U.S. patent application number 11/009008 was filed with the patent office on 2005-05-12 for mammalian cdp-diacylglycerol synthase.
This patent application is currently assigned to CELL THERAPEUTICS, INC.. Invention is credited to Leung, David W..
Application Number | 20050100949 11/009008 |
Document ID | / |
Family ID | 23080549 |
Filed Date | 2005-05-12 |
United States Patent
Application |
20050100949 |
Kind Code |
A1 |
Leung, David W. |
May 12, 2005 |
Mammalian CDP-diacylglycerol synthase
Abstract
There is disclosed cDNA sequences and polypeptides having the
enzyme CDP-diacylglycerol synthase (CDS) activity. CDS is also
known as CTP:phosphatidate cytidylyltransferase. There is further
disclosed methods for isolation and production of polypeptides
involved in phosphatidic acid metabolism and signaling in mammalian
cells, in particular, the production of purified forms of CDS.
Inventors: |
Leung, David W.; (Mercer
Island, WA) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
CELL THERAPEUTICS, INC.
|
Family ID: |
23080549 |
Appl. No.: |
11/009008 |
Filed: |
December 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11009008 |
Dec 13, 2004 |
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10136517 |
May 2, 2002 |
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10136517 |
May 2, 2002 |
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09282218 |
Mar 31, 1999 |
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6503700 |
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Current U.S.
Class: |
435/6.14 ;
435/193; 435/252.33; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1241 20130101;
C12Y 207/07041 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/193; 435/252.33; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/10; C12N 015/74 |
Claims
1-16. (canceled)
17. An isolated polynucleotide, comprising a polynucleotide
sequence selected from the group consisting of: (a) the DNA
sequence of SEQ ID NO. 11, (b) a DNA sequence, which encodes the
polypeptide of SEQ ID NO. 12 and any biologically active fragments
thereof, (c) a DNA sequence that has at least 85% sequence identity
to the sequence depicted in SEQ ID NO. 11, and (d) a DNA sequence
that has at least 85% sequence identity to a DNA sequence that
encodes the polypeptide of SEQ ID NO. 12 or any biologically active
fragments thereof, wherein said isolated polynucleotide encodes a
polypeptide having cytidine diphosphate diacylglycerol synthase
(CDS) activity.
18. A method of expressing a polypeptide from the polynucleotide
that has CDS activity, comprising (a) introducing into a cell the
polynucleotide of claim 17, wherein said polynucleotide is operably
linked to a promoter; and (b) maintaining or growing said cell
under conditions that result in the expression of a polypeptide
having CDS activity.
19. The isolated polynucleotide of claim 17, wherein said
polynucleotide comprises (a) the DNA sequence of SEQ ID NO. 11 or
(b) the DNA sequence encoding the polypeptide of SEQ ID NO. 12.
20. The isolated polynucleotide of claim 19, wherein said
polynucleotide comprises the DNA sequence of SEQ ID NO. 11.
21. The isolated polynucleotide of claim 19, wherein said
polynucleotide comprises the DNA sequence encoding the polypeptide
of SEQ ID NO. 12.
22. An isolated polynucleotide comprising a polynucleotide sequence
that hybridizes under high stringency conditions of either (i)
incubation in 5.times.SSC at 65.degree. C., followed by washing in
0.1.times.SSC for 30 minutes, or (ii) incubation in 50% formamide
and 5.times.SSC at 42.degree. C. to the coding region of the DNA
sequence of claim 19, wherein said polynucleotide encodes a protein
having CDS activity.
23. An isolated polynucleotide comprising a polynucleotide sequence
that hybridizes under high stringency conditions of either (i)
incubation in 5.times.SSC at 65.degree. C., followed by washing in
0.1.times.SSC for 30 minutes, or (ii) incubation in 50% formamide
and 5.times.SSC at 42.degree. C. to the complement of the DNA
sequence depicted in SEQ ID NO. 11, wherein said polynucleotide
encodes a protein having CDS activity.
24. The isolated polynucleotide of claim 17, wherein said
polynucleotide encodes a full-length CDS polypeptide.
Description
TECHNICAL FILED OF THE INVENTION
[0001] This present invention provides cDNA sequences and
polypeptides having the enzyme CDP-diacylglycerol synthase (CDS)
activity. CDS is also known as CTP:phosphatidate cytidyltransferase
(EC2.7.7.41). The present invention further provides for isolation
and production of polypeptides involved in phosphatidic acid
metabolism and signaling in mammalian cells, in particular, the
production of purified forms of CDS.
BACKGROUND OF THE INVENTION
[0002] CDP-diacylglycerol (DAG) is an important branch point
intermediate just downstream of phosphatidic acid (PA) in the
pathways for biosynthesis of glycerophosphate-based phospholipids
(Kent, Anal. Rev. Biochem. 64: 315-343, 1995). In eukaryotic cells,
PA, the precursor molecule for all glycerophospholipid, is
converted either to CDP-DAG by CDP-DAG synthase (CDS) or to DAG by
a phosphohydrolase. In mammalian cells, CDP-DAG is the precursor to
phosphatidylinositol (PI), phosphatidylglycerol (PG), and
cardiolipin (CL). Diacylglycerol is the precursor to
triacylglycerol, phosphatidylethanolamine, and phosphatidylcholine
in eukaryotic cells. Therefore, the partitioning of phosphatidic
acid between CDP-diacylglycerol and diacylglycerol must be an
important regulatory point in eukaryotic phospholipid metabolism
(Shen et al., J. Biol. Chem. 271:789-795, 1996). In eukaryotic
cells, CDP-diacylglycerol is required in the mitochondria for
phosphatidylglycerol and cardiolipin synthesis and in the
endoplasmic reticulum and possibly other organelles for the
synthesis of phosphatidylinositol (PI). PI, in turn, is the
precursor for the synthesis of a series of lipid second messengers,
such as phosphatidylinositol-4,5-bisphosphate (PIP.sub.2), DAG and
inositol-1,4,5-trisphosphate (IP.sub.3). Specifically, PIP.sub.2 is
the substrate for phospholipase C that is activated in response to
a wide variety of extracellular stimuli, leading to the generation
of two lipid second messengers; namely, DAG for the activation of
protein kinase C and IP.sub.3 for the release of Ca.sup.++ from
internal stores (Dowhan, Anal. Rev. Biochem. 66: 199-232,
1997).
[0003] The genes coding for CDS have been identified in E. coli
(Icho et al, J. Biol. Chem. 260:12078-12083, 1985), in yeast (Shen
et al., J. Biol. Chem. 271:789-795, 1996), and in Drosophila (Wu et
al., Nature 373:216-222, 1995). A human cDNA coding for CDS (hCDS1)
is described by us herein and has been reported in Weeks et al.,
DNA Cell Biol. 16: 281-289, 1997. Moreover, Heacock et al., J.
Neurochem. 67: 2200-2203, 1997 report cloning of a CDS1 from a
human neuronal cell line. Furthermore, Lykidis et al., J. Biol.
Chem 272:33402-33409, 1997 and Halford et al., Genomics 54:140-144,
1998 both report DNA sequences suspected to encode a human cds2
protein, but these references fail to disclose either biological
activity or an intact N-terminal region for the putative
proteins.
[0004] It is of interest to isolate polynucleotides coding for
human CDS and express them in mammalian cells to determine the
potential roles of this enzyme in cellular function and use this
enzyme as a target for the development of specific compounds that
are modulators of its activity. With the advance in the
understanding of disease processes, it has been found that many
diseases result from the malfunction of intracellular signaling.
This recognition has led to research and development of therapies
based on the interception of signaling pathways in diseases
(Levitzki, Curr. Opin. Cell Biol. 8:239-244, 1996). Compounds that
modulate CDS activity, and hence modulate generation of a variety
of lipid second messengers and signals involved in cell activation,
are therefore of therapeutic interest generally, and of particular
interest in the areas of inflammation and oncology.
SUMMARY OF THE INVENTION
[0005] The present invention provides cDNA sequences, polypeptide
sequences, and transformed cells for producing isolated recombinant
mammalian CDS. The present invention provides two novel human
polypeptides and fragment thereof, having CDS activity. The
polypeptides discovered herein are novel and will be called hCDS1
(human CDS1) and hCDS2 (human CDS2). CDS catalyzes the conversion
of phosphatidic acid (PA) to CDP-diacylglycerol (CDP-DAG), which in
turn is the precursor to phosphatidylinositol (PI),
phosphatidylglycerol (PG) and cardiolipin (CL).
[0006] The present invention further provides nucleic acid
sequences coding for expression of the novel CDS polypeptides and
active fragments thereof The invention further provides purified
CDS mRNAs and antisense oligonucleotides for modulation of
expression of the genes coding for CDS polypeptides. Assays for
screening test compounds for their ability to modulate CDS activity
are also provided.
[0007] Recombinant CDS is useful for screening candidate drug
compounds that modulate CDS activity, particularly those compounds
that activate or inhibit CDS activity. The present invention
provides cDNA sequences encoding a polypeptide having CDS activity
and comprising the DNA sequence set forth in SEQ ID NO. 1 (hCDS1),
the DNA sequence set forth in FIG. 8 (hCDS2), shortened fragments
thereof, or additional cDNA sequences which due to the degeneracy
of the genetic code encode a polypeptide of SEQ ID NO. 2 (hCDS1), a
polypeptide of FIG. 8 (hCDS2), or biologically active fragments
thereof, or a sequence hybridizing thereto under high stringency
conditions. The present invention further provides a polypeptide
having CDS activity and comprising the amino acid sequence of SEQ
ID NO. 2 (hCDS1), the amino acid sequence of FIG. 8 (hCDS2), or
biologically active fragments thereof.
[0008] Also provided by the present invention are vectors
containing a DNA sequence encoding a mammalian CDS enzyme in
operative association with an expression control sequence. Host
cells, transformed with such vectors for use in producing
recombinant CDS are also provided with the present invention. The
inventive vectors and transformed cells are employed in a process
for producing recombinant mammalian CDS. In this process, a cell
line transformed with a cDNA sequence encoding a CDS enzyme in
operative association with an expression control sequence, is
cultured. The claimed process may employ a number of known cells as
host cells for expression of the CDS polypeptide, including, for
example, mammalian cells, yeast cells, insect cells and bacterial
cells.
[0009] Another aspect of this invention provides a method for
identifying a pharmaceutically-active compound by determining if a
selected compound modulates the activity of CDS for converting PA
to CDP-DAG. A compound having such activity is capable of
modulating signaling kinase pathways and being a pharmaceutical
compound useful for augmenting trilineage hematopoiesis after
cytoreductive therapy and for anti-inflammatory activity in
inhibiting the inflammatory cascade following hypoxia and
reoxygenation injury (e.g., sepsis, trauma, ARDS, etc.).
[0010] The present invention further provides a transformed cell
that expresses active mammalian CDS and further comprises a means
for determining if a drug candidate compound is therapeutically
active by modulating recombinant CDS activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the cDNA sequence encoding hCDS1. The
nucleotide sequence analysis and restriction mapping of the cDNA
clone revealed a 5'-untranslated region of 149 base pairs, an open
reading frame capable of encoding a 461 amino acid polypeptide that
spans nucleotide positions 150 to 1535 and a 3'-untranslated region
of 520 base pairs.
[0012] FIG. 2 shows the translated amino acid sequence of
hCDS1.
[0013] FIG. 3 shows the amino acid sequence of hCDS1.
[0014] FIG. 4 shows the sequence homology among the hCDS1 coding
sequence, the yeast CDS coding sequence, E. coli CDS coding
sequence, and the Drosophila CDS coding sequence. This comparison
shows that hCDS1 has the greatest extended homology with amino
acids 109 to 448 of Drosophila CDS. The hCDS1 protein and the CDS
protein from Drosophila, yeast, and E. coli have 45%, 21% and 7%
overall match in amino acid sequence, respectively.
[0015] FIG. 5 shows the results of in vitro hCDS1 activity assays
on cell fractions from stable transfectants of NCI-H460 cells. CDS
activity was assessed by conversion of (.alpha.-.sup.32P)CTP to
(.sup.32P)CDP-DAG in in vitro reactions that required addition of
an exogenous PA substrate. This is a representative histogram
comparing the radiolabel incorporated into various cell fractions
(membranes, cytosol, and nuclei/unbroken cells) from NCI-H460 cells
stably transfected with the hCDS1 cDNA (pCE2.hCDS) or vector only
(pCE2). In all fractions, the hCDS1 cDNA increased radiolabel in
the organic phase of the reactions. Total CDS activity was much
greater in membrane fractions, as would be expected for membrane
associated CDS, compared to cytosol fractions. Activity in unbroken
cells masked the activity specific to nuclei.
[0016] FIG. 6 is a representative phosphorimage of
[.sup.32P]phospholipids from membrane fraction CDS assay reactions
after the second dimension of ffTLC. FIG. 6 confirms that the
radiolabeled product found in the membrane fractions does migrate
with a CDP-DAG standard on TLC. The identities of labeled bands
were determined by migration of phospholipid standards visualized
by UV or FL imaging on the STORM after primulin staining. Lanes 1-3
represent triplicate samples derived from membranes of NCI-H460
cells transfected with the hCDS1 expression vector, and lanes 4-6
represent triplicate samples from transfectants with the control
vector. Cells transfected with the hCDS1 cDNA showed 1.6-2.4 fold
more CDS activity in membrane fractions than vector transfectants.
The relative CDS activity between hCDS1 transfectants and vector
transfectants was similar when determined by scintillation counting
or TLC analysis. These data indicate that the hCDS1 cDNA clone of
SEQ ID NO. 1 does encode CDS activity.
[0017] FIGS. 7A and 7B show, respectively, that production of
TNF-.alpha. (tumor necrosis factor alpha) and IL-6 in ECV304 cells
stably transfected with a hCDS1 expression vector increases by
greater than five fold relative to ECV304 cells stably transfected
with control vector after equal stimulation with IL-1.beta.
