct ncttccgagt 240gtgcaaagna agtgaagcca gccag
265115334DNAMus musculus 115cgtttgtgga ggaaacggtt ccacatgcaa
gaagatgtca ggaatagtca ctagtacaag 60acctgggtat catgacattg tcacaattcc
tgctggagcc accaacattg aagtgaaaca 120tcggaatcaa agggggtcca
gaaacaatgg cagctttctg gctattagag ccgctgatgg 180tacctatatt
ctgaatggaa acttcactct gtccacacta gagcaagacc tcacctacaa
240aggtactgtc ttaaggtaca gtggttcctc ggctgcgctg gagagaatcc
gcagctttag 300tccactcaaa gaacccttaa ccatccaggt tctt 334116528DNAMus
musculus 116agaattcctg gatgatggtc atggtaattg cttccgtggt aggtctagca
aacaattacc 60atgaccatca tccaggaatt ctgtgatggt ggctgacgtg catttggacc
agggcttgga 120tgcatcgatg ctggtaagga ttgaagacat taaacgcttg
tcttctgtag taccgaagtt 180ctcttcacag aatttggaat cgtcatgaga
aaggccaagt agatgcccaa tttcatgagc 240cacagtgaag gctgcatgga
ggccatcatc ttcaatcact gcacagctgc gctccggaga 300acatatggtc
ccaacgtctg ccattcccag ggtgtcacat gaatgatgcc cacataaatc
360ctctcgggtg aacaggatgg ctgcatcgta gtgctcttcg tgatcatccc
ctagctggtt 420atgttggtgc tgccatttgc aaaagttctt gagggtcgtg
gccgcattct tgctcacctc 480cagactcgtg tccttgtccg tcagcaccac
caccttcacc accgccag 528117438DNAHomo sapiensMisc_feature(389)N is
any nucleic acid 117atttgatagg atagttagtg aggattatta acctctgagc
agtgatatag cataataaag 60ccccgggcat tattattatt atttcttttg ttacatctat
tacaagttta gaaaaaacaa 120agcaattgtc aaaaaaagtt agaactatta
caacccctgt ttcctggtac ttatcaaata 180cttagtatca tgggggttgg
gaaatgaaaa gtaggagaaa agtgagattt tactaagacc 240tgttttactt
tacctcacta acaatggggg gagaaaggag tacaaatagg atctttgacc
300agcactgttt atgggctgct atgggtttca gaggaatgtt tatacattat
ttctacccga 360ggatttaaaa cttcagattg ttccaaccng gaggggaagg
gcttccggcc aacgtggaat 420taaccggcaa tnggcctt 438118455DNAHomo
sapiensMisc_feature(452)N is any nucleic acid 118atttgatagg
atagttagtg aggattatta acctctgagc agtgatatag cataataaag 60ccccgggcat
tattattatt atttcttttg ttacatctat tacaagttta gaaaaaacaa
120agcaattgtc aaaaaaagtt agaactatta caacccctgt ttcctggtac
ttatcaaata 180cttagtatca tgggggttgg gaaatgaaaa gtaggagaaa
agtgagattt tactaagacc 240tgttttactt tacctcacta acaatggggg
gagaaaggag tacaaatagg atctttgacc 300agcactgttt atggctgcta
tggtttcaga gaatgtttat acattatttc taccgaggat 360taaaacttcc
agattgtttc aacatggaga ggaaaggctc aggcaacgtg gaaataacgc
420aaatgggctt cctcttttcc tttttgggac cntct 455119380DNAHomo
sapiensMisc_feature(25)N is any nucleic acid 119aatttgatag
gatagttagt gaggnttatt aacctctgag cagtgatata gcataataaa 60gccccgggca
ttattattat tattnctttt gttacatcta ttacaagttt agaaaaaaca
120aagcaattgt caaaaaaagt tagaactatt acaacccctg tttcctggta
