U.S. patent application number 09/861205 was filed with the patent office on 2002-06-20 for human rnase h and compositions and uses thereof.
Invention is credited to Crooke, Stanley T., Lima, Walter, Wu, Hongjiang.
Application Number | 20020076712 09/861205 |
Document ID | / |
Family ID | 22076123 |
Filed Date | 2002-06-20 |
United States Patent
Application |
20020076712 |
Kind Code |
A1 |
Crooke, Stanley T. ; et
al. |
June 20, 2002 |
Human RNase H and compositions and uses thereof
Abstract
The present invention provides polynucleotides and polypeptides
encoded thereby of human Type 2 RNase H. Methods of using these
polynucleotides and polypeptides in enhancing antisense
oligonucleotide therapies are also provided.
Inventors: |
Crooke, Stanley T.;
(Carlsbad, CA) ; Lima, Walter; (San Diego, CA)
; Wu, Hongjiang; (Carlsbad, CA) |
Correspondence
Address: |
LICATLA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
22076123 |
Appl. No.: |
09/861205 |
Filed: |
May 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09861205 |
May 18, 2001 |
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09684254 |
Oct 6, 2000 |
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09684254 |
Oct 6, 2000 |
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09343809 |
Jun 30, 1999 |
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09343809 |
Jun 30, 1999 |
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09203716 |
Dec 2, 1998 |
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60067458 |
Dec 4, 1997 |
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Current U.S.
Class: |
435/6.18 ;
435/199; 435/320.1; 435/325; 435/6.1; 435/69.1; 514/44A;
536/23.2 |
Current CPC
Class: |
C12N 9/22 20130101; C12N
15/113 20130101; A61K 38/00 20130101; C12N 2310/11 20130101 |
Class at
Publication: |
435/6 ; 435/199;
435/325; 435/320.1; 536/23.2; 435/69.1; 514/44 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/22; A61K 048/00; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polynucleotide encoding the human Type 2 RNase H
polypeptide encoded by the nucleic acid sequence of the cDNA
contained within ATCC Deposit No. 98536.
2. A vector comprising a nucleic acid encoding the human Type 2
RNase H polypeptide encoded by the nucleic acid sequence of the
cDNA contained within ATCC Deposit No. 98536.
3. A host cell comprising the vector of claim 2.
4. A composition comprising a vector comprising a nucleic acid
encoding the human Type 2 RNase H polypeptide encoded by the
nucleic acid sequence of the cDNA contained within ATCC Deposit No.
98536.
5. The composition of claim 4 further comprising an antisense
oligonucleotide.
6. A composition comprising an antisense oligonucleotide and a
human Type 2 RNase H polypeptide, wherein the human Type 2 RNase H
polypeptide is the human Type 2 RNase H polypeptide encoded by the
nucleic acid sequence of the cDNA contained within ATCC Deposit No.
98536.
7. A human Type 2 RNase H--his-tag fusion polypeptide, wherein the
human Type 2 RNase H polypeptide is the human Type 2 RNase H
polypeptide encoded by the nucleic acid sequence of the cDNA
contained within ATCC Deposit No. 98536.
8. A method of screening oligonucleotides to identify effective
antisense oligonucleotides for inhibition of expression of a
selected target protein comprising: (a) contacting the human Type 2
RNase H polypeptide encoded by the nucleic acid sequence of the
cDNA contained within ATCC Deposit No. 98536 with an RNA encoding
the selected target protein and an oligonucleotide complementary to
at least a portion of the RNA under conditions in which an
RNA-oligonucleotide duplex is formed; (b) detecting cleavage of the
RNA of the RNA-oligonucleotide duplex wherein cleavage is
indicative of antisense efficacy.
9. The method of claim 8 further comprising determining the site on
the RNA at which cleavage occurs, whereby said site is identified
as a Type 2 RNase H-sensitive site.
10. The method of claim 9 further comprising identifying an
effective antisense oligonucleotide which hybridizes to said Type 2
RNase H-sensitive site.
11. The method of claim 8 wherein the oligonucleotide is one of a
mixture or library of oligonucleotides.
12. A method of identifying agents which increase or decrease
activity or levels of the human Type 2 RNase H polypeptide encoded
by the nucleic acid sequence of the cDNA contained within ATCC
Deposit No. 98536 in a host cell comprising: (a) contacting a cell
in vitro expressing the human Type 2 RNase H polypeptide with an
agent suspected of increasing or decreasing activity or levels of
the human RNase H polypeptide; and (b) measuring the activity or
levels of the human RNase H polypeptide in the presence and absence
of the agent so that an increase or decrease in the activity or
levels of the human RNase H polypeptide can be determined.