(interleukin-1 beta).
[0018] There was little effect on basal level of cytokine release.
These data indicate that overexpression of hCDS1 amplified the
cytokine signaling response in these cells, as opposed to enhancing
steady state, basal signals.
[0019] FIG. 8 shows the DNA and amino acid sequence of hCDS2.
[0020] FIG. 9 shows an amino acid sequence alignment of the hCDS2
coding sequence with the hCDS1 coding sequence. The amino acids
that are identical between the two sequences are highlighted.
[0021] FIG. 10 shows the results of a TLS analysis of hCDS2
production of [32P]CDP-DAG after TLC analysis
[0022] FIG. 11 shows expression of hCDS1 and hCDS2 mRNAs in cancer
versus normal prostate tissues.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides novel, isolated, biologically
active mammalian CDS enzymes. The term "isolated" means any CDS
polypeptide of the present invention, or any other gene encoding
CDS polypeptide, which is essentially free of other polypeptides or
genes, respectively, or of other contaminants with which the CDS
polypeptide or gene might normally be found in nature.
[0024] The invention includes a biologically active polypeptide,
CDS, and biologically active fragments thereof As used herein, the
term "biologically active polypeptide" refers to a polypeptide
which possesses a biological function or activity which is
identified through a biological assay, preferably cell-based, and
which results in the formation of CDS-DAG species from PA. A
"biologically active polynucleotide" denotes a polynucleotide which
encodes a biologically active polypeptide. The term "biologically
active fragment," as used herein, refers to a nucleotide or
polypeptide sequence in which one or more amino acids or
nucleotides has been deleted but which retains CDS activity.
[0025] Minor modification of the CDS primary amino acid sequence
may result in proteins which have substantially equivalent activity
as compared to the sequenced CDS polypeptide described herein. Such
modifications may be deliberate, as by site-directed mutagenesis,
or may be spontaneous. All of the polypeptides produced by these
modifications are included herein as long as the activity of CDS is
present. This can lead to the development of a smaller active
molecule which would have broader utility. For example, the present
invention includes removal of one or more amino, carboxy terminal,
or internal amino acids from the CDS polypeptide, so long as such
amino acids are not required for CDS activity.
[0026] The CDS polypeptide of the present invention also includes
conservative variations of the polypeptide sequence. The term
"conservative variation" denotes the replacement of an amino acid
residue by another, biologically active similar residue. Examples
of conservative variations include the substitution of one
hydrophobic residue, such as isoleucine, valine, leucine or
methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acids, or glutamine for asparagine, and the
like. The term "conservative variation" also includes the use of a
substituted amino acid in place of parent amino acid provided that
antibodies raised to the substituted polypeptide also
immunologically react with the unsubstituted polypeptide.
[0027] The present invention further includes allelic variations
(naturally-occurring base changes in the species population which
may or may not result in an amino acid change) of the DNA sequences
herein encoding active CDS polypeptides and active fragments
thereof
[0028] The inventive DNA sequences further comprise those sequences
which hybridize under high stringency conditions (see, for example,
Maniatis et al, Molecular Cloning (A Laboratory Manual), Cold
Spring Harbor Laboratory, pages 387-389, 1982) to the coding region
of hCDS1 (e.g., nucleotide #150 to nucleotide #1535 in SEQ ID NO.
1) or the coding region of hCDS2 (FIG. 8) and which have CDS
activity. High stringency conditions include 5.times.SSC at
65.degree. C., followed by washing in 0.1.times.SSC at 65.degree.
C. for thirty minutes or, alternatively, 50% formamide, 5.times.SSC
at 42.degree. C.
[0029] The present invention also includes nucleotide sequences
having at least an 85%, at least a 90%, or at least a 95% sequence
identity to the nucleotide sequence of the coding region of hCDS2
(FIG. 8) and which have CDS activity. The present invention further
includes a polypeptide having at least an 85%, at least a 90%, or
at least a 95% sequence identity to the hCDS2 polypeptide shown in
FIG. 8 and which have CDS activity. As used herein, the term
"sequence identity" denotes the "match percentage" calculated by
the DNASIS computer program (Version 2.5 for Windows; available
from Hitachi Software Engineering Co., Ltd., South San Francisco,
Calif.) using standard defaults as described in the reference
manual accompanying the software, which is incorporated herein by
reference.
[0030] With regard to the above-described fragments of hCDS2,
sequences that hybridize to hCDS2, and sequences having sequence
identity to hCDS2, the invention includes embodiments where these
sequences have an intact CDS N-terminal region.
[0031] The present invention further includes DNA sequences which
code for CDS polypeptides having CDS activity but differ in codon
sequence due to degeneracy of the genetic code. Variations in the
DNA sequences which are caused by point mutations or by induced
modifications of the sequence of SEQ ID NO. 1 or FIG. 8, which
enhance the activity of the encoded polypeptide or production of
the encoded CDS polypeptide are also encompassed by the present
invention.
[0032] CDS Sequence Discovery
[0033] hCDS1
[0034] A homology search of the Genbank database (Boguski, et al.,
Science 265:1993-1994, 1994) of expressed sequence tags (dbEST)
using Drosophila CDS protein sequence as a probe came up with
several short stretches of cDNA sequence with homology to the
Drosophila CDS protein sequence. These cDNA sequences were derived
from single-run partial sequencing of random human cDNA clones
carried out mainly by I.M.A.G.E. Consortium [LLNL] cDNA clones
program. An example of the amino acid sequence homology between the
Drosophila CDS and a human cDNA clone (IMAGE Clone ID #135630) is
shown below:
1 371 KRAFKIKDFGDMIPGHGGIMDRFDCQFLMATFVNVYIS 408 KRAFKIKDF +
IPGHGGIMDRFDCQ+LMATFV+VYI+ 11
KRAFKIKDFANTIPGHGGIMDRFDCQYLMATFVHVYIT 124
[0035] The top line (SEQ ID NO. 3) refers to the Drosophila CDS
sequence from amino acids 371 to 408 and the bottom line (SEQ ID
NO. 4) refers to a homologous region from IMAGE Clone ID #135630
translated using reading frame +2. Identical amino acids between
these two sequences are shown on the middle line with the "+" signs
indicating conservative amino acid changes. In order to determine
if such cDNA clones with this level of homology to the Drosophila
CDS sequence encoded human CDS sequence, it was necessary to
isolate the full-length cDNA clone, insert it into an expression
vector, and test if cells transfected with the cDNA expression
vector will produce more CDS activity.
[0036] Accordingly, a synthetic oligonucleotide (o.h.cds.1R),
5'-CCCACCATGG CCAGGAATGG TATTTGC-3' (SEQ ID NO. 5), was made based
on the complement sequence of the amino acid region, ANTIPGHGG, of
IMAGE Clone ID #135630 for the isolation of a putative human cDNA
clone from a SuperScript human leukocyte cDNA library (Life
Technologies, Gaithersburg, Md.) using the GeneTrapper cDNA
positive selection system (Life Technologies, Gaithersburg, Md.).
The colonies obtained from positive selection were screened with a
[.gamma.-.sup.32P]-ATP labeled synthetic oligonucleotide
(o.h.cds.1), 5'-AGTGATGTGA ATTCCTTCGT GACAG-3' (SEQ ID NO. 6),
corresponding to nucleotides 144-168 of IMAGE Clone ID #133825. Of
the few cDNA clones that hybridized with the o.h.cds.1 probe, clone
LK64 contained the largest cDNA insert with a size of 1700 base
pairs. DNA sequence analysis of LK64 showed the translated sequence
of its largest open reading frame from the 5' end contained
extensive homology with amino acids 109 to 448 of the Drosophila
CDS protein sequence. Clone LK64 did not appear to contain a
full-length cDNA insert for CDS. It was missing the coding region
corresponding to the first 110 amino acids from the N-terminus. A
second homology search of the Genbank database (Boguski, et al.,
Science 265:1993-1994, 1994) using the 3'-untranslated sequence of
LK64 as a probe came up with more short stretches of cDNA sequences
with perfect homology to the 3' end of the putative human CDS clone
LK64. Restriction mapping and DNA sequence analysis of IMAGE Clone
ID #145253 (Genome Systems, St. Louis, Mo.), derived from a
placental cDNA library, showed it contained extensive sequence
homology with the N-terminal coding region of the Drosophila CDS
and overlapped with the sequence obtained from clone LK64.
[0037] To assemble the putative full-length human CDS cDNA clone, a
500 base pair Pst I-Nco I fragment from of IMAGE Clone ID #145253
and a 1500 base pair Nco I-Not I fragment from LK64 were isolated.
These two fragments were inserted into a Pst I and Not I digested
vector pBluescriptII SK(-) vector via a three-part ligation to
generate pSK.hcds.
[0038] FIG. 1 shows the cDNA sequence of hCDS1. The nucleotide
sequence analysis and restriction mapping of the cDNA clone
revealed a 5'-untranslated region of 149 base pairs, an open
reading frame encoding a 461 amino acids polypeptide that spans
nucleotide positions 150 to 1535 and a 3'-untranslated region of
520 base pairs (FIG. 2). The ATG initiation site for translation
was identified at nucleotide positions 150-152 and fulfilled the
requirement for an adequate initiation site. (Kozak, Critical Rev.
Biochem. Mol. Biol. 27:385-402, 1992). There was another upstream
ATG at positions 4-6 but it was followed by an in-phase stop codon
at positions 19-20. The calculated molecular weight of hCDS1 is
53,226 daltons with a predicted pI of 7.57.
[0039] The sequence of the 461 amino acid open reading frame (FIG.
3) was used as the query sequence to search for homologous
sequences in protein databases. A search of Genbank Release 92 from
the National Center for Biotechnology Information (NCBI) using the
BLAST program showed that this protein was most homologous to the
Drosophila CDS, the yeast CDS, and the E. coli CDS. FIG. 4 shows
amino acid sequence alignment of this putative human CDS coding
sequence with the Drosophila CDS, the yeast CDS, and the E. coli
coding sequences, showing that the human CDS is most homologous to
the Drosophila CDS.
[0040] hCDS2
[0041] A homology search of the Genbank database (Boguski, et al.,
Science 265:1993-1994, 1994) of expressed sequence tags (dbEST)
using the hCDS1 protein sequence (Weeks et al, DNA Cell Biol. 16:
281-289, 1997) as probe came up with several short stretches of
human cDNA sequences that were homologous but distinct from the
hCDS1 sequence. These cDNA sequences were derived from single-run
partial sequencing of random human cDNA clones projects carried out
mainly by I.M.A.G.E. Consortium [LLNL] cDNA clones program.
[0042] Of these sequences, IMAGE Clone ID#485825 was found to have
the following homology to the coding region of hCDS1 from amino
acids 227-271:
2 10 20 30 40 QSHLVIHNLFEGMIWFIVPISCVICNDIMAYMFGFFFGRTPLIKL
X:::::.:::::::::.::::.:::::: ::.:::::::::::::
QSHLVIQNLFEGMIWFLVPISSVICNDITAYLFGFFFGRTPLIKL 230 240 250 260
270
[0043] The top line refers to IMAGE Clone ID#485825 translated
using reading frame +3 and the bottom line refers to the coding
region of hCDS1 from amino acids 227-271. Identical amino acids
between these two sequences are shown on the middle line as ":" and
with the "." signs indicating conservative amino acid changes.
Since the 5'-end of the cDNA insert of IMAGE Clone ID#485825
corresponded to amino acid 227 of hCDS1, this clone therefore does
not appear to contain a full-length cDNA insert for CDS, most
likely missing the coding region corresponding to the first 220
amino acids from the N-terminus. A second homology search of the
Genbank database (Boguski, et al., Science 265:1993-1994, 1994) of
expressed sequence tags (dbEST) using the sequence of IMAGE Clone
ID#485825 as probe came up with a clone with a longer cDNA insert
(clone ID#663789) from the Genbank database with perfect homology
to the IMAGE Clone ID#485825. Restriction mapping and DNA sequence
analysis of IMAGE Clone ID#663789 (Genome Systems, St. Louis, Mo.)
showed it to be a longer cDNA clone with extensive sequence
homology with the coding region of hCDS1 but still missing the
first 60 amino acids in the coding region. To isolate the 5'-coding
region of hCDS2 cDNA, a synthetic oligonucleotide, 5'-AGGACGCATA
TGAGTGGTAG AC-3' (oCDS2.sub.--2R), complementary to a region
spanning the Nde I site near the 5' portion of clone ID#663789 was
used in combination with a forward vector primer (o.sport.1),
5'-GACTCTAGCC TAGGCTTTTG C-3' for amplification of the 5'-region
from a pCMV.SPORT human leukocyte cDNA library (Life Technologies,
Gaithersburg, Md.). PCR fragments generated that were >400 bp
were inserted into the pGEM-T vector (Promega, Madison, Wis.) for
further analysis. Restriction mapping and DNA sequence analysis
showed one of the clones, pCDS2.H7, to be homologous to the
N-terminal coding region of hCDS1.
[0044] To assemble the putative full-length human CDS cDNA clone,
the 420 bp Acc65 I-Nde I fragment from pCDS2.H7 and the 1200 bp Nde
I-Xba I fragment from clone ID#663789 were isolated. These two
fragments were inserted into a Acc65 I and Xba I digested vector
pBluescript SK(-)II vector via a three-part ligation to generate
pSK.CDS2.
[0045] FIG. 8 shows the DNA sequence ID of the hCDS2. The
nucleotide sequence analysis and restriction mapping of the cDNA
clone revealed a 5'-untranslated region of 24 bp, an open reading
frame capable of encoding a 445 amino acid polypeptide that spans
nucleotide positions 25 to 1362 and a 3'-untranslated region of
1126 bp. The ATG initiation site for translation was localized at
nucleotide positions 25-27 and fulfilled the requirement for an
adequate initiation site according to Kozak (Kozak, Critical Rev.
Biochem. Mol. Biol. 27:385-402, 1992).