cttatcaaat 180acttagtatn atgggggttg ggaaatgaaa agtaggagaa
aagtgagatt ttactaagac 240ctgttttact ttacctcact aacaatgggg
ggagaaagga gtacanatag gatctttgac 300cagcactgtt tatggctgct
atggtttcag aggaatgttt atacattatt tctaccgaga 360nttaaaactt
cagattgttc 380120199DNAMus musculus 120caatggcagc ttgctggcta
taatagccgc tgatggtacc tatatactga atggaaactt 60cactctgtcc acactagagc
aagacctcac ctacgaatgt actgtcttaa ggtacagtgg 120ttcctcggct
gcgcaggaaa gagtccgcag ctttagtcca ctcaaataac ccttaaccat
180ccaggttctt atggtagga 199121439DNAHomo sapiensMisc_feature(198)N
is any nucleic acid 121atttaacctc tgagcagtga tatagcataa taaagccccg
ggcattatta ttattatttc 60ttttgttaca tctattacaa gtttagaaaa aacaaagcaa
ttgtcaaaaa aagttagaac 120tattacaacc cctgtttcct ggtacttatc
aaatacttag tatcatgggg gttgggaaat 180gaaaagtagg aggaaagnng
agnttttact aagacctgtt ttacctttac ctcactaaca 240atggggggag
aaaggagtac aaataggatc tttgaccagc actgtttatg gctgctatgg
300tttcagagaa tgtttataca ttatttctac cgagaattaa aacttcagat
tgttcaacat 360ggagagaaag gctcagcaac gtggaaataa cgcaaatggg
cttccccctt tccctttttt 420gggaccatct caggtcctt 439122471DNAHomo
sapiens 122cagagtaaac tgccagttgc aaatttgata ggatagttag tgaggattat
taacctctga 60gcagtgatat agcataataa agccccgggc attattatta ttattatttc
ttttgttaca 120tctattacaa gtttagaaaa aacaaagcaa ttgtcaaaaa
aagttagaac tattacaacc 180cctgtttcct ggtacttatc aaatacttag
tatcatgggg gttgggaaat gaaaagtagg 240agaaaagtga gattttacta
agacctgttt tacttttcct cactaacaat ggggggagaa 300aggagtacaa
ataggatctt tgaccagcac tgtttatggc tgctatggtt tcagagaatg
360tttatacatt atttctaccc gagaattaaa acttcagatt ggttcaacat
gagagaaagg 420ctccagcaac gtgaaattaa cgccaatggc ttcctccttc
ccttttttgg a 471123424DNAHomo sapiensMisc_feature(39)N is any
nucleic acid 123cgtgaggatt attaacctct gagcagtgat atagcatant
aaagccccgg nattattatt 60attatttctt ttgttacatc tattacaagt ttagaaaaaa
caaagcaatt gtcaaaaaaa 120gttagaacta ttacaacccc tgtttcctgg
tacttatcaa atacttagta tcatgggggt 180tgggaaatga aaagtaggag
aaaagtgaga ttttactaag acctgtttta ctttacctca 240ctaacaatgg
ggggagaaag gagtacaaat aggatctttg accagcactg tttatggctg
300ctaatggttt cagagaatgt ttatacatta tttctacccg agaattaaaa
cttcagattg 360ttcaacctga gagaaaggct cagcaacgtg aaatnacgcc
aatggcttcc tctttccctt 420tttg 424124458DNAHomo
sapiensMisc_feature(453)N is any nucleic acid 124tacatctatt
acaagtttag aaaaaacaaa gcaattgtca aaaaaagtta gaactattac 60aacccctgtt
tcctggtact tatcaaatac ttagtatcat gggggttggg aaatgaaaag
120taggagaaaa gtgagatttt actaagacct gttttacttt acctcactaa
caatgggggg 180agaaaggagt acaaatagga tctttgacca gcactgttta