Description
[0001] This application is a continuation of U.S. Ser. No.
09/684,254, filed on Oct. 6, 2000, which is a continuation of U.S.
Ser. No. 09/343,809, filed Jun. 30, 1999, which is a continuation
of U.S. Ser. No. 09/203,716, filed Dec. 2, 1998 and issued as U.S.
Pat. No. 6,001,653 on Dec. 14, 1999 which claims the benefit of
priority of U.S. Provisional Application Serial No. 60/067,458,
filed Dec. 4, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates to a human Type 2 RNase H
which has now been cloned, expressed and purified to
electrophoretic homogeneity and human RNase H and compositions and
uses thereof.
BACKGROUND OF THE INVENTION
[0003] RNase H hydrolyzes RNA in RNA-DNA hybrids. This enzyme was
first identified in calf thymus but has subsequently been described
in a variety of organisms (Stein, H. and Hausen, P., Science, 1969,
166, 393-395; Hausen, P. and Stein, H., Eur. J. Biochem., 1970, 14,
278-283). RNase H activity appears to be ubiquitous in eukaryotes
and bacteria (Itaya, M. and Kondo K. Nucleic Acids Res., 1991, 19,
4443-4449; Itaya et al., Mol. Gen. Genet., 1991 227, 438-445;
Kanaya, S., and Itaya, M., J. Biol. Chem., 1992, 267, 10184-10192;
Busen, W., J. Biol. Chem., 1980, 255, 9434-9443; Rong, Y. W. and
Carl, P. L., 1990, Biochemistry 29, 383-389; Eder et al.,
Biochimie, 1993 75, 123-126). Although RNases H constitute a family
of proteins of varying molecular weight, nucleolytic activity and
substrate requirements appear to be similar for the various
isotypes. For example, all RNase Hs studied to date function as
endonucleases, exhibiting limited sequence specificity and
requiring divalent cations (e.g., Mg.sup.2+, Mn.sup.2+) to produce
cleavage products with 5' phosphate and 3' hydroxyl termini
(Crouch, R. J., and Dirksen, M. L., Nuclease, Linn, S, M., &
Roberts, R. J., Eds., Cold Spring Harbor Laboratory Press,
Plainview, N.Y. 1982, 211-241).
[0004] In addition to playing a natural role in DNA replication,
RNase H has also been shown to be capable of cleaving the RNA
component of certain oligonucleotide-RNA duplexes. While many
mechanisms have been proposed for oligonucleotide mediated
destabilization of target RNAs, the primary mechanism by which
antisense oligonucleotides are believed to cause a reduction in
target RNA levels is through this RNase H action. Monia et al., J.
Biol. Chem., 1993, 266:13, 14514-14522. In vitro assays have
demonstrated that oligonucleotides that are not substrates for
RNase H can inhibit protein translation (Blake et al.,
Biochemistry, 1985, 24, 6139-4145) and that oligonucleotides
inhibit protein translation in rabbit reticulocyte extracts that
exhibit low RNase H activity. However, more efficient inhibition
was found in systems that supported RNase H activity (Walder, R. Y.
and Walder, J. A., Proc. Nat'l Acad. Sci. USA, 1988, 85, 5011-5015;
Gagnor et al., Nucleic Acid Res., 1987, 15, 10419-10436; Cazenave
et al., Nucleic Acid Res., 1989, 17, 4255-4273; and Dash et al.,
Proc. Nat'l Acad. Sci. USA, 1987, 84, 7896-7900.
[0005] Oligonucleotides commonly described as "antisense
oligonucleotides" comprise nucleotide sequences sufficient in
identity and number to effect specific hybridization with a
particular nucleic acid. This nucleic acid or the protein(s) it
encodes is generally referred to as the "target." Oligonucleotides
are generally designed to bind either directly to mRNA transcribed
from, or to a selected DNA portion of, a preselected gene target,
thereby modulating the amount of protein translated from the mRNA
or the amount of mRNA transcribed from the gene, respectively.
Antisense oligonucleotides may be used as research tools,
diagnostic aids, and therapeutic agents.
[0006] "Targeting" an oligonucleotide to the associated nucleic
acid, in the context of this invention, also refers to a multistep
process which usually begins with the identification of the nucleic
acid sequence whose function is to be modulated. This may be, for
example, a cellular gene (or mRNA transcribed from the gene) whose
expression is associated with a particular disorder or disease
state, or a foreign nucleic acid from an infectious agent. The
targeting process also includes determination of a site or sites
within this gene for the oligonucleotide interaction to occur such
that the desired effect, either detection or modulation of
expression of the protein, will result.