[0046] Amino acid sequence alignment of the hCDS2 coding sequence
with the human CDS1 shows 64% identity (FIG. 9). The amino acids
that are identical between the two sequences are highlighted.
[0047] Expression of Human CDS cDNA in Mammalian Cells
[0048] hCDS1
[0049] To see if overexpression of hCDS1 would have any effect on
mammalian cells, the entire cDNA insert (.about.2,000 base pairs)
from pSK.hcds was cleaved with Asp718 I and Not I for insertion
into the mammalian expression vector pCE2 to generate pCE2.hCDS.
The plasmid pCE2 was derived from pREP7b (Leung et al. Proc. Natl.
Acad. Sci. USA, 92:4813-4817, 1995) with the RSV promoter region
replaced by the CMV enhancer and the elongation factor-1.alpha.
(EF-1.alpha.) promoter and intron. The CMV enhancer came from a 380
base pair Xba I-Sph I fragment produced by PCR from pCEP4
(Invitrogen, San Diego, Calif.) using the primers 5'-GGCTCTAGAT
ATTAATAGTA ATCAATTAC-3' (SEQ ID NO. 7) and 5'-CCTCACGCAT GCACCATGGT
AATAGC-3' (SEQ ID NO. 8). The EF-1.alpha. promoter and intron
(Uetsuki et al., J. Biol. Chem., 264:5791-5798, 1989) came from a
1200 base pair Sph I-Asp718 I fragment produced by PCR from human
genomic DNA using the primers 5'-GGTGCATGCG TGAGGCTCCG GTGC-3' (SEQ
ID NO. 9) and 5'-GTAGTTTTCA CGGTACCTGA AATGGAAG-3' (SEQ ID NO. 10).
These 2 fragments were ligated into a Xba I/Asp718 I digested
vector derived from pREP7b to generate pCE2.
[0050] A second clone, pCE2.hCDS2, was constructed that lacked the
human CDS 3'-UT region (520 nt). An Asp718 I (in the multiple
cloning site)/NcoI fragment and a NcoI/BamHI fragment from pSK.hCDS
were combined in a three-part ligation with Asp718 I/BamHI digested
pCE2. Northern blot analysis of 293-EBNA human embryonic kidney
cells transiently transfected with CDS cDNA expression plasmids
(pCE2.hCDS or pCE2.hCDS2) showed that deletion of the entire 3'-UT
region had little effect on CDS steady-state mRNA levels.
[0051] The CDS activity in transfected cell fractions (membranes,
cytosol, nuclei/unbroken cells) was determined by incorporation of
(.alpha.-.sup.32P)CTP into (.sup.32P)CDP-DAG in the presence of
exogenously added PA substrate. Cells were fractionated by
resuspending previously frozen cell pellets in cold hypotonic lysis
buffer (HLB; 10 mM KCl, 1.5 mM MgCl.sub.2, 10 mM Tris, pH 7.4, 2 mM
benzamidine HCl, and 10 .mu.g/ml each leupeptin, soybean trypsin
inhibitor, and pepstatin A) at approx. 5.times.10.sup.7 cells/ml.
After 10 min. on ice, cells were dounced (Wheaton pestle A) 40
strokes, then spun 500.times.g, 10 min. at 4.degree. C. to remove
nuclei and unbroken cells. The resuspension of the pellet,
incubation, and low speed spin were repeated twice. The final
"nuclei/unbroken cells" pellet was resuspended in 50-100 .mu.l HLB.
Supernatants were spun at 109,000.times.g, 30 min. at 4.degree. C.
generating "cytosol" supernates and "membrane" pellets. The pellets
were resuspended in 150-225 .mu.l HLB. An aliquot of each fraction
was removed for determination of protein concentration by a BCA
assay. Fractions were stored at -70.degree. C. All assays were done
on fractions after one thaw.
[0052] The in vitro CDS activity assay conditions were a
modification of methods described previously (Mok et al., FEBS
Letters 312:236-240, 1992; and Wu et al., Nature 373:216-222,1995).
Briefly, each 0.3 ml reaction combined 0.23 mM PA (Sigma; from egg
yolk lecithin), 50 mM Tris-maleate, pH 7.0, 1.5% Triton X-100, 0.5
mM DTT, 75-500 .mu.g protein from cell fractions, 30 mM MgCl.sub.2,
and 2 .mu.Ci (.alpha.-.sup.32P)CTP. MgCl.sub.2 and
(.alpha.-.sup.32P)CTP were added just prior to a 10 min. incubation
at 37.degree. C. The reactions were terminated with 4 ml
chloroform:methanol (1:1) and vortexing. The organic phase was
extracted three times with 1.8 ml 0.1N HCl with 1 M NaCl, and
vortexing. Radioactivity in the organic phase was determined by
scintillation counting or TLC.
[0053] A flip-flop TLC (ffTLC) system (Gruchalla et al., J.
Immunol. 144:2334-2342, 1990) was modified for the separation of
CDP-DAG and PA. Specifically, 200 ml of organic phase was dried and
brought up in 20 .mu.L CHCl.sub.3:MeOH (2:1) and spotted in the
center of a 20.times.20 cm TLC plate (Analtech Silica Gel HP-HLF).
TLC was run in CHCl.sub.3:MeOH:NH.sub.4OH:H.sub.2O (65:30:4:1)
until the solvent had reached the top of the plate. In this solvent
system, neutral and cationic lipids migrate, whereas PA, CDP-DAG
and other anionic lipids stay near the origin. The plate was dried
and visualized by UV with 0.05% primulin stain (Sigma, St. Louis,
Mo.) in 80% acetone. The plate was cut below the PC standard, and
the bottom half of the plate was rotated 180.degree. and run in
CHCl.sub.3:MeOH:Acetic Acid:H.sub.2O (80:25:15:5) to enable
migration of the anionic lipids until the solvent reached the top
of the plate. The radioactive bands on the TLC plate were
quantified using a STORM.RTM. phosphorimager (Molecular Dynamics,
Sunnyvale, Calif.). Non-radiolabeled lipid standards were stained
with primulin and visualized by fluorescence using the
STORM.RTM..
[0054] FIG. 5 shows the results of in vitro CDS activity assays on
cell fractions from stable transfectants of NCI-H460 cells. CDS
activity was assessed by conversion of (.alpha.-.sup.32P)CTP to
(.sup.32P)CDP-DAG in in vitro reactions that required addition of
an exogenous PA substrate. This is a representative histogram
comparing the radiolabel incorporated into various cell fractions
(membranes, cytosol, and nuclei/unbroken cells) from NCI-H460 cells
stably transfected with the hCDS1 cDNA (pCE2.hCDS) or vector only
(pCE2). In all fractions, the CDS cDNA increased radiolabel in the
organic phase of the reactions. Total CDS activity was much greater
in membrane fractions, as would be expected for membrane associated
CDS, compared to cytosol fractions. Activity in unbroken cells
masked the activity specific to nuclei.
[0055] FIG. 6 is a representative phosphorimage of
[.sup.32P]phospholipids from membrane fraction CDS assay reactions
after the second dimension of ffTLC. FIG. 6 confirms that the
radiolabeled product found in the membrane fractions does migrate
with a CDP-DAG standard on TLC. The identities of labeled bands
were determined by migration of phospholipid standards visualized
by UV or FL imaging on the STORM after primulin staining. Lanes 1-3
represent triplicate samples derived from membranes of NCI-H460
cells transfected with the hCDS1 expression vector, and lanes 4-6
represent triplicate samples from transfectants with the control
vector. Cells transfected with the hCDS1 cDNA showed 1.6-2.4 fold
more CDS activity in membrane fractions than vector transfectants.
The relative CDS activity between CDS transfectants and vector
transfectants was similar when determined by scintillation counting
or TLC analysis. Similar CDS activity was seen in two different
transfected human cell lines, NCI-H460 and ECV304. The average
specific activity of CDS in membranes of CDS transfectants was 2.7
fmol/min/mg protein compared to 1.4 fmol/min/mg protein in
membranes of vector transfectants. These results demonstrated that
overexpression of the human CDS cDNA clone lead to an increase in
CDS activity in cell fractions and that activity in an in vitro
assay was completely dependent on the addition of PA. These data
indicate that the human cDNA clone of SEQ ID NO. 1 does encode CDS
activity.
[0056] hCDS2
[0057] To see if overexpression of hCDS2 has an effect in mammalian
cells, the entire cDNA insert (.about.1,900 bp) from pSK.CDS2 was
cleaved with Asp718 I and Xba I for insertion into a mammalian
inducible expression vector pIND (Invitrogen, San Diego, Calif.) to
generate pI_CDS2.
[0058] pI_CDS2 DNA and pVgRXR (Invitrogen, San Diego, Calif.) DNA
were co-transfected into ECV304 cells (American Type Culture
Collection, Rockville, Md.) with a Cell-Porator.TM. (Life
Technologies, Gaithersburg, Md.) using conditions described
previously (Cachianes, et al., Biotechniques 15:255-259, 1993).
After adherence of the transfected cells 24 hours later, the cells
were grown in the presence of 500 .mu.g/ml G418 (Life Technologies,
Gaithersburg, Md.) and 100 .mu.g/ml Zeocin (Invitrogen, San Diego,
Calif.) to select for cells that had incorporated both plasmids.
G418 and Zeocin resistant clones that expressed CDS2 mRNA at a
level more than 10 fold higher in the presence of muristerone A
(Invitrogen, San Diego, Calif.) relative to uninduced or
untranfected cells based on Northern Blot analysis (Kroczek, et
al., Anal. Biochem. 184: 90-95, 1990) were selected for further
study.
[0059] The CDS activity in ECV304 cells transfected with pI_CDS2
DNA and pVgRXR DNA with or without muristerone A induction was
compared using a TLC assay (Weeks et al, DNA Cell Biol. 16:
281-289, 1997).
[0060] FIG. 10 shows an example of hCDS2 assay results by measuring
the production of [32P]CDP-DAG after TLC analysis. The identities
of labeled bands were determined based on Rf values obtained for
standard phospholipids visualized by primulin staining. The left
two bars represent triplicate samples derived from ECV304 cells
transfected with pVgRXR and the control vector pIND in the absence
or presence of the inducer muristerone A. The enzyme activity found
here represents endogenous CDS activity found in ECV304 cells, as
cells without or with muristerone A treatment produced similar
activity. The right two bars represent triplicate samples derived
from ECV304 cells transfected with pVgRXR and the inducible CDS2
vector pI_CDS2 in the absence or presence of the inducer
muristerone A. Quantitation of the radioactive bands corresponding
to CDP-DAG shows cells transfected with the inducible hCDS2
expression plasmid have an approximately two fold increase in
activity after induction with muristerone A compared to same cells
without induction or to vector control cells either with or without
induction, showing that the hCDS2 cDNA clone encode a protein
having CDS activity.
[0061] Complementation of Yeast cds1 Mutant with hCDS1
[0062] As the yeast CDS gene is essential for growth (Shen et al.,
J. Biol. Chem. 271:789-795, 1996), another way to show that the
cDNA does encode CDS activity was to determine if the human CDS
cDNA will complement the growth defect of a mutant yeast strain
with a deletion in the endogenous yeast CDS gene. Accordingly, the
hCDS1 cDNA was cloned downstream of a GAL1 promoter in a yeast
expression vector. Specifically, a Hind III-Sac I fragment from
pSK.hCDS was inserted into pYES.LEU vector to generate pYES.hCDS.
pYES.LEU was derived from pYES2 (Invitrogen, San Diego, Calif.) by
inserting a BspH I fragment containing a LEU2 marker from pRS315
(Sikorski et al., Genetics 122:19-27, 1989) into the Nco I of
pYES2. pYES.hCDS was introduced into a null cds1 strain of yeast,
YSD90A (Shen et al., J. Biol. Chem. 271:789-795, 1996), with a
covering plasmid, pSDG1, carrying the functional yeast CDS1. The
latter plasmid was cured from cells by growth in media lacking
leucine but containing uracil and galactose. PCR analysis confirmed
the absence of the yeast CDS1 gene and Northern blot analysis
verified expression of the hCDS1 cDNA. This strain was found to be
absolutely dependent on galactose for growth. Galactose activates
the GAL1 promoter for the production of human CDS protein. When the
carbon source was switched to glucose, which would shut down the
GAL1 promoter, growth stopped completely in less than a generation.
These data show the human CDS was able to complement the growth
defect of a yeast cds1 mutant.
[0063] The cells grown on galactose were lysed and assayed for CDS
activity according to the assay method described (Shen et al., J.
Biol. Chem. 271:789-795, 1996). The specific activity using yeast
conditions showed activity at 20% of single copy CDS1 wild type
activity. This is consistent with the above plasmid in a wild type
background showing approximately 1.3 fold increase in activity when
grown on galactose versus glucose.
[0064] The following experiment found that hCDS1 over-expression
enhanced cytokine induced signaling in cells. Over-expression of
CDS was expected to alter the cellular level of various lipid
second messengers such as PA, IP.sub.3 and DAG (Kent, Anal. Rev.
Biochem. 64:315-343, 1995) and hence modulates cytokine induced
signaling response in cells. To test this hypothesis, a hCDS1
expression plasmid (pCE2.hCDS), or vector (pCE2) were stably
transfected into ECV304 cells (American Type Culture Collection,
Rockville, Md.), an endothelial cell line that produces IL-6 and
TNF-.alpha. upon stimulation with IL- 1.beta.. FIG. 7 shows that
the secretion of TNF-.alpha. IL-6 in ECV304 cells stably
transfected with CDS expression vector increased by >5 fold
relative to ECV304 cells stably transfected with control vector
after stimulation with 1 ng/ml IL-1.beta.. However, there was
little effect on the basal level of cytokine release, suggesting
that over-expression of CDS amplified the cytokine signaling
response, as opposed to enhancing the steady-state, basal signal,
in these cells.