tggctgctat ggtttcagag 240aatgtttata cattatttct accgagaatt
aaaacttcag attgttcaac atgagagaaa 300ggctcagcaa cgtgaaataa
cgcaaatggc ttcctctttc cttttttgga ccacagccag 360ccttggtctc
cttgcagtgg ctacatgatt acatcatttc tggataatag tcatggggaa
420tgtttgatgg acaagctcag
aatcccatac agntccca 4581254014DNAHomo sapiensCDS(466)..(3366)
125cccacgcgtc cgcccacgcg tccggcggct ccgagccagg ggctattgca
aagccagggt 60gcgctaccgg acggagaggg gagagccctg agcagagtga gcaacatcgc
agccaaggcg 120gaggccgaag aggggcgcca ggcaccaatc tccgcgttgc
ctcagccccg gaggcgcccc 180agagcgcttc ttgtcccagc agagccactc
tgcctgcgcc tgcctctcag tgtctccaac 240tttgcgctgg aagaaaaact
tcccgcgcgc cggcagaact gcagcgcctc ctcttagtga 300ctccgggagc
ttcggctgta gccggctctg cgcgcccttc caacgaataa tagaaattgt
360taattttaac aatccagagc aggccaacga ggctttgctc tcccgacccg
aactaaagct 420ccctcgctcc gtgcgctgct acgagcggtg tctcctgggg ctcca atg
cag cga gct 477 Met Gln Arg Ala 1gtg ccc gag ggg ttc gga agg cgc
aag ctg ggc agc gac atg ggg aac 525Val Pro Glu Gly Phe Gly Arg Arg
Lys Leu Gly Ser Asp Met Gly Asn5 10 15 20gcg gag cgg gct ccg ggg
tct cgg agc ttt ggg ccc gta ccc acg ctg 573Ala Glu Arg Ala Pro Gly
Ser Arg Ser Phe Gly Pro Val Pro Thr Leu 25 30 35ctg ctg ctc gcc gcg
gcg cta ctg gcc gtg tcg gac gca ctc ggg cgc 621Leu Leu Leu Ala Ala
Ala Leu Leu Ala Val Ser Asp Ala Leu Gly Arg 40 45 50ccc tcc gag gag
gac gag gag cta gtg gtg ccg gag ctg gag cgc gtc 669Pro Ser Glu Glu
Asp Glu Glu Leu Val Val Pro Glu Leu Glu Arg Val 55 60 65ccg gga cac
ggg acc acg cgc ctc cgc ctg cac gcc ttt gac cag cag 717Pro Gly His
Gly Thr Thr Arg Leu Arg Leu His Ala Phe Asp Gln Gln 70 75 80ctg gat
ctg gac gtg ccg ccc gac agc agc ttt ttg gcg ccc ggc ttc 765Leu Asp
Leu Asp Val Pro Pro Asp Ser Ser Phe Leu Ala Pro Gly Phe85 90 95
100acg ctc cag aac gtg ggg cgc aaa tcc ggg tcc gac acc ccg ctt ccg
813Thr Leu Gln Asn Val Gly Arg Lys Ser Gly Ser Asp Thr Pro Leu Pro
105 110 115gaa acc gac ctg gcg cac tgc ttc tac tcc ggc acc gtg aat
ggc gat 861Glu Thr Asp Leu Ala His Cys Phe Tyr Ser Gly Thr Val Asn
Gly Asp 120 125 130ccc agc tcg gct gcc gcc ctc agc ctc tgc gag ggc
gtg cgc ggc gcc 909Pro Ser Ser Ala Ala Ala Leu Ser Leu Cys Glu Gly
Val Arg Gly Ala 135 140 145ttc tac ctg ctg ggg gag gcg tat ttc atc
cag ccg ctg ccc gcc gcc 957Phe Tyr Leu Leu Gly Glu Ala Tyr Phe Ile
Gln Pro Leu Pro Ala Ala 150 155 160agc gag cgc ctc gcc acc gcc gcc
cca ggg gag aag ccg ccg gca cca 1005Ser Glu Arg Leu Ala Thr Ala Ala
Pro Gly Glu Lys Pro Pro Ala Pro165 170 175 180cta cag ttc cac ctc