[0007] RNase HI from E. coli is the best-characterized member of
the RNase H family. The 3-dimensional structure of E. coli RNase HI
has been determined by x-ray crystallography, and the key amino
acids involved in binding and catalysis have been identified by
site-directed mutagenesis (Nakamura et al., Proc. Natl. Acad. Sci.
USA, 1991, 88, 11535-11539; Katayanagi et al., Nature, 1990, 347,
306-309; Yang et al., Science, 1990, 249, 1398-1405; Kanaya et al.,
J. Biol. Chem., 1991, 266, 11621-11627). The enzyme has two
distinct structural domains. The major domain consists of four
.alpha. helices and one large .beta. sheet composed of three
antiparallel .beta. strands. The Mg.sup.2+ binding site is located
on the .beta. sheet and consists of three amino acids, Asp-10,
Glu-48, and Gly-11 (Katayanagi et al., Proteins: Struct., Funct.,
Genet., 1993, 17, 337-346). This structural motif of the Mg.sup.2+
binding site surrounded by .beta. strands is similar to that in
DNase I (Suck, D., and Oefner, C., Nature, 1986, 321, 620-625). The
minor domain is believed to constitute the predominant binding
region of the enzyme and is composed of an .alpha. helix
terminating with a loop. The loop region is composed of a cluster
of positively charged amino acids that are believed to bind
electrostatistically to the minor groove of the DNA/RNA
heteroduplex substrate. Although the conformation of the RNA/DNA
substrate can vary, from A-form to B-form depending on the sequence
composition, in general RNA/DNA heteroduplexes adopt an A-like
geometry (Pardi et al., Biochemistry, 1981, 20, 3986-3996; Hall, K.
B., and Mclaughlin, L. W., Biochemistry, 1991, 30, 10606-10613;
Lane et al., Eur. J. Biochem., 1993, 215, 297-306). The entire
binding interaction appears to comprise a single helical turn of
the substrate duplex. Recently the binding characteristics,
substrate requirements, cleavage products and effects of various
chemical modifications of the substrates on the kinetic
characteristics of E. coli RNase HI have been studied in more
detail (Crooke, S. T. et al., Biochem. J., 1995, 312, 599-608;
Lima, W. F. and Crooke, S. T., Biochemistry, 1997, 36, 390-398;
Lima, W. F. et al., J. Biol. Chem., 1997, 272, 18191-18199; Tidd,
D. M. and Worenius, H. M., Br. J. Cancer, 1989, 60, 343; Tidd, D.
M. et al., Anti-Cancer Drug Des., 1988, 3, 117.
[0008] In addition to RNase HI, a second E. coli RNase H, RNase HII
has been cloned and characterized (Itaya, M., Proc. Natl. Acad.
Sci. USA, 1990, 87, 8587-8591). It is comprised of 213 amino acids
while RNase HI is 155 amino acids long. E. coli RNase HII displays
only 17% homology with E. coli RNase HI. An RNase H cloned from S.
typhimurium differed from E. coli RNase HI in only 11 positions and
was 155 amino acids in length (Itaya, M. and Kondo K., Nucleic
Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet.,
1991, 227, 438-445). An enzyme cloned from S. cerevisiae was 30%
homologous to E. coli RNase HI (Itaya, M. and Kondo K., Nucleic
Acids Res., 1991, 19, 4443-4449; Itaya et al., Mol. Gen. Genet.,
1991, 227, 438-445). Thus, to date, no enzyme cloned from a species
other than E. coli has displayed substantial homology to E. coli
RNase H II.
[0009] Proteins that display RNase H activity have also been cloned
and purified from a number of viruses, other bacteria and yeast
(Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many
cases, proteins with RNase H activity appear to be fusion proteins
in which RNase H is fused to the amino or carboxy end of another
enzyme, often a DNA or RNA polymerase. The RNase H domain has been
consistently found to be highly homologous to E. coli RNase HI, but
because the other domains vary substantially, the molecular weights
and other characteristics of the fusion proteins vary widely.