[0065] Expression of hCDS1 and hCDS2 mRNA in Cancer Versus Nnormal
Prostate Tissue
[0066] To examine if CDS mRNA expression in cancer versus normal
tissues, RT-PCR was performed on specimens of prostate cancer
tissues and the corresponding normal prostate tissues in the
surgical margins from four independent patients. FIG. 11 shows
hCDS1 mRNA was elevated in prostate cancer in 2 out of 4 patients,
whereas hCDS2 mRNA was elevated in prostate cancer in 3 out of 4
patients. A housekeeping gene .beta.2-microglobulin mRNA level was
found to be similar in normal and cancer prostate tissues. ETS-2, a
transcription factor reported to be elevated in prostate cancer
(Liu et al., Prostate 30: 145-153, 1997), was found to be elevated
in the same 3 out of 4 patients examined here, suggesting hCDS2,
like ETS-2, may be a target for drug intervention in cancer
therapy.
[0067] CDS Polypeptide Synthesis
[0068] Polypeptides of the present invention can be synthesized by
such commonly used methods as t-BOC or FMOC protection of
alpha-amino groups. Both methods involve step-wise syntheses
whereby a single amino acid is added at each step starting from the
C-terminus of the peptide (Coligan et al., Current Protocols in
Immunology, Wiley Interscience, Unit 9, 1991). In addition,
polypeptides of the present invention can also be synthesized by
solid phase synthesis methods (e.g., Merrifield, J. Am. Chem. Soc.
85:2149, 1962; and Steward and Young, Solid Phase Peptide
Synthesis, Freeman, San Francisco pp. 27-62, 1969) using copolyol
(styrene-divinylbenzene) containing 0.1-1.0 mM amines/g polymer. On
completion of chemical synthesis, the polypeptides can be
deprotected and cleaved from the polymer by treatment with liquid
HF 10% anisole for about 15-60 min at 0.degree. C. After
evaporation of the reagents, the peptides are extracted from the
polymer with 1% acetic acid solution, which is then lyophilized to
yield crude material. This can normally be purified by such
techniques as gel filtration of Sephadex G-15 using 5% acetic acid
as a solvent. Lyophilization of appropriate fractions of the column
will yield a homogeneous polypeptide or polypeptide derivatives,
which are characterized by such standard techniques as amino acid
analysis, thin layer chromatography, high performance liquid
chromatography, ultraviolet absorption spectroscopsy, molar
rotation, solubility and quantitated by solid phase Edman
degradation.
[0069] CDS Polynucleotides
[0070] The invention also provides polynucleotides which encode the
CDS polypeptide of the invention. As used herein, "polynucleotide"
refers to a polymer of deoxyribonucleotides or ribonucleotides in
the form of a separate fragment or as a component of a larger
construct. DNA encoding the polypeptide of the invention can be
assembled from cDNA fragments or from oligonucleotides which
provide a synthetic gene which is capable of being expressed in a
recombinant transcriptional unit. Polynucleotide sequences of the
invention include DNA, RNA and cDNA sequences. Preferably, the
nucleotide sequence encoding CDS is the sequence of SEQ ID NO. 1 or
of FIG. 8. DNA sequences of the present invention can be obtained
by several methods. For example, the DNA can be isolated using
hybridization procedures which are known in the art. Such
hybridization procedures include, for example, hybridization of
probes to genomic or cDNA libraries to detect shared nucleotide
sequences, antibody screening of expression libraries to detect
common antigenic epitopes or shared structural features and
synthesis by the polymerase chain reaction (PCR). Such
hybridization includes hybridization under high stringency
conditions as described above.
[0071] Hybridization procedures are useful for screening
recombinant clones by using labeled mixed synthetic
oligonucleotides probes, wherein each probe is potentially the
complete complement of a specific DNA sequence in a hybridization
sample which includes a heterogeneous mixture of denatured
double-stranded DNA. For such screening, hybridization is
preferably performed on either single-stranded DNA or denatured
double-stranded DNA. Hybridization is particularly useful for
detection of cDNA clones derived from sources where an extremely
low amount of mRNA sequences relating to the polypeptide of
interest are present. Using stringent hybridization conditions to
avoid non-specific binding, it is possible to allow an
autoradiographic visualization of a specific genomic DNA or cDNA
clone by the hybridization of the target DNA to a radiolabeled
probe, which is its complement (Wallace et al. Nucl. Acid Res.
9:879, 1981). Specific DNA sequences encoding CDS can also be
obtained by isolation and cloning of double-stranded DNA sequences
from the genomic DNA, chemical manufacture of a DNA sequence to
provide the necessary codons for the complete polypeptide of
interest or portions of the sequence for use in PCR to obtain the
complete sequence, and in vitro synthesis of a double-stranded DNA
sequence by reverse transcription of mRNA isolated from a
eukaryotic donor cell. In the latter case, a double-stranded DNA
complement of mRNA is eventually formed which is generally referred
to as cDNA. Of these three methods for developing specific DNA
sequences for use in recombinant procedures, the isolation of cDNA
clones is the most useful. This is especially true when it is
desirable to obtain the microbial expression of mammalian
polypeptides since the presence of introns in genomic DNA clones
can prevent accurate expression.
[0072] The synthesis of DNA sequences is sometimes a method that is
preferred when the entire sequence of amino acids residues of the
desired polypeptide product is known. When the entire sequence of
amino acid residues of the desired polypeptide is not known, direct
synthesis of DNA sequences is not possible and it is desirable to
synthesize cDNA sequences. cDNA sequence isolation can be done, for
example, by formation of plasmid- or phage-carrying cDNA libraries
which are derived from reverse transcription of mRNA. mRNA is
abundant in donor cells that have high levels of genetic
expression. In the event of lower levels of expression, PCR
techniques can be used to isolate and amplify the cDNA sequence of
interest. Using synthesized oligonucleotides corresponding exactly,
or with some degeneracy, to known CDS amino acid or nucleotide
sequences, one can use PCR to obtain and clone the sequence between
the oligonucleotides. The oligonucleotide may represent invariant
regions of the CDS sequence and PCR may identify sequences
(isoforms) with variations from SEQ ID NO. 1 or FIG. 8.
[0073] A cDNA expression library, such as lambda gtl1, can be
screened indirectly for the CDS polypeptide, using antibodies
specific for CDS. Such antibodies can be either polyclonal or
monoclonal, derived from the entire CDS protein or fragments
thereof, and used to detect and isolate expressed proteins
indicative of the presence of CDS cDNA.
[0074] A polynucleotide sequence can be deduced from an amino acid
sequence by using the genetic code, however the degeneracy of the
code must be taken into account. Polynucleotides of this invention
include variant polynucleotide sequences which code for the same
amino acids as a result of degeneracy in the genetic code. There
are 20 natural amino acids, most of which are specified by more
that one codon (a three base sequence). Therefore, as long as the
amino acid sequence of CDS results in a biologically active
polypeptide (at least, in the case of the sense polynucleotide
strand), all degenerate nucleotide sequences are included in the
invention. The polynucleotide sequence for CDS also includes
sequences complementary to the polynucleotides encoding CDS
(antisense sequences). Antisense nucleic acids are DNA, and RNA
molecules that are complementary to at least a portion of a
specific mRNA molecule (Weintraub, Sci. Amer. 262:40, 1990). The
invention embraces all antisense polynucleotides capable of
inhibiting the production of CDS polypeptide. In the cell, the
antisense nucleic acids hybridize to the corresponding mRNA,
forming a double-stranded molecule. The antisense nucleic acids
interfere with the translation of mRNA since the cell cannot
translate mRNA that is double-stranded. Antisense oligomers of
about 15 nucleotides are preferred, since they are easily
synthesized and are less likely to cause problems than larger
molecules when introduced into the target CDS-producing cell. The
use of antisense methods to inhibit translation of genes is known
(e.g., Marcus-Sakura, Anal. Biochem. 172:289, 1988).
[0075] In addition, ribozyme nucleotide sequences for CDS are
included in this invention. Ribozymes are hybrid RNA:DNA molecules
possessing an ability to specifically cleave other single-stranded
RNA in a manner analogous to DNA restriction endonucleases. Through
the modification of nucleotide sequences which encode such RNAs, it
is possible to engineer molecules that recognize specific
nucleotide sequences in an RNA molecule and cleave it (Cech, J.
Amer. Med. Assn. 260:3030, 1988). An advantage of this approach is
that only mRNAs with particular sequences are inactivated because
they are sequence-specific.
[0076] The CDS DNA sequence may be inserted into an appropriate
recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle that has been
manipulated by insertion or incorporation of the genetic sequences.
Such expression vectors contain a promoter sequence which
facilitates efficient transcription of the inserted genetic
sequence in the host. The expression vector typically contains an
origin of replication, a promoter, as well as specific genes which
allow phenotypic selection of the transformed cells. Vectors
suitable for use in the present invention include, for example,
vectors with a bacterial promoter and ribosome binding site for
expression in bacteria (Gold, Meth. Enzymol. 185:11, 1990),
expression vectors with mammalian or viral promoter and enhancer
for expression in mammalian cells (Kaufman, Meth. Enzymol. 185:487,
1990) and baculovirus-derived vectors for expression in insect
cells (Luckow et al., J. Virol. 67:4566, 1993). The DNA segment can
be present in the vector operably linked to regulatory elements,
for example, constitutive or inducible promoters (e.g., T7,
metallothionein I, CMV, or polyhedren promoters).
[0077] The vector may include a phenotypically selectable marker to
identify host cells which contain the expression vector. Examples
of markers typically used in prokaryotic expression vectors include
antibiotic resistance genes for ampicillin (.beta.-lactamases),
tetracycline and chloramphenicol (chloramphenicol
acetyltransferase). Examples of such markers typically used in
mammalian expression vectors include the gene for adenosine
deaminase (ADA), aminoglycoside phosphotransferase (neo, G418),
dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase
(HPH), thymidine kinase (TK), and xanthine guanine
phosphoriboseyltransferase (XGPRT, gpt).
[0078] In another preferred embodiment, the expression system used
is one driven by the baculovirus polyhedrin promoter. The gene
encoding the polypeptide can be manipulated by standard techniques
in order to facilitate cloning into the baculovirus vector. A
preferred baculovirus vector is the pBlueBac vector (Invitrogen,
Sorrento, Calif.). The vector carrying the gene for the polypeptide
is transfected into Spodoptera frigiperda (Sf9) cells by standard
protocols, and the cells are cultured and processed to produce the
recombinant polypeptide. See Summers et al., A Manual for Methods
of Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station.
[0079] Once the entire coding sequence of the gene for the
polypeptides has been determined, the gene can be expressed in any
number of different recombinant DNA expression systems to generate
large amounts of polypeptide. Included within the present invention
are polypeptides having native glycosylation sequences, and
deglycosylated or unglycosylated polypeptides prepared by the
methods described below. Examples of expression systems known to
the skilled practitioner in the art include bacteria such as E.
coli, yeast such as Pichia pastoris, baculovirus, and mammalian
expression systems such as in COS or CHO cells.
[0080] The gene or gene fragment encoding the desired polypeptide
can be inserted into an expression vector by standard subcloning
techniques. In a preferred embodiment, an E. coli expression vector
is used which produces the recombinant protein as a fusion protein,
allowing rapid affinity purification of the protein. Examples of
such fusion protein expression systems are the glutathione
S-transferase system (Pharmacia, Piscataway, N.J.), the maltose
binding protein system (NEB, Beverley, Mass.), the thiofusion
system (Invotrogen, San Diego, Calif.), the FLAG system (IBI, New
Haven, Conn.), and the 6.times.His system (Qiagen, Chatsworth,
Calif.). Some of these systems produce recombinant polypeptides
bearing only a small number of additional amino acids, which are
unlikely to affect the CDS activity of the recombinant polypeptide.
For example, both the FLAG system and the 6.times.His system add
only short sequences, both of which are known to be poorly
antigenic and which do not adversely affect folding of the
polypeptide to its native conformation. Other fusion systems
produce proteins where it is desirable to excise the fusion partner
from the desired protein. In a preferred embodiment, the fusion
partner is linked to the recombinant polypeptide by a peptide
sequence containing a specific recognition sequence for a protease.
Examples of suitable sequences are those recognized by the Tobacco
Etch Virus protease (Life Technologies, Gaithersburg, Md.) or
Factor Xa (New England Biolabs, Beverley, Mass.) or enterokinase
(Invotrogen, San Diego, Calif.).
[0081] Production of Polypeptides
[0082] Polynucleotide sequences encoding CDS polypeptides of the
invention can be expressed in either prokaryotes or eukaryotes.
Hosts can include microbial (bacterial), yeast, insect and
mammalian organisms. Methods of expressing DNA sequences inserted
downstream of prokaryotic or viral regulatory sequences in
prokaryotes are known in the art (Makrides, Microbio. Rev. 60:512,
1996). Biologically functional viral and plasmid DNA vectors
capable of expression and replication in a eukaryotic host are
known in the art (Cachianes, Biotechniques 15:255, 1993). Such
vectors are used to incorporate DNA sequences of the invention. DNA
sequences encoding the inventive polypeptides can be expressed in
vitro by DNA transfer into a suitable host using known methods of
transfection.
[0083] Sequences encoding CDS polypeptides may be inserted into a
recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle that has been
manipulated by inserting or incorporating genetic sequences. Such
expression vectors contain a promoter sequence which facilitates
efficient transcription of the inserted genetic sequence of the
host. The expression vector typically contains an origin of
replication and a promoter, as well as specific genes which allow
phenotypic selection of the transformed cells. The DNA segment can
be present in the vector, operably linked to regulatory elements,
for example, a promoter (e.g., T7, metallothionein I, or polyhedren
promoters). Vectors suitable for use in the present invention
include, for example, bacterial expression vectors, with bacterial
promoter and ribosome binding sites, for expression in bacteria
(Gold, Meth. Enzymol. 185:11, 1990), expression vector with animal
promoter and enhancer for expression in mammalian cells (Kaufman,
Meth. Enzymol. 185:487, 1990) and baculovirus-derived vectors for
expression in insect cells (Luckow et al., J. Virol:67:4566,
1993).