ctg cgg cgg aat cgg cag ggc gac gta ggc ggc 1053Leu Gln Phe His Leu
Leu Arg Arg Asn Arg Gln Gly Asp Val Gly Gly 185 190 195acg tgc ggg
gtc gtg gac gac gag ccc cgg ccg act ggg aaa gcg gag 1101Thr Cys Gly
Val Val Asp Asp Glu Pro Arg Pro Thr Gly Lys Ala Glu 200 205 210acc
gaa gac gag gac gaa ggg act gag ggc gag gac gaa ggg cct cag 1149Thr
Glu Asp Glu Asp Glu Gly Thr Glu Gly Glu Asp Glu Gly Pro Gln 215 220
225tgg tcg ccg cag gac ccg gca ctg caa ggc gta gga cag ccc aca gga
1197Trp Ser Pro Gln Asp Pro Ala Leu Gln Gly Val Gly Gln Pro Thr Gly
230 235 240act gga agc ata aga aag aag cga ttt gtg tcc agt cac cgc
tat gtg 1245Thr Gly Ser Ile Arg Lys Lys Arg Phe Val Ser Ser His Arg
Tyr Val245 250 255 260gaa acc atg ctt gtg gca gac cag tcg atg gca
gaa ttc cac ggc agt 1293Glu Thr Met Leu Val Ala Asp Gln Ser Met Ala
Glu Phe His Gly Ser 265 270 275ggt cta aag cat tac ctt ctc acg ttg
ttt tcg gtg gca gcc aga ttg 1341Gly Leu Lys His Tyr Leu Leu Thr Leu
Phe Ser Val Ala Ala Arg Leu 280 285 290tac aaa cac ccc agc att cgt
aat tca gtt agc ctg gtg gtg gtg aag 1389Tyr Lys His Pro Ser Ile Arg
Asn Ser Val Ser Leu Val Val Val Lys 295 300 305atc ttg gtc atc cac
gat gaa cag aag ggg ccc gaa gtg acc tcc aat 1437Ile Leu Val Ile His
Asp Glu Gln Lys Gly Pro Glu Val Thr Ser Asn 310 315 320gct gcc ctc
act ctg cgg aac ttt tgc aac tgg cag aag cag cac aac 1485Ala Ala Leu
Thr Leu Arg Asn Phe Cys Asn Trp Gln Lys Gln His Asn325 330 335
340cca ccc agt gac cgg gat gca gag cac tat gac aca gca att ctt ttc
1533Pro Pro Ser Asp Arg Asp Ala Glu His Tyr Asp Thr Ala Ile Leu Phe
345 350 355acc aga cag gac ttg tgt ggg tcc cag aca tgt gat act ctt
ggg atg 1581Thr Arg Gln Asp Leu Cys Gly Ser Gln Thr Cys Asp Thr Leu
Gly Met 360 365 370gct gat gtt gga act gtg tgt gat ccg agc aga agc
tgc tcc gtc ata 1629Ala Asp Val Gly Thr Val Cys Asp Pro Ser Arg Ser
Cys Ser Val Ile 375 380 385gaa gat gat ggt tta caa gct gcc ttc acc
aca gcc cat gaa tta ggc 1677Glu Asp Asp Gly Leu Gln Ala Ala Phe Thr
Thr Ala His Glu Leu Gly 390 395 400cac gtg ttt aac atg cca cat gat
gat gca aag cag tgt gcc agc ctt 1725His Val Phe Asn Met Pro His Asp
Asp Ala Lys Gln Cys Ala Ser Leu405 410 415 420aat ggt gtg aac cag
gat tcc cac atg atg gcg tca atg ctt tcc aac 1773Asn Gly Val Asn Gln
Asp Ser His Met Met Ala Ser Met Leu Ser Asn 425 430 435ctg gac cac
agc cag cct tgg tct cct tgc agt ggc tac atg att aca 1821Leu Asp His
Ser Gln Pro Trp Ser Pro Cys Ser Gly Tyr Met Ile Thr 440 445 450tca
ttt ctg gat aat ggt cat ggg gaa tgt ttg atg gac aag cct cag 1869Ser
Phe Leu Asp Asn Gly His Gly Glu Cys Leu Met Asp Lys Pro Gln 455 460