[0010] In higher eukaryotes two classes of RNase H have been
defined based on differences in molecular weight, effects of
divalent cations, sensitivity to sulfhydryl agents and
immunological cross-reactivity (Busen et al., Eur. J. Biochem.,
1977, 74, 203-208). RNase H Type 1 enzymes are reported to have
molecular weights in the 68-90 kDa range, be activated by either
Mn.sup.2+ or Mg.sup.2+ and be insensitive to sulfhydryl agents. In
contrast, RNase H Type 2 enzymes have been reported to have
molecular weights ranging from 31-45 kDa, to require Mg.sup.2+, to
be highly sensitive to sulfhydryl agents and to be inhibited by
Mn.sup.2+ (Busen, W., and Hausen, P., Eur. J. Biochem., 1975, 52,
179-190; Kane, C. M., Biochemistry, 1988, 27, 3187-3196; Busen, W.,
J. Biol. Chem., 1982, 257, 7106-7108.).
[0011] An enzyme with Type 2 RNase H characteristics has been
purified to near homogeneity from human placenta (Frank et al.,
Nucleic Acids Res., 1994, 22, 5247-5254). This protein has a
molecular weight of approximately 33 kDa and is active in a pH
range of 6.5-10, with a pH optimum of 8.5-9. The enzyme requires
Mg.sup.2+ and is inhibited by Mn.sup.2+ and n-ethyl maleimide. The
products of cleavage reactions have 3' hydroxyl and 5' phosphate
termini.
[0012] Despite the substantial information about members of the
RNase family and the cloning of a number of viral, prokaryotic and
yeast genes with RNase H activity, until now, no mammalian RNase H
had been cloned. This has hampered efforts to understand the
structure of the enzyme(s), their distribution and the functions
they may serve.
[0013] In the present invention, cDNA of human RNase H with Type 2
characteristics and protein expressed thereby are provided.
SUMMARY OF THE INVENTION
[0014] The present invention provides polypeptides which have been
identified as novel human Type 2 RNases H by homology between the
amino acid sequence set forth in FIG. 1 and known amino acid
sequences of chicken, yeast and E. coli RNase H1 as well as an EST
deduced mouse RNase H homolog. In accordance with this aspect of
the present invention, as a preferred embodiment, a sample of E.
coli DH5.alpha. containing a BLUESCRIPT.sup.7 plasmid containing a
human cDNA nucleic acid molecule encoding a human Type 2 RNase H
polypeptide has been deposited as ATCC Deposit No. ATCC 98536.
[0015] The present invention also provides polynucleotides that
encode human Type 2 RNase H, vectors comprising nucleic acids
encoding human RNase H, host cells containing such vectors,
antibodies targeted to human Type 2 RNase H, human Type 2 RNase
H--his-tag fusion peptides, nucleic acid probes capable of
hybridizing to a nucleic acid encoding a human RNase H polypeptide.
Pharmaceutical compositions which include a human Type 2 RNase H
polypeptide or a vector encoding a human Type 2 RNase H polypeptide
are also provided. These compositions may additionally contain an
antisense oligonucleotide.
[0016] The present invention is also directed to methods of
enhancing antisense inhibition of expression of a target protein
via use of human Type 2 RNase H. Methods of screening for effective
antisense oligonucleotides and of producing effective antisense
oligonucleotides using human Type 2 RNase H are also provided.
[0017] Yet another object of the present invention is to provide
methods for identifying agents which modulate activity and/or
levels of human Type 2 RNase H. In accordance with this aspect, the
polynucleotides and polypeptides of the present invention are
useful for research, biological and clinical purposes. For example,
the polynucleotides and polypeptides are useful in defining the
interaction of human Type 2 RNase H and antisense oligonucleotides
and identifying means for enhancing this interaction so that
antisense oligonucleotides are more effective at inhibiting their
target mRNA.
[0018] Yet another object of the present invention is to provide a
method of prognosticating efficacy of antisense therapy of a
selected disease which comprises measuring the level or activity of
human RNase H in a target cell of the antisense therapy. Similarly,
oligonucleotides can be screened to identify those oligonucleotides
which are effective antisense agents by measuring binding of the
oligonucleotide to the human Type 2 RNase H.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 provides a human Type 2 RNase H primary sequence (286
amino acids; SEQ ID NO: 1) and sequence comparisons with chicken
(293 amino acids; SEQ ID NO: 2), yeast (348 amino acids; SEQ ID NO:
3) and E. coli RNase H1 (155 amino acids; SEQ ID NO: 4) as well as
an EST deduced mouse RNase H homolog (GenBank accession no.
AA389926 and AA518920; SEQ ID NO: 5). Boldface type indicates amino
acid residues identical to human. "@" indicates the conserved amino
acid residues implicated in E. coli RNase H1 Mg.sup.2+ binding site
and catalytic center (Asp-10, Gly-11, Glu-48 and Asp-70). "*"
indicates the conserved residues implicated in E. coli RNases H1
for substrate binding.
DETAILED DESCRIPTION OF THE INVENTION
[0020] A Type 2 human RNase H has now been cloned and expressed.