[0084] The vector may include a phenotypically selectable marker to
identify host cells which contain the expression vector. Examples
of markers typically used in prokaryotic expression vectors include
antibiotic resistance genes for ampicillin (.beta.-lactamases),
tetracycline and chloramphenicol (chloramphenicol
acetyltransferase).
[0085] Examples of such markers typically used in mammalian
expression vectors include the gene for adenosine deaminase (ADA),
aminoglycoside phosphotransferase (neo, G418), dihydrofolate
reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine
kinase (TK), and xanthine guanine phosphoriboseyltransferase
(XGPRT, gpt).
[0086] In another preferred embodiment, the expression system used
is one driven by the baculovirus polyhedrin promoter. The
polynucleotide encoding CDS can be manipulated by standard
techniques in order to facilitate cloning into the baculovirus
vector. See Ausubel et al., supra. A preferred baculovirus vector
is the pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector
carrying a polynucleotide encoding CDS is transfected into
Spodoptera frugiperda (Sf9) cells by standard protocols, and the
cells are cultured and processed to produce the recombinant
polypeptide. See Summers et al., A Manual for Methods of
Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station.
[0087] The polynucleotides of the present invention can be
expressed in any number of different recombinant DNA expression
systems to generate large amounts of polypeptide. Included within
the present invention are CDS polypeptides having native
glycosylation sequences, and deglycosylated or unglycosylated
polypeptides prepared by the methods described below. Examples of
expression systems known to the skilled practitioner in the art
include bacteria such as E. coli, yeast such as Pichia pastoris,
baculovirus, and mammalian expression systems such as in Cos or CHO
cells.
[0088] The polynucleotides of the present invention can be inserted
into an expression vector by standard subcloning techniques. In a
preferred embodiment, an E. coli expression vector is used which
produces the recombinant protein as a fusion protein, allowing
rapid affinity purification of the protein. Examples of such fusion
protein expression systems are the glutathione S-transferase system
(Pharmacia, Piscataway, N.J.), the maltose binding protein system
(NEB, Beverley, Mass.), the thiofusion system (Invitrogen, San
Diego, Calif.), the Strep-tag II system (Genosys, Woodlands, Tex.),
the FLAG system (IBL New Haven, Conn.), and the 6.times.His system
(Qiagen, Chatsworth, Calif.). Some of these systems produce
recombinant polypeptides bearing only a small number of additional
amino acids, which are unlikely to affect the CDS ability of the
recombinant polypeptide. For example, both the FLAG system and the
6.times.His system add only short sequences, both of which are
known to be poorly antigenic and which do not adversely affect
folding of the polypeptide to its native conformation. Other fusion
systems produce proteins where it is desirable to excise the fusion
partner from the desired protein. In a preferred embodiment, the
fusion partner is linked to the recombinant polypeptide by a
peptide sequence containing a specific recognition sequence for a
protease. Examples of suitable sequences are those recognized by
the Tobacco Etch Virus protease (Life Technologies, Gaithersburg,
Md.) or Factor Xa (New England Biolabs, Beverley, Mass.) or
enterokinase (Invitrogen, San Diego, Calif.).
[0089] In an embodiment of the present invention, the
polynucleotides encoding CDS are analyzed to detect putative
transmembrane sequences. Such sequences are typically very
hydrophobic and are readily detected by the use of standard
sequence analysis software, such as MacDNASIS (Hitachi, San Bruno,
Calif.). The presence of transmembrane sequences is often
deleterious when a recombinant protein is synthesized in many
expression systems, especially in E. coli, as it leads to the
production of insoluble aggregates which are difficult to renature
into the native conformation of the polypeptide.
[0090] Accordingly, deletion of one or more of the transmembrane
sequences may be desirable. Deletion of transmembrane sequences
typically does not significantly alter the conformation or activity
of the remaining polypeptide structure. However, one can determine
whether deletion of one or more of the transmembrane sequences has
effected the biological activity of the CDS protein by, for
example, assaying the activity of the CDS protein containing one or
more deleted sequences and comparing this activity to that of
unmodified CDS. Examples of assays for CDS activity are described
above.
[0091] Moreover, transmembrane sequences, being by definition
embedded within a membrane, are inaccessible as antigenic
determinants to a host immune system. Antibodies to these sequences
will not, therefore, provide immunity to the host and, hence,
little is lost in terms of generating monoclonal or polyclonal
antibodies by omitting such sequences from the recombinant
polypeptides of the invention. Deletion of transmembrane-encoding
sequences from the polynucleotide used for expression can be
achieved by standard techniques. See Ausubel et al., supra, Chapter
8. For example, fortuitously-placed restriction enzyme sites can be
used to excise the desired gene fragment, or the PCR can be used to
amplify only the desired part of the gene.
[0092] Transformation of a host cell with recombinant DNA may be
carried out by conventional techniques. When the host is
prokaryotic, such as E. coli, competent cells which are capable of
DNA uptake can be prepared from cells harvested after exponential
growth phases and subsequently treated by a CaCl.sub.2 method using
standard procedures. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell or by electroporation.
[0093] When the host is a eukaryote, methods of transfection of
DNA, such as calcium phosphate co-precipitates, conventional
mechanical procedures, (e.g., microinjection), electroporation,
liposome-encased plasmids, or virus vectors may be used. Eukaryotic
cells can also be cotransformed with DNA sequences encoding CDS
polypeptides of the present invention, and a second foreign DNA
molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method uses a eukaryotic
viral vector, such as simian virus 40 (SV40) or bovine papilloma
virus to transiently infect or transform eukaryotic cells and
express the CDS polypeptides.
[0094] Expression vectors that are suitable for production of CDS
polypeptides preferably contain (1) prokaryotic DNA elements coding
for a bacterial replication origin and an antibiotic resistance
marker to provide for the growth and selection of the expression
vector in a bacterial host; (2) eukaryotic DNA elements that
control initiation of transcription, such as a promoter; and (3)
DNA elements that control the processing of transcripts, such as a
transcription termination/polyadenylation sequence. CDS
polypeptides of the present invention preferably are expressed in
eukaryotic cells, such as mammalian, insect and yeast cells.
Mammalian cells are especially preferred eukaryotic hosts because
mammalian cells provide suitable post-translational modifications
such as glycosylation. Examples of mammalian host cells include
Chinese hamster ovary cells (CHO-K1; ATCC CCL61), rat pituitary
cells (GH.sub.1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rat
hepatoma cells (H-4-II-E; ATCC CRL1548) SV40-transformed monkey
kidney cells (COS-1; ATCC CRL 1650) and murine embryonic cells
(NIH-3T3; ATCC CRL 1658). For a mammalian host, the transcriptional
and translational regulatory signals may be derived from viral
sources, such as adenovirus, bovine papilloma virus, simian virus,
or the like, in which the regulatory signals are associated with a
particular gene which has a high level of expression. Suitable
transcriptional and translational regulatory sequences also can be
obtained from mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0095] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet
1:273,1982); the TK promoter of Herpes virus (McKnight, Cell 31:
355, 1982); the SV40 early promoter (Benoist et al., Nature
290:304, 1981); the Rous sarcoma virus promoter (Gorman et al.,
Proc. Nat'l. Acad Sci. USA 79:6777, 1982); and the cytomegalovirus
promoter (Foecking et al, Gene 45:101, 1980). Alternatively, a
prokaryotic promoter, such as the bacteriophage T3 RNA polymerase
promoter, can be used to control fusion gene expression if the
prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et
al., Mol. Cell. Biol. 10:4529, 1990; Kaufman et al., Nucl. Acids
Res. 19:4485, 1991).
[0096] An expression vector can be introduced into host cells using
a variety of techniques including calcium phosphate transfection,
liposome-mediated transfection, electroporation, and the like.
Preferably, transfected cells are selected and propagated wherein
the expression vector is stably integrated in the host cell genome
to produce stable transformants. Techniques for introducing vectors
into eukaryotic cells and techniques for selecting stable
transformants using a dominant selectable marker are described, for
example, by Ausubel and by Murray (ed.), Gene Transfer and
Expression Protocols (Humana Press 1991). Examples of mammalian
host cells include COS, BHK, 293 and CHO cells.
[0097] Purification of Recombinant Polypeptides.
[0098] The polypeptide expressed in recombinant DNA expression
systems can be obtained in large amounts and tested for biological
activity. The recombinant bacterial cells, for example E. coli, are
grown in any of a number of suitable media, for example LB, and the
expression of the recombinant polypeptide induced by adding IPTG to
the media or switching incubation to a higher temperature. After
culturing the bacteria for a further period of between 2 and 24
hours, the cells are collected by centrifugation and washed to
remove residual media. The bacterial cells are then lysed, for
example, by disruption in a cell homogenizer and centrifuged to
separate the dense inclusion bodies and cell membranes from the
soluble cell components. This centrifugation can be performed under
conditions whereby the dense inclusion bodies are selectively
enriched by incorporation of sugars such as sucrose into the buffer
and centrifugation at a selective speed. If the recombinant
polypeptide is expressed in the inclusion, these can be washed in
any of several solutions to remove some of the contaminating host
proteins, then solubilized in solutions containing high
concentrations of urea (e.g., 8 M) or chaotropic agents such as
guanidine hydrochloride in the presence of reducing agents such as
.beta.-mercaptoethanol or DTT (dithiothreitol). At this stage it
may be advantageous to incubate the polypeptide for several hours
under conditions suitable for the polypeptide to undergo a
refolding process into a conformation which more closely resembles
that of the native polypeptide. Such conditions generally include
low polypeptide (concentrations less than 500 mg/ml), low levels of
reducing agent, concentrations of urea less than 2 M and often the
presence of reagents such as a mixture of reduced and oxidized
glutathione which facilitate the interchange of disulphide bonds
within the protein molecule. The refolding process can be
monitored, for example, by SDS-PAGE or with antibodies which are
specific for the native molecule. Following refolding, the
polypeptide can then be purified further and separated from the
refolding mixture by chromatography on any of several supports
including ion exchange resins, gel permeation resins or on a
variety of affinity columns.
[0099] Isolation and purification of host cell expressed
polypeptide, or fragments thereof may be carried out by
conventional means including, but not limited to, preparative
chromatography and immunological separations involving monoclonal
or polyclonal antibodies.
[0100] These polypeptides may be produced in a variety of ways,
including via recombinant DNA techniques, to enable large scale
production of pure, active CDS useful for screening compounds for
trilineage hematopoietic and anti-inflammatory therapeutic
applications, and developing antibodies for therapeutic, diagnostic
and research use.
[0101] Screening Assays Using CDS Polypeptides
[0102] The CDS polypeptide of the present invention is useful in a
screening methodology for identifying compounds or compositions
which affect cellular signaling of an inflammatory response. This
method comprises incubating the CDS polypeptides or a cell
transfected with cDNA encoding CDS, with a suitable substrate, for
example, PA, under conditions sufficient to allow the components to
interact, and then measuring the effect of the compound or
composition on CDS activity. See, for example, above, and Weeks et
al., DNA Cell Biol. 16: 281-289, 1997. The observed effect on CDS
may be either inhibitory or stimulatory. Such compounds or
compositions to be tested can be selected from a combinatorial
chemical library or any other suitable source (Hogan, Jr., Nat.
Biotechnology 15:328, 1997).
[0103] Peptide Sequencing of Polypeptides
[0104] Substitutional variants typically contain the exchange of
one amino acid for another at one or more sites within the protein,
and are designed to modulate one or more properties of the
polypeptides such as stability against proteolytic cleavage.
Substitutions preferably are conservative, that is, one amino acid
is replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
the changes of alanine to serine; arginine to lysine; asparigine to
glutamine or histidine; aspartate to glutamate; cysteine to serine;
glutamine to asparigine; glutamate to aspartate; glycine to
proline; histidine to asparigine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine, glutamine, or glutamate; methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine;
serine to threonine; threonine to serine; tryptophan to tyrosine;
tyrosine to tryptophan or phenylalanine; and valine to isoleucine
or leucine. Insertional variants contain fusion proteins such as
those used to allow rapid purification of the polypeptide and also
can include hybrid polypeptides containing sequences from other
proteins and polypeptides which are homologues of the inventive
polypeptide. For example, an insertional variant could include
portions of the amino acid sequence of the polypeptide from one
species, together with portions of the homologous polypeptide from
another species. Other insertional variants can include those in
which additional amino acids are introduced within the coding
sequence of the polypeptides. These typically are smaller
insertions than the fusion proteins described above and are
introduced, for example, to disrupt a protease cleavage site.
[0105] Anti-CDS Antibodies
[0106] Antibodies to human CDS protein can be obtained using the
product of a CDS expression vector or synthetic peptides derived
from the CDS coding sequence coupled to a carrier (Pasnett et al.,
J. Biol. Chem. 263:1728, 1988) as an antigen. The preparation of
polyclonal antibodies is well-known to those of skill in the art.
See, for example, Green et al., "Production of Polyclonal
Antisera," in Immunochemical Protocols (Manson, ed.), pages 1-5
(Humana Press 1992). Alternatively, a CDS antibody of the present
invention may be derived as a rodent monoclonal antibody (MAb).
Rodent monoclonal antibodies to specific antigens may be obtained
by methods known to those skilled in the art. See, for example,
Kohler and Milstein, Nature 256:495, 1975, and Coligan et al.
(eds.), Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley
& Sons 1991). Briefly, monoclonal antibodies can be obtained by
injecting mice with a composition comprising an antigen, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0107] MAbs can be isolated and purified from hybridoma cultures by
a variety of well-established techniques. Such isolation techniques
include affinity chromatography with Protein-A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages
2.9.1-2.9.3. Also, see Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, 10:79-104
Humana Press, Inc. 1992. A CDS antibody of the present invention
may also be derived from a subhuman primate. General techniques for
raising therapeutically useful antibodies in baboons may be found,
for example, in Goldenberg et al., international patent publication
No. WO 91/11465 (1991), and in Losman et al., Int. J. Cancer
46:310, 1990.