465aat ccc ata cag ctc cca ggc gat ctc cct ggc acc tcg tac gat gcc
1917Asn Pro Ile Gln Leu Pro Gly Asp Leu Pro Gly Thr Ser Tyr Asp Ala
470 475 480aac cgg cag tgc cag ttt aca ttt ggg gag gac tcc aaa cac
tgc cct 1965Asn Arg Gln Cys Gln Phe Thr Phe Gly Glu Asp Ser Lys His
Cys Pro485 490 495 500gat gca gcc agc aca tgt agc acc ttg tgg tgt
acc ggc acc tct ggt 2013Asp Ala Ala Ser Thr Cys Ser Thr Leu Trp Cys
Thr Gly Thr Ser Gly 505 510 515ggg gtg ctg gtg tgt caa acc aaa cac
ttc ccg tgg gcg gat ggc acc 2061Gly Val Leu Val Cys Gln Thr Lys His
Phe Pro Trp Ala Asp Gly Thr 520 525 530agc tgt gga gaa ggg aaa tgg
tgt atc aac ggc aag tgt gtg aac aaa 2109Ser Cys Gly Glu Gly Lys Trp
Cys Ile Asn Gly Lys Cys Val Asn Lys 535 540 545aac cac aga aag cat
ttt gat acg cct ttt cat gga agc tgg gga atg 2157Asn His Arg Lys His
Phe Asp Thr Pro Phe His Gly Ser Trp Gly Met 550 555 560tgg ggg cct
tgg gga gac tgt tcg aga acg tgc ggt gga gga gtc cag 2205Trp Gly Pro
Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln565 570 575
580tac acg atg agg gaa tgt gac aac cca gtc cca aag aat gga ggg aag
2253Tyr Thr Met Arg Glu Cys Asp Asn Pro Val Pro Lys Asn Gly Gly Lys
585 590 595tac tgt gaa ggc aaa cga gtg cgc tac aga tcc tgt aac ctt
gag gac 2301Tyr Cys Glu Gly Lys Arg Val Arg Tyr Arg Ser Cys Asn Leu
Glu Asp 600 605 610tgt cca gac aat aat gga aaa acc ttt aga gag gaa
caa tgt gaa gca 2349Cys Pro Asp Asn Asn Gly Lys Thr Phe Arg Glu Glu
Gln Cys Glu Ala 615 620 625cac aac gag ttt tca aaa gct tcc ttt ggg
agt ggg cct gcg gtg gaa 2397His Asn Glu Phe Ser Lys Ala Ser Phe Gly
Ser Gly Pro Ala Val Glu 630 635 640tgg att ccc aag tac gct ggc gtc
tca cca aag gac agg tgc aag ctc 2445Trp Ile Pro Lys Tyr Ala Gly Val
Ser Pro Lys Asp Arg Cys Lys Leu645 650 655 660atc tgc caa gcc aaa
ggc att ggc tac ttc ttc gtt ttg cag ccc aag 2493Ile Cys Gln Ala Lys
Gly Ile Gly Tyr Phe Phe Val Leu Gln Pro Lys 665 670 675gtt gta gat
ggt act cca tgt agc cca gat tcc acc tct gtc tgt gtg 2541Val Val Asp
Gly Thr Pro Cys Ser Pro Asp Ser Thr Ser Val Cys Val 680 685 690caa
gga cag tgt gta aaa gct ggt tgt gat cgc atc ata gac tcc aaa 2589Gln
Gly Gln Cys Val Lys Ala Gly Cys Asp Arg Ile Ile Asp Ser Lys 695 700
705aag aag ttt gat aaa tgt ggt gtt tgc ggg gga aat gga tct act tgt
2637Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly Asn Gly Ser Thr Cys
710 715 720aaa aaa ata tca gga tca gtt act agt gca aaa cct gga tat
cat gat 2685Lys Lys Ile Ser Gly Ser Val Thr Ser Ala Lys Pro Gly Tyr