The enzyme encoded by this cDNA is inactive against single-stranded
RNA, single-stranded DNA and double-stranded DNA. However, this
enzyme cleaves the RNA in an RNA/DNA duplex and cleaves the RNA in
a duplex comprised of RNA and a chimeric oligonucleotide with 2'
methoxy flanks and a 5-deoxynucleotide center gap. The rate of
cleavage of the RNA duplexed with this so-called "deoxy gapmer" was
significantly slower than observed with the full RNA/DNA duplex.
These properties are consistent with those reported for E.coli
RNase H1 (Crooke et al., Biochem. J., 1995, 312, 599-608; Lima, W.
F. and Crooke, S. T., Biochemistry, 1997, 36, 390-398). They are
also consistent with the properties of a human Type 2 RNase H
protein purified from placenta, as the molecular weight (32 kDa) is
similar to that reported by Frank et al., Nucleic Acids Res., 1994,
22, 5247-5254) and the enzyme is inhibited by Mn.sup.2+.
Accordingly, we refer to the newly cloned human RNase H as Type 2
RNase H or human RNase H1.
[0021] Thus, in accordance with one aspect of the present
invention, there are provided isolated polynucleotides which encode
human Type 2 RNase H polypeptides. By "polynucleotides" it is meant
to include any form of RNA or DNA such as mRNA or cDNA or genomic
DNA, respectively, obtained by cloning or produced synthetically by
well known chemical techniques. DNA may be double- or
single-stranded. Single-stranded DNA may comprise the coding or
sense strand or the non-coding or antisense strand.
[0022] Methods of isolating a polynucleotide of the present
invention via cloning techniques are well known. For example, to
obtain the cDNA contained in ATCC Deposit No. 98536, primers based
on a search of the XREF database were used. An approximately 1 Kb
cDNA corresponding to the carboxy terminal portion of the protein
was cloned by 3' RACE. Seven positive clones were isolated by
screening a liver cDNA library with this 1 Kb cDNA. The two longest
clones were 1698 and 1168 base pairs. They share the same 5'
untranslated region and protein coding sequence but differ in the
length of the 3' UTR. A single reading frame encoding a 286 amino
acid protein (calculated mass: 32029.04 Da) was identified. The
proposed initiation codon is in agreement with the mammalian
translation initiation consensus sequence described by Kozak, M.,
J. Cell Biol., 1989, 108, 229-241, and is preceded by an in-frame
stop codon. Efforts to clone cDNA's with longer 5' UTR's from both
human liver and lymphocyte cDNA=s by 5=RACE failed, indicating that
the 1698-base-pair clone was full length.
[0023] In a preferred embodiment, the polynucleotide of the present
invention comprises the nucleic acid sequence of the cDNA contained
within ATCC Deposit No. 98536. The deposit of E. coli DH5.alpha.
containing a BLUESCRIPT.sup.7 plasmid containing a human Type 2
RNase H cDNA was made with the American Type Culture Collection,
12301 Park Lawn Drive, Rockville, Md. 20852, USA, on Sep. 4, 1997
and assigned ATCC Deposit No. 98536. The deposited material is a
culture of E. coli DH5.alpha. containing a BLUESCRIPT.sup.7 plasmid
(Stratagene, La Jolla Calif.) that contains the full-length human
Type 2 RNase H cDNA. The deposit has been made under the terms of
the Budapest Treaty on the international recognition of the deposit
of micro-organisms for the purposes of patent procedure. The
culture will be released to the public, irrevocably and without
restriction to the public upon issuance of this patent. The
sequence of the polynucleotide contained in the deposited material
and the amino acid sequence of the polypeptide encoded thereby are
controlling in the event of any conflict with the sequences
provided herein. However, as will be obvious to those of skill in
the art upon this disclosure, due to the degeneracy of the genetic
code, polynucleotides of the present invention may comprise other
nucleic acid sequences encoding the polypeptide and derivatives,
variants or active fragments thereof.
[0024] Another aspect of the present invention relates to the
polypeptides encoded by the polynucleotides of the present
invention. A polypeptide of the present invention comprises the
deduced amino acid sequence of human Type 2 RNase H provided in
FIG. 1 as SEQ ID NO: 1. However, by "polypeptide" it is also meant
to include fragments, derivatives and analogs which retain
essentially the same biological activity and/or function as human
Type 2 RNase H. Alternatively, polypeptides of the present
invention may retain their ability to bind to an antisense-RNA
duplex even though they do not function as active RNase H enzymes
in other capacities. In another embodiment, polypeptides of the
present invention may retain nuclease activity but without
specificity for the RNA portion of an RNA/DNA duplex. Polypeptides
of the present invention include recombinant polypeptides, isolated
natural polypeptides and synthetic polypeptides, and fragments
thereof which retain one or more of the activities described
above.