[0108] Alternatively, a therapeutically useful CDS antibody may be
derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light chain
variable regions of the mouse antibody into a human antibody
variable domain, and then, substituting human residues in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by the
publication of Orlandi et al., Proc. Nat'l. Acad Sci. USA 86:3833,
1989. Techniques for producing humanized MAbs are described, for
example, by Jones et al., Nature 321:522, 1986; Riechmann et al.,
Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988;
Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu,
Crit. Rev. Biotech. 12: 437, 1992; and Singer et al., J. Immun.
150:2844, 1993.
[0109] As an alternative, a CDS antibody of the present invention
may be derived from human antibody fragments isolated from a
combinatorial immunoglobulin library. See, for example, Barbas et
al., METHODS: A Companion to Methods in Enzymology 2:119 1991, and
Winter et al., Ann. Rev. Immunol. 12:433, 1994. Cloning and
expression vectors that are useful for producing a human
immunoglobulin phage library can be obtained, for example, from
STRATAGENE Cloning Systems (La Jolla, Calif.). In addition, a CDS
antibody of the present invention may be derived from a human
monoclonal antibody. Such antibodies are obtained from transgenic
mice that have been "engineered" to produce specific human
antibodies in response to antigenic challenge. In this technique,
elements of the human heavy and light chain loci are introduced
into strains of mice derived from embryonic stem cell lines that
contain targeted disruptions of the endogenous heavy chain and
light chain loci. The transgenic mice can synthesize human
antibodies specific for human antigens, and the mice can be used to
produce human antibody-secreting hybridomas. Methods for obtaining
human antibodies from transgenic mice are described by Green et
al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856,
1994; and Taylor et al., Int. Immun. 6:579, 1994.
Sequence CWU 1
1
19 1 2051 DNA Homo sapiens CDS (150)..(1532) 1 tctatggtgg
ggccgcgtta gtggctgcgg ctccgcggga ctccagggcg cggctgcgag 60
gtggcggggc gccccgcctg cagaaccctg cttgcagctc aggtttcggg gtgcttgagg
120 aggccgccac ggcagcgcgg gagcggaag atg ttg gag ctg agg cac cgg gga
173 Met Leu Glu Leu Arg His Arg Gly 1 5 agc tgc ccc ggc ccc agg gaa
gcg gtg tcg ccg cca cac cgc gag gga 221 Ser Cys Pro Gly Pro Arg Glu
Ala Val Ser Pro Pro His Arg Glu Gly 10 15 20 gag gcg gcc ggc ggc
gac cac gaa acc gag agc acc agc gac aaa gaa 269 Glu Ala Ala Gly Gly
Asp His Glu Thr Glu Ser Thr Ser Asp Lys Glu 25 30 35 40 aca gat att
gat gac aga tat gga gat ttg gat tcc aga aca gat tct 317 Thr Asp Ile
Asp Asp Arg Tyr Gly Asp Leu Asp Ser Arg Thr Asp Ser 45 50 55 gat
att ccg gaa att cca cca tcc tca gat aga acc cct gag att ctc 365 Asp
Ile Pro Glu Ile Pro Pro Ser Ser Asp Arg Thr Pro Glu Ile Leu 60 65
70 aaa aaa gct cta tct ggt tta tct tca agg tgg aaa aac tgg tgg ata
413 Lys Lys Ala Leu Ser Gly Leu Ser Ser Arg Trp Lys Asn Trp Trp Ile
75 80 85 cgt gga att ctc act cta act atg atc tcg ttg ttt ttc ctg
atc atc 461 Arg Gly Ile Leu Thr Leu Thr Met Ile Ser Leu Phe Phe Leu
Ile Ile 90 95 100 tat atg gga tcc ttc atg ctg atg ctt ctt gtt ctg
ggc atc caa gtg 509 Tyr Met Gly Ser Phe Met Leu Met Leu Leu Val Leu
Gly Ile Gln Val 105 110 115 120 aaa tgc ttc cat gaa att atc act ata
ggt tat aga gtc tat cat tct 557 Lys Cys Phe His Glu Ile Ile Thr Ile
Gly Tyr Arg Val Tyr His Ser 125 130 135 tat gat cta cca tgg ttt aga
aca cta agt tgg tac ttt cta ttg tgt 605 Tyr Asp Leu Pro Trp Phe Arg
Thr Leu Ser Trp Tyr Phe Leu Leu Cys 140 145 150 gta aac tac ttt ttc
tat gga gag act gta gct gat tat ttt gct aca 653 Val Asn Tyr Phe Phe
Tyr Gly Glu Thr Val Ala Asp Tyr Phe Ala Thr 155 160 165 ttt gtt caa
aga gaa gaa caa ctt cag ttc ctc att cgc tac cat aga 701 Phe Val Gln
Arg Glu Glu Gln Leu Gln Phe Leu Ile Arg Tyr His Arg 170 175 180 ttt
ata tca ttt gcc ctc tat ctg gca ggt ttc tgc atg ttt gta ctg 749 Phe
Ile Ser Phe Ala Leu Tyr Leu Ala Gly Phe Cys Met Phe Val Leu 185 190
195 200 agt ttg gtg aag gaa cat tat cgt ctg cag ttt tat atg ttc gca
tgg 797 Ser Leu Val Lys Glu His Tyr Arg Leu Gln Phe Tyr Met Phe Ala
Trp 205 210 215 act cat gtc act tta ctg ata act gtc act cag tca cac
ctt gtc atc 845 Thr His Val Thr Leu Leu Ile Thr Val Thr Gln Ser His
Leu Val Ile 220 225 230 caa aat ctg ttt gaa ggc atg ata tgg ttc ctt
gtt cca ata tca agt 893 Gln Asn Leu Phe Glu Gly Met Ile Trp Phe Leu
Val Pro Ile Ser Ser 235 240 245 gtt atc tgc aat gac ata act gct tac
ctt ttt gga ttt ttt ttt ggg 941 Val Ile Cys Asn Asp Ile Thr Ala Tyr
Leu Phe Gly Phe Phe Phe Gly 250 255 260 aga act cca tta att aag ttg
tct cct aaa aag act tgg gaa gga ttc 989 Arg Thr Pro Leu Ile Lys Leu
Ser Pro Lys Lys Thr Trp Glu Gly Phe 265 270 275 280 att ggt ggt ttc
ttt tcc aca gtt gtg ttt gga ttc att gct gcc tat 1037 Ile Gly Gly
Phe Phe Ser Thr Val Val Phe Gly Phe Ile Ala Ala Tyr 285 290 295 gtg
tta tcc aaa tac cag tac ttt gtc tgc cca gtg gaa tac cga agt 1085
Val Leu Ser Lys Tyr Gln Tyr Phe Val Cys Pro Val Glu Tyr Arg Ser 300
305 310 gat gta aac tcc ttc gtg aca gaa tgt gag ccc tca gaa ctt ttc
cag 1133 Asp Val Asn Ser Phe Val Thr Glu Cys Glu Pro Ser Glu Leu
Phe Gln 315 320 325 ctt cag act tac tca ctt cca ccc ttt cta aag gca
gtc ttg aga cag 1181 Leu Gln Thr Tyr Ser Leu Pro Pro Phe Leu Lys
Ala Val Leu Arg Gln 330 335 340 gaa aga gtg agc ttg tac cct ttc cag
atc cac agc att gca ctg tca 1229 Glu Arg Val Ser Leu Tyr Pro Phe
Gln Ile His Ser Ile Ala Leu Ser 345 350 355 360 acc ttt gca tct tta
att ggc cca ttt gga ggc ttc ttt gct agt gga 1277 Thr Phe Ala Ser
Leu Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser Gly 365 370 375 ttc aaa
aga gcc ttc aaa atc aag gat ttt gca aat acc att cct gga 1325 Phe
Lys Arg Ala Phe Lys Ile Lys Asp Phe Ala Asn Thr Ile Pro Gly 380 385
390 cat ggt ggg ata atg gac aga ttt gat tgt cag tat ttg atg gca act
1373 His Gly Gly Ile Met Asp Arg Phe Asp Cys Gln Tyr Leu Met Ala
Thr 395 400 405 ttt gta cat gtg tac atc aca agt ttt ata agg ggc cca
aat ccc agc 1421 Phe Val His Val Tyr Ile Thr Ser Phe Ile Arg Gly
Pro Asn Pro Ser 410 415 420 aaa gtg cta cag cag ttg ttg gtg ctt caa
cct gaa cag cag tta aat 1469 Lys Val Leu Gln Gln Leu Leu Val Leu
Gln Pro Glu Gln Gln Leu Asn 425 430 435 440 ata tat aaa acc ctg aag
act cat ctc att gag aaa gga atc cta caa 1517 Ile Tyr Lys Thr Leu
Lys Thr His Leu Ile Glu Lys Gly Ile Leu Gln 445 450 455 ccc acc ttg
aag gta taactggatc cagagaggga aggactgaca agaaggaatt 1572 Pro Thr
Leu Lys Val 460 attcagaaaa acactgacag atgttttata aattgtacag
aaaaatagtt aaaaatgcaa 1632 taggttgaag ttttggagat atgtttctct
ctgaaattac tgtgaatatt taacaaacac 1692 ttacttgatc tatgttatga
aataagtagc aaattgccag caaaatgtct tgtacctttt 1752 ctaaagtgta
ttttctgatg tgaacttcct tccccttact tgctaggttt cataatttaa 1812
aagactggta tttaaaagag tcaaacacta taaaatgagt aagttgacga tgttttaaga
1872 ttgcacctgg cagtgtgcct ttttgcacaa atatttactt ttgcacttgg
agctgctttt 1932 aattttagca aaatgtttta tgcaaggcac aataggaagt
cagttctcct gcacttcctc 1992 ctcatgtagt ctggagtact ttctaaaggg
cttagttgga tttaaaaaaa aaaaaaaaa 2051 2 461 PRT Homo sapiens 2 Met
Leu Glu Leu Arg His Arg Gly Ser Cys Pro Gly Pro Arg Glu Ala 1 5 10
15 Val Ser Pro Pro His Arg Glu Gly Glu Ala Ala Gly Gly Asp His Glu
20 25 30 Thr Glu Ser Thr Ser Asp Lys Glu Thr Asp Ile Asp Asp Arg
Tyr Gly 35 40 45 Asp Leu Asp Ser Arg Thr Asp Ser Asp Ile Pro Glu
Ile Pro Pro Ser 50 55 60 Ser Asp Arg Thr Pro Glu Ile Leu Lys Lys
Ala Leu Ser Gly Leu Ser 65 70 75 80 Ser Arg Trp Lys Asn Trp Trp Ile
Arg Gly Ile Leu Thr Leu Thr Met 85 90 95 Ile Ser Leu Phe Phe Leu
Ile Ile Tyr Met Gly Ser Phe Met Leu Met 100 105 110 Leu Leu Val Leu
Gly Ile Gln Val Lys Cys Phe His Glu Ile Ile Thr 115 120 125 Ile Gly
Tyr Arg Val Tyr His Ser Tyr Asp Leu Pro Trp Phe Arg Thr 130 135 140
Leu Ser Trp Tyr Phe Leu Leu Cys Val Asn Tyr Phe Phe Tyr Gly Glu 145
150 155 160 Thr Val Ala Asp Tyr Phe Ala Thr Phe Val Gln Arg Glu Glu
Gln Leu 165 170 175 Gln Phe Leu Ile Arg Tyr His Arg Phe Ile Ser Phe
Ala Leu Tyr Leu 180 185 190 Ala Gly Phe Cys Met Phe Val Leu Ser Leu
Val Lys Glu His Tyr Arg 195 200 205 Leu Gln Phe Tyr Met Phe Ala Trp
Thr His Val Thr Leu Leu Ile Thr 210 215 220 Val Thr Gln Ser His Leu
Val Ile Gln Asn Leu Phe Glu Gly Met Ile 225 230 235 240 Trp Phe Leu
Val Pro Ile Ser Ser Val Ile Cys Asn Asp Ile Thr Ala 245 250 255 Tyr
Leu Phe Gly Phe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu Ser 260 265
270 Pro Lys Lys Thr Trp Glu Gly Phe Ile Gly Gly Phe Phe Ser Thr Val
275 280 285 Val Phe Gly Phe Ile Ala Ala Tyr Val Leu Ser Lys Tyr Gln
Tyr Phe 290 295 300 Val Cys Pro Val Glu Tyr Arg Ser Asp Val Asn Ser
Phe Val Thr Glu 305 310 315 320 Cys Glu Pro Ser Glu Leu Phe Gln Leu
Gln Thr Tyr Ser Leu Pro Pro 325 330 335 Phe Leu Lys Ala Val Leu Arg
Gln Glu Arg Val Ser Leu Tyr Pro Phe 340 345 350 Gln Ile His Ser Ile