His Asp725 730 735 740atc atc aca att cca act gga gcc acc aac atc
gaa gtg aaa cag cgg 2733Ile Ile Thr Ile Pro Thr Gly Ala Thr Asn Ile
Glu Val Lys Gln Arg 745 750 755aac cag agg gga tcc agg aac aat ggc
agc ttt ctt gcc atc aaa gct 2781Asn Gln Arg Gly Ser Arg Asn Asn Gly
Ser Phe Leu Ala Ile Lys Ala 760 765 770gct gat ggc aca tat att ctt
aat ggt gac tac act ttg tcc acc tta 2829Ala Asp Gly Thr Tyr Ile Leu
Asn Gly Asp Tyr Thr Leu Ser Thr Leu 775 780 785gag caa gac att atg
tac aaa ggt gtt gtc ttg agg tac agc ggc tcc 2877Glu Gln Asp Ile Met
Tyr Lys Gly Val Val Leu Arg Tyr Ser Gly Ser 790 795 800tct gcg gca
ttg gaa aga att cgc agc ttt agc cct ctc aaa gag ccc 2925Ser Ala Ala
Leu Glu Arg Ile Arg Ser Phe Ser Pro Leu Lys Glu Pro805 810 815
820ttg acc atc cag gtt ctt act gtg ggc aat gcc ctt cga cct aaa att
2973Leu Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu Arg Pro Lys Ile
825 830 835aaa tac acc tac ttc gta aag aag aag aag gaa tct ttc aat
gct atc 3021Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys Glu Ser Phe Asn
Ala Ile 840 845 850ccc act ttt tca gca tgg gtc att gaa gag tgg ggc
gaa tgt tct aag 3069Pro Thr Phe Ser Ala Trp Val Ile Glu Glu Trp Gly
Glu Cys Ser Lys 855 860 865tca tgt gaa ttg ggt tgg cag aga aga ctg
gta gaa tgc cga gac att 3117Ser Cys Glu Leu Gly Trp Gln Arg Arg Leu
Val Glu Cys Arg Asp Ile 870 875 880aat gga cag cct gct tcc gag tgt
gca aag gaa gtg aag cca gcc agc 3165Asn Gly Gln Pro Ala Ser Glu Cys
Ala Lys Glu Val Lys Pro Ala Ser885 890 895 900acc aga cct tgt gca
gac cat ccc tgc ccc cag tgg cag ctg ggg gag 3213Thr Arg Pro Cys Ala
Asp His Pro Cys Pro Gln Trp Gln Leu Gly Glu 905 910 915tgg tca tca
tgt tct aag acc tgt ggg aag ggt tac aaa aaa aca agc 3261Trp Ser Ser
Cys Ser Lys Thr Cys Gly Lys Gly Tyr Lys Lys Thr Ser 920 925 930ttg
aag tgt ctg tcc cat gat gga ggg gtg tta tct cat gac agc tgt 3309Leu
Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser His Asp Ser Cys 935 940
945gat cct tta aag aaa cct aaa cat ttc ata gac ttt tgc aca atg gca
3357Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp Phe Cys Thr Met Ala
950 955 960gaa tgc agt taagtggttt aagtggtgtt agctttgagg gcaaggcaaa
3406Glu Cys Ser965gtgaggaagg gctggtgcag ggaaagcaag aaggctggag
ggatccagcg tatcttccca 3466gtaaccagtg aggtgtatca gtaaggtggg
attatggggg tagatagaaa aggagttgaa 3526tcatcagagt aaactgccag
ttgcaaattt gataggatag ttagtgagga ttattaacct 3586ctgagcagtg
atatagcata ataaagcccc gggcattatt attattattt cttttgttac
3646atctactaca agtttagaaa aaacaaagca attgtcaaaa aaagttagaa
ctattacaac 3706ccctgcttcc tggtacttat caaatactta gtatcatggg