[0025] In a preferred embodiment, the polypeptide is prepared
recombinantly, most preferably from the culture of E. coli of ATCC
Deposit No. 98536. Recombinant human RNase H fused to histidine
codons (his-tag; in the present embodiment six histidine codons
were used) expressed in E.coli can be conveniently purified to
electrophoretic homogeneity by chromatography with Ni-NTA followed
by C4 reverse phase HPLC. The polypeptide of SEQ ID NO: 1 is highly
homologous to E.coli RNase H, displaying nearly 34% amino acid
identity with E.coli RNase H1. FIG. 1 compares a protein sequence
deduced from human RNase H cDNA (SEQ ID NO: 1) with those of
chicken (SEQ ID NO: 2), yeast (SEQ ID NO: 3) and E.coli RNase HI
(GenBank accession no. 1786408; SEQ ID NO: 4), as well as an EST
deduced mouse RNase H homolog (GenBank accession no. AA389926 and
AA518920; SEQ ID NO: 5). The deduced amino acid sequence of human
RNase H (SEQ ID NO: 1) displays strong homology with yeast (21.8%
amino acid identity), chicken (59%), E.coli RNase HI (33.6%) and
the mouse EST homolog (84.3%). They are all small proteins (<40
KDa) and their estimated pIs are all 8.7 and greater. Further, the
amino acid residues in E.coli RNase HI thought to be involved in
the Mg.sup.2+ binding site, catalytic center and substrate binding
region are completely conserved in the cloned human RNase H
sequence (FIG. 1).
[0026] The human Type 2 RNase H is expressed ubiquitously. Northern
blot analysis demonstrated that the transcript was abundant in all
tissues and cell lines except the MCR-5 line. Northern blot
analysis of total RNA from human cell lines and Poly A containing
RNA from human tissues using the 1.7 kb full length probe or a
332-nucleotide probe that contained the 5' UTR and coding region of
human RNase H cDNA revealed two strongly positive bands with
approximately 1.2 and 5.5 kb in length and two less intense bands
approximately 1.7 and 4.0 kb in length in most cell lines and
tissues. Analysis with the 332-nucleotide probe showed that the 5.5
kb band contained the 5' UTR and a portion of the coding region,
which suggests that this band represents a pre-processed or
partially processed transcript, or possibly an alternatively
spliced transcript. Intermediate sized bands may represent
processing intermediates. The 1.2 kb band represents the full
length transcripts. The longer transcripts may be processing
intermediates or alternatively spliced transcripts.
[0027] RNase H is expressed in most cell lines tested; only MRC5, a
breast cancer cell line, displayed very low levels of RNase H.
However, a variety of other malignant cell lines including those of
bladder (T24), breast (T-47D, HS578T), lung (A549), prostate
(LNCap, DU145), and myeloid lineage (HL-60), as well as normal
endothelial cells (HUVEC), expressed RNase H. Further, all normal
human tissues tested expressed RNase H. Again, larger transcripts
were present as well as the 1.2 kb transcript that appears to be
the mature mRNA for RNase H. Normalization based on G3PDH levels
showed that expression was relatively consistent in all of the
tissues tested.
[0028] The Southern blot analysis of EcoRI digested human and
various mammalian vertebrate and yeast genomic DNAs probed with the
1.7 kb probe shows that four EcoRI digestion products of human
genomic DNA (2.4, 4.6, 6.0, 8.0 Kb) hybridized with the 1.7 kb
probe. The blot re-probed with a 430 nucleotide probe corresponding
to the C-terminal portion of the protein showed only one 4.6 kbp
EcoRI digestion product hybridized. These data indicate that there
is only one gene copy for RNase H and that the size of the gene is
more than 10 kb. Both the full length and the shorter probe
strongly hybridized to one EcoRI digestion product of yeast genomic
DNA (about 5 kb in size), indicating a high degree of conservation.
These probes also hybridized to the digestion product from monkey,
but none of the other tested mammalian genomic DNAs including the
mouse which is highly homologous to the human RNase H sequence.
[0029] A recombinant human RNase H (his-tag fusion protein)
polypeptide of the present invention was expressed in E.coli and
purified by Ni-NTA agarose beads followed by C4 reverse phase
column chromatography. A 36 kDa protein copurified with activity
measured after renaturation. The presence of the his-tag was
confirmed by Western blot analyses with an anti-penta-histidine
antibody (Qiagen, Germany).