Ala Leu Ser Thr Phe Ala Ser Leu Ile Gly Pro 355 360 365 Phe Gly Gly
Phe Phe Ala Ser Gly Phe Lys Arg Ala Phe Lys Ile Lys 370 375 380 Asp
Phe Ala Asn Thr Ile Pro Gly His Gly Gly Ile Met Asp Arg Phe 385 390
395 400 Asp Cys Gln Tyr Leu Met Ala Thr Phe Val His Val Tyr Ile Thr
Ser 405 410 415 Phe Ile Arg Gly Pro Asn Pro Ser Lys Val Leu Gln Gln
Leu Leu Val 420 425 430 Leu Gln Pro Glu Gln Gln Leu Asn Ile Tyr Lys
Thr Leu Lys Thr His 435 440 445 Leu Ile Glu Lys Gly Ile Leu Gln Pro
Thr Leu Lys Val 450 455 460 3 38 PRT Drosophila 3 Lys Arg Ala Phe
Lys Ile Lys Asp Phe Gly Asp Met Ile Pro Gly His 1 5 10 15 Gly Gly
Ile Met Asp Arg Phe Asp Cys Gln Phe Leu Met Ala Thr Phe 20 25 30
Val Asn Val Tyr Ile Ser 35 4 38 PRT Homo sapiens 4 Lys Arg Ala Phe
Lys Ile Lys Asp Phe Ala Asn Thr Ile Pro Gly His 1 5 10 15 Gly Gly
Ile Met Asp Arg Phe Asp Cys Gln Tyr Leu Met Ala Thr Phe 20 25 30
Val His Val Tyr Ile Thr 35 5 27 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 5 cccaccatgg
ccaggaatgg tatttgc 27 6 25 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 6 agtgatgtga
attccttcgt gacag 25 7 29 DNA Artificial Sequence Description of
Artificial Sequence Primer 7 ggctctagat attaatagta atcaattac 29 8
26 DNA Artificial Sequence Description of Artificial Sequence
Primer 8 cctcacgcat gcaccatggt aatagc 26 9 24 DNA Artificial
Sequence Description of Artificial Sequence Primer 9 ggtgcatgcg
tgaggctccg gtgc 24 10 28 DNA Artificial Sequence Description of
Artificial Sequence Primer 10 gtagttttca cggtacctga aatggaag 28 11
2488 DNA Homo sapiens CDS (25)..(1359) 11 cgacgtcggg ccgattttcc
cagg atg aca gag ctg agg cag agg gtg gcc 51 Met Thr Glu Leu Arg Gln
Arg Val Ala 1 5 cat gag ccg gtt gcg cca ccc gag gac aag gag tca gag
tca gaa gca 99 His Glu Pro Val Ala Pro Pro Glu Asp Lys Glu Ser Glu
Ser Glu Ala 10 15 20 25 aag gta gat gga gag act gca tcg gac agt gag
agc cag gca gaa tcc 147 Lys Val Asp Gly Glu Thr Ala Ser Asp Ser Glu
Ser Gln Ala Glu Ser 30 35 40 gca ccc ctg cca gtc tct gca gat gat
acc ccg gag gtc ctc aat agg 195 Ala Pro Leu Pro Val Ser Ala Asp Asp
Thr Pro Glu Val Leu Asn Arg 45 50 55 gcc ctt tcc aac ttg tct tca
aga tgg aag gac tgg tgg gtg aga ggc 243 Ala Leu Ser Asn Leu Ser Ser
Arg Trp Lys Asp Trp Trp Val Arg Gly 60 65 70 atc ctg act ttg gcc
atg att gca ttt ttc ttc atc atc att tac ctg 291 Ile Leu Thr Leu Ala
Met Ile Ala Phe Phe Phe Ile Ile Ile Tyr Leu 75 80 85 gga cca atg
gtt ttg atg ata atc gtg atg tgc gtt cag att aag tgt 339 Gly Pro Met
Val Leu Met Ile Ile Val Met Cys Val Gln Ile Lys Cys 90 95 100 105
ttc cat gag ata atc act att ggc tac aac gtc tac cac tca tat gat 387
Phe His Glu Ile Ile Thr Ile Gly Tyr Asn Val Tyr His Ser Tyr Asp 110
115 120 ctg ccc tgg ttc agg acg ctc agc tgg tac ttt ctc ctg tgt gta
aac 435 Leu Pro Trp Phe Arg Thr Leu Ser Trp Tyr Phe Leu Leu Cys Val
Asn 125 130 135 tat ttc ttc tat ggt gag aca gtg acg gat tac ttc ttc
acc ctg gtc 483 Tyr Phe Phe Tyr Gly Glu Thr Val Thr Asp Tyr Phe Phe
Thr Leu Val 140 145 150 cag aga gaa gag cct ttg cgg att ctc agt aaa
tac cac cgg ttc att 531 Gln Arg Glu Glu Pro Leu Arg Ile Leu Ser Lys
Tyr His Arg Phe Ile 155 160 165 tcc ttt act ctc tat cta ata gga ttc
tgc atg ttt gta ctg agt ctg 579 Ser Phe Thr Leu Tyr Leu Ile Gly Phe
Cys Met Phe Val Leu Ser Leu 170 175 180 185 gtc aag aag cat tat cga
ctg cag ttc tac atg ttt ggc tgg acc cat 627 Val Lys Lys His Tyr Arg
Leu Gln Phe Tyr Met Phe Gly Trp Thr His 190 195 200 gtg aca ttg ctg
att gtt gta aca cag tca cat ctt gtt atc cac aac 675 Val Thr Leu Leu
Ile Val Val Thr Gln Ser His Leu Val Ile His Asn 205 210 215 cta ttt
gaa gga atg atc tgg ttc att gtc ccc ata tct tgt gtg atc 723 Leu Phe
Glu Gly Met Ile Trp Phe Ile Val Pro Ile Ser Cys Val Ile 220 225 230
tgt aat gac atc atg gcc tat atg ttt ggc ttt ttc ttt ggt cgg acc 771
Cys Asn Asp Ile Met Ala Tyr Met Phe Gly Phe Phe Phe Gly Arg Thr 235
240 245 cca ctc atc aag ctg tcc ccg aag aag acc tgg gaa ggc ttc att
ggg 819 Pro Leu Ile Lys Leu Ser Pro Lys Lys Thr Trp Glu Gly Phe Ile
Gly 250 255 260 265 ggc ttc ttt gct act gtg gtg ttt ggc ctt ctg ctg
tcc tat gtg atg 867 Gly Phe Phe Ala Thr Val Val Phe Gly Leu Leu Leu
Ser Tyr Val Met 270 275 280 tcc ggg tac aga tgc ttt gtc tgc cct gtg
gag tac aac aat gac acc 915 Ser Gly Tyr Arg Cys Phe Val Cys Pro Val
Glu Tyr Asn Asn Asp Thr 285 290 295 aac agc ttc act gtg gac tgt gag
ccc tcg gac ctg ttt cgc ctg cag 963 Asn Ser Phe Thr Val Asp Cys Glu
Pro Ser Asp Leu Phe Arg Leu Gln 300 305 310 gag tac aac att cct ggg
gtg atc cag tca gtc att ggc tgg aaa acg 1011 Glu Tyr Asn Ile Pro
Gly Val Ile Gln Ser Val Ile Gly Trp Lys Thr 315 320 325 gtc cgg atg
tac ccc ttc cag att cac agc atc gct ctc tcc acc ttt 1059 Val Arg
Met Tyr Pro Phe Gln Ile His Ser Ile Ala Leu Ser Thr Phe 330 335 340
345 gcc tcg ctc att ggc ccc ttt gga gga ttc ttc gca agt gga ttc aaa
1107 Ala Ser Leu Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser Gly Phe
Lys 350 355 360 cga gcc ttt aaa atc aaa gac ttt gcc aat acc att cct
ggc cat gga 1155 Arg Ala Phe Lys Ile Lys Asp Phe Ala Asn Thr Ile
Pro Gly His Gly 365 370 375 ggc atc atg gat cgc ttt gac tgc cag tat
ctg atg gcc acc ttt gtc 1203 Gly Ile Met Asp Arg Phe Asp Cys Gln
Tyr Leu Met Ala Thr Phe Val 380 385 390 aat gta tac atc gcc agt ttt
atc aga ggc cct aac cca agc aaa ctg 1251 Asn Val Tyr Ile Ala Ser
Phe Ile Arg Gly Pro Asn Pro Ser Lys Leu 395 400 405 att cag cag ttc
ctg act tta cgg cca gat cag cag ctc cac atc ttc 1299 Ile Gln Gln
Phe Leu Thr Leu Arg Pro Asp Gln Gln Leu His Ile Phe 410 415 420 425
aac acg ctg cgg tct cat ctg atc gac aaa ggg atg ctg aca tcc acc
1347 Asn Thr Leu Arg Ser His Leu Ile Asp Lys Gly Met Leu Thr Ser
Thr 430 435 440 aca gag gac gag taggggccac ccagggccag gagaacagga
acagaactga 1399 Thr Glu Asp Glu 445 gcaggggcag gtctccaagg
caagcccagc tggtgtgact tagacaatga cgaggcttca 1459 actcactgtc
tttttttttt tttttttttt ggagggtatt ttttatttgt gggttcaaaa 1519
aatctgtata tacagtctat gtgtttagaa tttgtgttgt aagtaaacta cagctttgag
1579 ttggaaagaa gtcacgggtt gtaaaaccat ttggattttt ttaaaacaaa
agtattaata 1639 atctggaaga cagtgttgcc caggtcagga gtgttttctt
ggtggttcca gcccccatca 1699 attgaactgt ttctgggctc agtcagacac
agacattcat ctgtgtctga ccaaatcagg 1759 ggacttcccc acctgtggtg
ggaggcacag cttagatgtt ttgtacacct ggtcttttct 1819 agaaatccct
gcttggagct gcagaagggt tgccttctgt aggtcggagg aatggaggct 1879
tactaaccag gtaagccttc tatgcatcca caccaaaatc ctgcagaatg taagtaagct
1939 ctgctttata agatgggttc accttcatcg cagactgaaa gtttcagttt
ttattttttt 1999 cagaaagcac gaaaaattat ttataatagt ctggagaaaa
aacacactgt aatatttcaa 2059 gtgtatgcag tagaatgtac tgtaactgag
ccctttccca catgtctagg ctccaatgtc 2119 tcctgtaggt ccacctaact
gtgtgttttc agggacaatg ccatccatgt ttgtgctgta 2179 gacttgctgc
tgctgaatcc tttctgggga ctttctcatc gggcagggag cagagggctt 2239
ctcgttcatg caccctttgc ctgaacaccc atgtagctgc tgtgttgtgt atatattact
2299 cttaagagga gtgtgtgtgt ctgtgtttgt tttaaaagtc acttatttct
tacagtgatt 2359 tcaattgcac catgacttct tcactaaaac cacaaagtcc
tgcttaaaac tatggaaaac
2419 ctaacctgat tagagccttg actattttga agattaaatg cacacttttt
atataaaaaa 2479 aaaaaaaaa 2488 12 445 PRT Homo sapiens 12 Met Thr
Glu Leu Arg Gln Arg Val Ala His Glu Pro Val Ala Pro Pro 1 5 10 15
Glu Asp Lys Glu Ser Glu Ser Glu Ala Lys Val Asp Gly Glu Thr Ala 20
25 30 Ser Asp Ser Glu Ser Gln Ala Glu Ser Ala Pro Leu Pro Val Ser
Ala 35 40 45 Asp Asp Thr Pro Glu Val Leu Asn Arg Ala Leu Ser Asn
Leu Ser Ser 50 55 60 Arg Trp Lys Asp Trp Trp Val Arg Gly Ile Leu
Thr Leu Ala Met Ile 65 70 75 80 Ala Phe Phe Phe Ile Ile Ile Tyr Leu
Gly Pro Met Val Leu Met Ile 85 90 95 Ile Val Met Cys Val Gln Ile
Lys Cys Phe His Glu Ile Ile Thr Ile 100 105 110 Gly Tyr Asn Val Tyr
His Ser Tyr Asp Leu Pro Trp Phe Arg Thr Leu 115 120 125 Ser Trp Tyr
Phe Leu Leu Cys Val Asn Tyr Phe Phe Tyr Gly Glu Thr 130 135 140 Val
Thr Asp Tyr Phe Phe Thr Leu Val Gln Arg Glu Glu Pro Leu Arg 145 150
155 160 Ile Leu Ser Lys Tyr His Arg Phe Ile Ser Phe Thr Leu Tyr Leu
Ile 165 170 175 Gly Phe Cys Met Phe Val Leu Ser Leu Val Lys Lys His
Tyr Arg Leu 180 185 190 Gln Phe Tyr Met Phe Gly Trp Thr His Val Thr
Leu Leu Ile Val Val 195 200 205 Thr Gln Ser His Leu Val Ile His Asn
Leu Phe Glu Gly Met Ile Trp 210 215 220 Phe Ile Val Pro Ile Ser Cys
Val Ile Cys Asn Asp Ile Met Ala Tyr 225 230 235 240 Met Phe Gly Phe
Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu Ser Pro 245 250 255 Lys Lys
Thr Trp Glu Gly Phe Ile Gly Gly Phe Phe Ala Thr Val Val 260 265 270
Phe Gly Leu Leu Leu Ser Tyr Val Met Ser Gly Tyr Arg Cys Phe Val 275
280 285 Cys Pro Val Glu Tyr Asn Asn Asp Thr Asn Ser Phe Thr Val Asp
Cys 290 295 300 Glu Pro Ser Asp Leu Phe Arg Leu Gln Glu Tyr Asn Ile
Pro Gly Val 305 310 315 320 Ile Gln Ser Val Ile Gly Trp Lys Thr Val
Arg Met Tyr Pro Phe Gln 325 330 335 Ile His Ser Ile Ala Leu Ser Thr
Phe Ala Ser Leu Ile Gly Pro Phe 340 345 350 Gly Gly Phe Phe Ala Ser
Gly Phe Lys Arg Ala Phe Lys Ile Lys Asp 355 360 365 Phe Ala Asn Thr
Ile Pro Gly His Gly Gly Ile Met Asp Arg Phe Asp 370 375 380 Cys Gln