ggttgggaaa tgaaaagtag 3766gagaaaagtg agattttact aagacctgtt
ttactttacc tcactaaaca atggggggag 3826aaaggagtac aaataggatc
ttttgaccag cactgtttat gggctgctat ggtttcagag 3886aacgtctata
cattatttct accgaggatt taaaacttcc agattgttcc aacatggaga
3946ggaaaggctc aggcaacgtg gaaataacgc aatgggcttc ccccttccct
ttttgggacc 4006cactccag 4014126967PRTHomo sapiens 126Met Gln Arg
Ala Val Pro Glu Gly Phe Gly Arg Arg Lys Leu Gly Ser1 5 10 15Asp Met
Gly Asn Ala Glu Arg Ala Pro Gly Ser Arg Ser Phe Gly Pro 20 25 30Val
Pro Thr Leu Leu Leu Leu Ala Ala Ala Leu Leu Ala Val Ser Asp 35 40
45Ala Leu Gly Arg Pro Ser Glu Glu Asp Glu Glu Leu Val Val Pro Glu
50 55 60Leu Glu Arg Val Pro Gly His Gly Thr Thr Arg Leu Arg Leu His
Ala65 70 75 80Phe Asp Gln Gln Leu Asp Leu Asp Val Pro Pro Asp Ser
Ser Phe Leu 85 90 95Ala Pro Gly Phe Thr Leu Gln Asn Val Gly Arg Lys
Ser Gly Ser Asp 100 105 110Thr Pro Leu Pro Glu Thr Asp Leu Ala His
Cys Phe Tyr Ser Gly Thr 115 120 125Val Asn Gly Asp Pro Ser Ser Ala
Ala Ala Leu Ser Leu Cys Glu Gly 130 135 140Val Arg Gly Ala Phe Tyr
Leu Leu Gly Glu Ala Tyr Phe Ile Gln Pro145 150 155 160Leu Pro Ala
Ala Ser Glu Arg Leu Ala Thr Ala Ala Pro Gly Glu Lys 165 170 175Pro
Pro Ala Pro Leu Gln Phe His Leu Leu Arg Arg Asn Arg Gln Gly 180 185
190Asp Val Gly Gly Thr Cys Gly Val Val Asp Asp Glu Pro Arg Pro Thr
195 200 205Gly Lys Ala Glu Thr Glu Asp Glu Asp Glu Gly Thr Glu Gly
Glu Asp 210 215 220Glu Gly Pro Gln Trp Ser Pro Gln Asp Pro Ala Leu
Gln Gly Val Gly225 230 235 240Gln Pro Thr Gly Thr Gly Ser Ile Arg
Lys Lys Arg Phe Val Ser Ser 245 250 255His Arg Tyr Val Glu Thr Met
Leu Val Ala Asp Gln Ser Met Ala Glu 260 265 270Phe His Gly Ser Gly
Leu Lys His Tyr Leu Leu Thr Leu Phe Ser Val 275 280 285Ala Ala Arg
Leu Tyr Lys His Pro Ser Ile Arg Asn Ser Val Ser Leu 290 295 300Val
Val Val Lys Ile Leu Val Ile His Asp Glu Gln Lys Gly Pro Glu305 310
315 320Val Thr Ser Asn Ala Ala Leu Thr Leu Arg Asn Phe Cys Asn Trp
Gln 325 330 335Lys Gln His Asn Pro Pro Ser Asp Arg Asp Ala Glu His
Tyr Asp Thr 340 345 350Ala Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly
Ser Gln Thr Cys Asp 355 360 365Thr Leu Gly Met Ala Asp Val Gly Thr
Val Cys Asp Pro Ser Arg Ser 370 375 380Cys Ser Val Ile Glu Asp Asp
Gly Leu Gln Ala Ala Phe Thr Thr Ala385 390 395 400His Glu Leu Gly
His Val Phe Asn Met Pro His Asp Asp Ala Lys Gln 405 410 415Cys Ala
Ser Leu Asn Gly Val Asn Gln Asp Ser His Met Met Ala Ser 420 425
430Met Leu Ser Asn Leu Asp His Ser Gln Pro Trp Ser Pro Cys Ser Gly