[0030] Renatured recombinant human RNase H displayed RNase H
activity. Incubation of 10 ng purified renatured RNase H with
RNA/DNA substrate for 2 hours resulted in cleavage of 40% of the
substrate. The enzyme also cleaved RNA in an oligonucleotide/RNA
duplex in which the oligonucleotide was a 2'-methoxy gapmer with a
5-deoxynucleotide gap, but at a much slower rate than the full
RNA/DNA substrate. This is consistent with observations with E.coli
RNase HI (Lima, W. F. and Crooke, S. T., Biochemistry, 1997, 36,
390-398). It was inactive against single-stranded RNA or
double-stranded RNA substrates and was inhibited by Mn.sup.2+. The
molecular weight (.about.36 kDa) and inhibition by Mn.sup.2+
indicate that the cloned enzyme is highly homologous to E.coli
RNase HI and has properties consistent with those assigned to Type
2 human RNase H.
[0031] The sites of cleavage in the RNA in the full RNA/DNA
substrate and the gapmer/RNA duplexes (in which the oligonucleotide
gapmer had a 5-deoxynucleotide gap) resulting from the recombinant
enzyme were determined. In the full RNA/DNA duplex, the principal
site of cleavage was near the middle of the substrate, with
evidence of less prominent cleavage sites 3' to the primary
cleavage site. The primary cleavage site for the gapmer/RNA duplex
was located across the nucleotide adjacent to the junction of the
2' methoxy wing and oligodeoxynucleotide gap nearest the 3' end of
the RNA. Thus, the enzyme resulted in a major cleavage site in the
center of the RNA/DNA substrate and less prominent cleavages to the
3' side of the major cleavage site. The shift of its major cleavage
site to the nucleotide in apposition to the DNA 2' methoxy junction
of the 2' methoxy wing at the 5' end of the chimeric
oligonucleotide is consistent with the observations for E. coli
RNase HI (Crooke et al. (1995) Biochem. J. 312, 599-608; Lima, W.
F. and Crooke, S. T. (1997) Biochemistry 36, 390-398). The fact
that the enzyme cleaves at a single site in a 5-deoxy gap duplex
indicates that the enzyme has a catalytic region of similar
dimensions to that of E.coli RNase HI.
[0032] Accordingly, expression of large quantities of a purified
human RNase H polypeptide of the present invention is useful in
characterizing the activities of a mammalian form of this enzyme.
In addition, the polynucleotides and polypeptides of the present
invention provide a means for identifying agents which enhance the
function of antisense oligonucleotides in human cells and
tissues.
[0033] For example, a host cell can be genetically engineered to
incorporate polynucleotides and express polypeptides of the present
invention. Polynucleotides can be introduced into a host cell using
any number of well known techniques such as infection,
transduction, transfection or transformation. The polynucleotide
can be introduced alone or in conjunction with a second
polynucleotide encoding a selectable marker. In a preferred
embodiment, the host comprises a mammalian cell. Such host cells
can then be used not only for production of human Type 2 RNase H,
but also to identify agents which increase or decrease levels of
expression or activity of human Type 2 RNase H in the cell. In
these assays, the host cell would be exposed to an agent suspected
of altering levels of expression or activity of human Type 2 RNase
in the cells. The level or activity of human Type 2 RNase in the
cell would then be determined in the presence and absence of the
agent. Assays to determine levels of protein in a cell are well
known to those of skill in the art and include, but are not limited
to, radioimmunoassays, competitive binding assays, Western blot
analysis and enzyme linked immunosorbent assays (ELISAs). Methods
of determining increase activity of the enzyme, and in particular
increased cleavage of an antisense-mRNA duplex can be performed in
accordance with the teachings of Example 5. Agents identified as
inducers of the level or activity of this enzyme may be useful in
enhancing the efficacy of antisense oligonucleotide therapies.
[0034] The present invention also relates to prognostic assays
wherein levels of RNase in a cell type can be used in predicting
the efficacy of antisense oligonucleotide therapy in specific
target cells. High levels of RNase in a selected cell type are
expected to correlate with higher efficacy as compared to lower
amounts of RNase in a selected cell type which may result in poor
cleavage of the mRNA upon binding with the antisense
oligonucleotide. For example, the MRC5 breast cancer cell line
displayed very low levels of RNase H as compared to other malignant
cell types. Accordingly, in this cell type it may be desired to use
antisense compounds which do not depend on RNase H activity for
their efficacy.
[0035] Similarly, oligonucleotides can be screened to identify
those which are effective antisense agents by contacting human Type
2 RNase H with an oligonucleotide and measuring binding of the
oligonucleotide to the human Type 2 RNase H. Methods of determining
binding of two molecules are well known in the art. For example, in
one embodiment, the oligonucleotide can be radiolabeled and binding
of the oligonucleotide to human Type 2 RNase H can be determined by
autoradiography. Alternatively, fusion proteins of human Type 2
RNase H with glutathione-S-transferase or small peptide tags can be
prepared and immobilized to a solid phase such as beads. Labeled or
unlabeled oligonucleotides to be screened for binding to this
enzyme can then be incubated with the solid phase. Oligonucleotides
which bind to the enzyme immobilized to the solid phase can then be
identified either by detection of bound label or by eluting
specifically the bound oligonucleotide from the solid phase.