Tyr Leu Met Ala Thr Phe Val Asn Val Tyr Ile Ala Ser Phe 385 390 395
400 Ile Arg Gly Pro Asn Pro Ser Lys Leu Ile Gln Gln Phe Leu Thr Leu
405 410 415 Arg Pro Asp Gln Gln Leu His Ile Phe Asn Thr Leu Arg Ser
His Leu 420 425 430 Ile Asp Lys Gly Met Leu Thr Ser Thr Thr Glu Asp
Glu 435 440 445 13 2103 DNA Homo sapiens 13 tctatggtgg ggccgcgtta
gtggctgcgg ctccgcggga ctccagggcg cggctgcgag 60 gtggcggggc
gccccgcctg cagaaccctg cttgcagctc aggtttcggg gtgcttgagg 120
aggccgccac ggcagcgcgg gagcggaaga tgttggagct gaggcaccgg ggaagctgcc
180 ccggccccag ggaagcggtg tcgccgccac accgcgaggg agaggcggcc
ggcggcgacc 240 acgaaaccga gagcaccagc gacaaagaaa cagatattga
tgacagatat ggagatttgg 300 attccagaac agattctgat attccggaaa
ttccaccatc ctcagataga acccctgaga 360 ttctcaaaaa agctctatct
ggtttatctt caaggtggaa aaactggtgg atacgtggaa 420 ttctcactct
aactatgatc tcgttgtttt tcctgatcat ctatatggga tccttcatgc 480
tgatgcttct tgttctgggc atccaagtga aatgcttcca tgaaattatc actataggtt
540 atagagtcta tcattcttat gatctaccat ggtttagaac actaagttgg
tactttctat 600 tgtgtgtaaa ctactttttc tatggagaga ctgtagctga
ttattttgct acatttgttc 660 aaagagaaga acaacttcag ttcctcattc
gctaccatag atttatatca tttgccctct 720 atctggcagg tttctgcatg
tttgtactga gtttggtgaa ggaacattat cgtctgcagt 780 tttatatgtt
cgcatggact catgtcactt tactgataac tgtcactcag tcacaccttg 840
tcatccaaaa tctgtttgaa ggcatgatat ggttccttgt tccaatatca agtgttatct
900 gcaatgacat aactgcttac ctttttggat ttttttttgg gagaactcca
ttaattaagt 960 tgtctcctaa aaagacttgg gaaggattca ttggtggttt
cttttccaca gttgtgtttg 1020 gattcattgc tgcctatgtg ttatccaaat
accagtactt tgtctgccca gtggaatacc 1080 gaagtgatgt aaactccttc
gtgacagaat gtgagccctc agaacttttc cagcttcaga 1140 cttactcact
tccacccttt ctaaaggcag tcttgagaca ggaaagagtg agcttgtacc 1200
ctttccagat ccacagcatt gcactgtcaa cctttgcatc tttaattggc ccatttggag
1260 gcttctttgc tagtggattc aaaagagcct tcaaaatcaa ggattttgca
aataccattc 1320 ctggacatgg tgggataatg gacagatttg attgtcagta
tttgatggca acttttgtac 1380 atgtgtacat cacaagtttt ataaggggcc
caaatcccag caaagtgcta cagcagttgt 1440 tggtgcttca acctgaacag
cagttaaata tatataaaac cctgaagact catctcattg 1500 agaaaggaat
cctacaaccc accttgaagg tataactgga tccagagagg gaaggactga 1560
caagaaggaa ttattcagaa aaacactgac agatgtttta taaattgtac agaaaaatag
1620 ttaaaaatgc aataggttga agttttggag atatgtttct ctctgaaatt
actgtgaata 1680 tttaacaaac acttacttga tctatgttat gaaataagta
gcaaattgcc agcaaaatgt 1740 cttgtacctt ttctaaagtg tattttctga
tgtgaacttc cttcccctta cttgctaggt 1800 ttcataattt aaaagactgg
tatttaaaag agtcaaacac tataaaatga gtaagttgac 1860 gatgttttaa
gattgcacct ggcagtgtgc ctttttgcac aaatatttac ttttgcactt 1920
ggagctgctt ttaattttag caaaatgttt tatgcaaggc acaataggaa gtcagttctc
1980 ctgcacttcc tcctcatgta gtctggagta ctttctaaag ggcttagttg
gatttaaaaa 2040 aaaaaaaaaa agggcggccg ctctagagga tccctcgagg
ggcccaagct tacgcgtgca 2100 tgc 2103 14 45 PRT Homo sapiens 14 Gln
Ser His Leu Val Ile His Asn Leu Phe Glu Gly Met Ile Trp Phe 1 5 10
15 Ile Val Pro Ile Ser Cys Val Ile Cys Asn Asp Ile Met Ala Tyr Met
20 25 30 Phe Gly Phe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu 35 40
45 15 22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 15 aggacgcata tgagtggtag ac 22 16 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 16
gactctagcc taggcttttg c 21 17 249 PRT E. coli 17 Met Leu Ala Ala
Trp Glu Trp Gly Gln Leu Ser Gly Phe Thr Thr Arg 1 5 10 15 Ser Gln
Arg Val Trp Leu Ala Val Leu Cys Gly Leu Leu Leu Ala Leu 20 25 30
Met Leu Phe Leu Leu Pro Glu Tyr His Arg Asn Ile His Gln Pro Leu 35
40 45 Val Glu Ile Ser Leu Trp Ala Ser Leu Gly Trp Trp Ile Val Ala
Leu 50 55 60 Leu Leu Val Leu Phe Tyr Pro Gly Ser Ala Ala Ile Trp
Arg Asn Ser 65 70 75 80 Lys Thr Leu Arg Leu Ile Phe Gly Val Leu Thr
Ile Val Pro Phe Phe 85 90 95 Trp Gly Met Leu Ala Leu Arg Ala Trp
His Tyr Asp Glu Asn His Tyr 100 105 110 Ser Gly Ala Ile Trp Leu Leu
Tyr Val Met Ile Leu Val Trp Gly Ala 115 120 125 Asp Ser Gly Ala Tyr
Met Phe Gly Lys Leu Phe Gly Lys His Lys Leu 130 135 140 Ala Pro Lys
Val Ser Pro Gly Lys Thr Trp Gln Gly Phe Ile Gly Gly 145 150 155 160
Leu Ala Thr Ala Ala Val Ile Ser Trp Gly Tyr Gly Met Trp Ala Asn 165
170 175 Leu Asp Val Ala Pro Val Thr Leu Leu Ile Cys Ser Ile Val Ala
Ala 180 185 190 Leu Ala Ser Val Leu Gly Asp Leu Thr Glu Ser Met Phe
Lys Arg Glu 195 200 205 Ala Gly Ile Lys Asp Ser Gly His Leu Ile Pro
Gly His Gly Gly Ile 210 215 220 Leu Asp Arg Ile Asp Ser Leu Thr Ala
Ala Val Pro Val Phe Ala Cys 225 230 235 240 Leu Leu Leu Leu Val Phe
Arg Thr Leu 245 18 457 PRT Yeast 18 Met Ser Asp Asn Pro Glu Met Lys
Pro His Gly Thr Ser Lys Glu Ile 1 5 10 15 Val Glu Ser Val Thr Asp
Ala Thr Ser Lys Ala Ile Asp Lys Leu Gln 20 25 30 Glu Glu Leu His
Lys Asp Ala Ser Glu Ser Val Thr Pro Val Thr Lys 35 40 45 Glu Ser
Thr Ala Ala Thr Lys Glu Ser Arg Lys Tyr Asn Phe Phe Ile 50 55 60
Arg Thr Val Trp Thr Phe Val Met Ile Ser Gly Phe Phe Ile Thr Leu 65
70 75 80 Ala Ser Gly His Ala Trp Cys Ile Val Leu Ile Leu Gly Cys
Gln Ile 85 90 95 Ala Thr Phe Lys Glu Cys Ile Ala Val Thr Ser Ala
Ser Gly Arg Glu 100 105 110 Lys Asn Leu Pro Leu Thr Lys Thr Leu Asn
Trp Tyr Leu Leu Phe Thr 115 120 125 Thr Ile Tyr Tyr Leu Asp Gly Lys
Ser Leu Phe Lys Phe Phe Gln Ala 130 135 140 Thr Phe Tyr Glu Tyr Pro
Val Leu Asn Phe Ile Val Thr Asn His Lys 145 150 155 160 Phe Ile Cys
Tyr Cys Leu Tyr Leu Met Gly Phe Val Leu Phe Val Cys 165 170 175 Ser
Leu Arg Lys Gly Phe Leu Lys Phe Gln Phe Gly Ser Leu Cys Val 180 185
190 Thr His Met Val Leu Leu Leu Val Val Phe Gln Ala His Leu Ile Ile
195 200 205 Lys Asn Val Leu Asn Gly Leu Phe Trp Phe Leu Leu Pro Cys
Gly Leu 210 215 220 Val Ile Val Asn Asp Ile Phe Ala Tyr Leu Cys Gly
Ile Thr Phe Gly 225 230 235 240 Lys Thr Lys Leu Ile Glu Ile Ser Pro
Lys Lys Thr Leu Glu Gly Phe 245 250 255 Leu Gly Ala Trp Phe Phe Thr
Ala Leu Ala Ser Ile Ile Leu Thr Arg 260 265 270 Ile Leu Ser Pro Tyr
Thr Tyr Leu Thr Cys Pro Val Glu Asp Leu His 275 280 285 Thr Asn Phe
Phe Ser Asn Leu Thr Cys Glu Leu Asn Pro Val Phe Leu 290 295 300 Pro
Gln Val Tyr Arg Leu Pro Pro Ile Phe Phe Asp Lys Val Gln Ile 305 310
315 320 Asn Ser Ile Thr Val Lys Pro Ile Tyr Phe His Ala Leu Asn Leu
Ala 325 330 335 Thr Phe Ala Ser Leu Phe Ala Pro Phe Gly Gly Phe Phe
Ala Ser Gly 340 345 350 Leu Lys Arg Thr Phe Lys Val Lys Asp Phe Gly
His Ser Ile Pro Gly 355 360 365 His Gly Gly Ile Thr Asp Arg Val Asp
Cys Gln Phe Ile Met Gly Ser 370 375 380 Phe Ala Asn Leu Tyr Tyr Glu
Thr Phe Ile Ser Glu His Arg Ile Thr 385 390 395 400 Val Asp Thr Val
Leu Ser Thr Ile Leu Met Asn Leu Asn Asp Lys Gln 405 410 415 Ile Ile
Glu Leu Ile Asp Ile Leu Ile Arg Phe Leu Ser Lys Lys Gly 420 425 430
Ile Ile Ser Ala Lys Asn Phe Glu Lys Leu Ala Asp Ile Phe Asn Val 435
440 445 Thr Lys Lys Ser Leu Thr Asn His Ser 450 455 19 446 PRT
Drosophila 19 Met Ala Glu Val Arg Arg Arg Lys Gly Glu Asp Glu Pro
Leu Glu Asp 1 5 10 15 Thr Ala Ile Ser Gly Ser Asp Ala Ala Asn Lys
Arg Asn Ser Ala Ala 20 25 30 Asp Ser Ser Asp His Val Asp Ser Glu
Glu Glu Lys Ile Pro Glu Glu 35 40 45 Lys Phe Val Asp Glu Leu Ala
Lys Asn Leu Pro Gln Gly Thr Asp Lys 50 55 60 Thr Pro Glu Ile Leu
Asp Ser Ala Leu Lys Asp Leu Pro Asp Arg Trp 65 70 75 80 Lys Asn Trp
Val Ile Arg Gly Ile Phe Thr Trp Ile Met Ile Cys Gly 85 90 95 Phe
Ala Leu Ile Ile Tyr Gly Gly Pro Leu Ala Leu Met Ile Thr Thr 100 105
110 Leu Leu Val Gln Val Lys Cys Phe Gln Glu Ile Ile Ser Ile Gly Tyr
115 120 125 Gln Val Tyr Arg Ile His Gly Leu Pro Trp Phe Arg Ser Leu
Ser Trp 130 135 140 Tyr Phe Leu Leu Thr Ser Asn Tyr Phe Phe Tyr Gly
Glu Asn Leu Val 145 150 155 160 Asp Tyr Phe Gly Val Val Ile Asn Arg
Val Glu Tyr Leu Lys Phe Leu 165 170 175 Val Thr Tyr His Arg Phe Leu
Ser Phe Ala Leu Tyr Ile Ile Gly Phe 180 185 190 Val Trp Phe Val Leu
Ser Leu Val Lys Lys Tyr Tyr Ile Lys Gln Phe 195 200 205 Ser Leu Phe
Ala Trp Thr His Val Ser Leu Leu Ile Val Val Thr Gln 210 215 220 Ser
Tyr Leu Ile Ile Gln Asn Ile Phe Glu Gly Leu Ile Trp Phe Ile 225 230
235 240 Val Pro Val Ser Met Ile Val Cys Asn Asp Val Met Ala Tyr Val
Phe 245 250 255 Gly Phe Phe Phe Gly Arg Thr Pro Leu Ile Lys Leu Ser
Pro Lys Lys 260 265 270 Thr Trp Glu Gly Phe Ile Gly Gly Gly Phe Ala
Thr Val Leu Phe Gly 275 280 285 Ile Leu Phe Ser Tyr Val Leu Cys Asn
Tyr Gln Tyr Phe Ile Cys Pro 290 295 300 Ile Gln Tyr Ser Glu Glu Gln
Gly Arg Met Thr Met Ser Cys Val Pro 305 310 315 320 Ser Tyr Leu Phe
Thr Pro Gln Glu Tyr Ser Leu Lys Leu Phe Gly Ile 325 330 335 Gly Lys
Thr Leu Asn Leu Tyr Pro Phe Ile Trp His Ser Ile Ser Leu 340 345 350
Ser Leu Phe Ser Ser Ile Ile Gly Pro Phe Gly Gly Phe Phe Ala Ser 355
360 365 Gly Phe Lys Arg Ala Phe Lys Ile Lys Asp Phe Gly Asp Met Ile
Pro 370 375 380 Gly His Gly Gly Ile Met Asp Arg Phe Asp Cys Gln Phe
Leu Met Ala 385 390 395 400 Thr Phe Val Asn Val Tyr Ile Ser Phe Ile
Arg Thr Pro Ser Pro Ala 405 410 415 Lys Leu Leu Thr Gln Ile Tyr Asn
Leu Lys Pro Asp Gln Gln Tyr Gln 420 425 430 Ile Tyr Gln Ser Leu Lys
Asp Asn Leu Gly His Met Leu Thr 435 440 445
* * * * *