435 440 445Tyr Met Ile Thr Ser Phe Leu Asp Asn Gly His Gly Glu Cys
Leu Met 450 455 460Asp Lys Pro Gln Asn Pro Ile Gln Leu Pro Gly Asp
Leu Pro Gly Thr465 470 475 480Ser Tyr Asp Ala Asn Arg Gln Cys Gln
Phe Thr Phe Gly Glu Asp Ser 485 490 495Lys His Cys Pro Asp Ala Ala
Ser Thr Cys Ser Thr Leu Trp Cys Thr 500 505 510Gly Thr Ser Gly Gly
Val Leu Val Cys Gln Thr Lys His Phe Pro Trp 515 520 525Ala Asp Gly
Thr Ser Cys Gly Glu Gly Lys Trp Cys Ile Asn Gly Lys 530 535 540Cys
Val Asn Lys Asn His Arg Lys His Phe Asp Thr Pro Phe His Gly545 550
555 560Ser Trp Gly Met Trp Gly Pro Trp Gly Asp Cys Ser Arg Thr Cys
Gly 565 570 575Gly Gly Val Gln Tyr Thr Met Arg Glu Cys Asp Asn Pro
Val Pro Lys 580 585 590Asn Gly Gly Lys Tyr Cys Glu Gly Lys Arg Val
Arg Tyr Arg Ser Cys 595 600 605Asn Leu Glu Asp Cys Pro Asp Asn Asn
Gly Lys Thr Phe Arg Glu Glu 610 615 620Gln Cys Glu Ala His Asn Glu
Phe Ser Lys Ala Ser Phe Gly Ser Gly625 630 635 640Pro Ala Val Glu
Trp Ile Pro Lys Tyr Ala Gly Val Ser Pro Lys Asp 645 650 655Arg Cys
Lys Leu Ile Cys Gln Ala Lys Gly Ile Gly Tyr Phe Phe Val 660 665
670Leu Gln Pro Lys Val Val Asp Gly Thr Pro Cys Ser Pro Asp Ser Thr
675 680 685Ser Val Cys Val Gln Gly Gln Cys Val Lys Ala Gly Cys Asp
Arg Ile 690 695
700Ile Asp Ser Lys Lys Lys Phe Asp Lys Cys Gly Val Cys Gly Gly
Asn705 710 715 720Gly Ser Thr Cys Lys Lys Ile Ser Gly Ser Val Thr
Ser Ala Lys Pro 725 730 735Gly Tyr His Asp Ile Ile Thr Ile Pro Thr
Gly Ala Thr Asn Ile Glu 740 745 750Val Lys Gln Arg Asn Gln Arg Gly
Ser Arg Asn Asn Gly Ser Phe Leu 755 760 765Ala Ile Lys Ala Ala Asp
Gly Thr Tyr Ile Leu Asn Gly Asp Tyr Thr 770 775 780Leu Ser Thr Leu
Glu Gln Asp Ile Met Tyr Lys Gly Val Val Leu Arg785 790 795 800Tyr
Ser Gly Ser Ser Ala Ala Leu Glu Arg Ile Arg Ser Phe Ser Pro 805 810
815Leu Lys Glu Pro Leu Thr Ile Gln Val Leu Thr Val Gly Asn Ala Leu
820 825 830Arg Pro Lys Ile Lys Tyr Thr Tyr Phe Val Lys Lys Lys Lys
Glu Ser 835 840 845Phe Asn Ala Ile Pro Thr Phe Ser Ala Trp Val Ile
Glu Glu Trp Gly 850 855 860Glu Cys Ser Lys Ser Cys Glu Leu Gly Trp
Gln Arg Arg Leu Val Glu865 870 875 880Cys Arg Asp Ile Asn Gly Gln
Pro Ala Ser Glu Cys Ala Lys Glu Val 885 890 895Lys Pro Ala Ser Thr
Arg Pro Cys Ala Asp His Pro Cys Pro Gln Trp 900 905 910Gln Leu Gly
Glu Trp Ser Ser Cys Ser Lys Thr Cys Gly Lys Gly Tyr 915 920 925Lys
Lys Thr Ser Leu Lys Cys Leu Ser His Asp Gly Gly Val Leu Ser 930 935
940His Asp Ser Cys Asp Pro Leu Lys Lys Pro Lys His Phe Ile Asp
Phe945 950 955 960Cys Thr Met Ala Glu Cys Ser965
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References