Another method involves screening of oligonucleotide libraries for
binding partners. Recombinant tagged or labeled human Type 2 RNase
H is used to select oligonucleotides from the library which
interact with the enzyme. Sequencing of the oligonucleotides leads
to identification of those oligonucleotides which will be more
effective as antisense agents.
[0036] The following nonlimiting examples are provided to further
illustrate the present invention.
EXAMPLES
Example 1
Rapid Amplification of 5'-cDNA end (5' BRACE) and 3'-cDNA end
(3'-RACE)
[0037] An internet search of the XREF database in the National
Center of Biotechnology Information (NCBI) yielded a 361 base pair
(bp) human expressed sequenced tag (EST, GenBank accession
#H28861), homologous to yeast RNase H (RNH1) protein sequenced tag
(EST, GenBank accession #Q04740) and its chicken homologue
(accession #D26340). Three sets of oligonucleotide primers encoding
the human RNase H EST sequence were synthesized. The sense primers
were ACGCTGGCCGGGAGTCGAAATGCTTC (H1: SEQ ID NO: 6),
CTGTTCCTGGCCCACAGAGTCGCCTTGG (H3: SEQ ID NO: 7) and
GGTCTTTCTGACCTGGAATGAGTGCAGAG (H5: SEQ ID NO: 8). The antisense
primers were CTTGCCTGGTTTCGCCCTCCGATTCTTGT (H2: SEQ ID NO: 9),
TTGATTTTCATGCCCTTCTGAAACTTCCG (H4; SEQ ID NO: 10) and
CCTCATCCTCTATGGCAAACTTCTTAAATCTGGC (H6; SEQ ID NO: 11). The human
RNase H 3' and 5' cDNAs derived from the EST sequence were
amplified by polymerase chain reaction (PCR), using human liver or
leukemia (lymphoblastic Molt-4) cell line Marathon ready cDNA as
templates, H1 or H3/AP1 as well as H4 or H6/AP2 as primers
(Clontech, Palo Alto, Calif.). The fragments were subjected to
agarose gel electrophoresis and transferred to nitrocellulose
membrane (Bio-Rad, Hercules, Calif.) for confirmation by Southern
blot, using .sup.32P-labeled H2 and 1 probes (for 3' and 5' RACE
products, respectively, in accordance with procedures described by
Ausubel et al., Current Protocols in Molecular Biology, Wiley and
Sons, New York, N.Y., 1988. The confirmed fragments were excised
from the agarose gel and purified by gel extraction (Qiagen,
Germany), then subcloned into Zero-blunt vector (Invitrogen,
Carlsbad, Calif.) and subjected to DNA sequencing.
Example 2
Screening of the cDNA Library, DNA Sequencing and Sequence
Analysis
[0038] A human liver cDNA lambda phage Uni-ZAP library (Stratagene,
La Jolla, Calif.) was screened using the RACE products as specific
probes. The positive cDNA clones were excised into the pBluescript
phagemid (Stratagene, La Jolla Calif.) from lambda phage and
subjected to DNA sequencing with an automatic DNA sequencer
(Applied Biosystems, Foster City, Calif.) by Retrogen Inc. (San
Diego, Calif.). The overlapping sequences were aligned and combined
by the assembling programs of MacDNASIS v3.0 (Hitachi Software
Engineering America, South San Francisco, Calif.). Protein
structure and subsequence analysis were performed by the program of
MacVector 6.0 (Oxford Molecular Group Inc., Campbell, Calif.). A
homology search was performed on the NCBI database by internet
E-mail.
Example 3
Northern Blot and Southern Blot Analysis
[0039] Total RNA from different human cell lines (ATCC, Rockville,
Md.) was prepared and subjected to formaldehyde agarose gel
electrophoresis in accordance with procedures described by Ausubel
et al., Current Protocols in Molecular Biology, Wiley and Sons, New
York, N.Y., 1988, and transferred to nitrocellulose membrane
(Bio-Rad, Hercules Calif.). Northern blot hybridization was carried
out in QuickHyb buffer (Stratagene, La Jolla, Calif.) with
.sup.32P-labeled probe of full length RNase H cDNA clone or primer
H1/H2 PCR-generated 322-base N-terminal RNase H cDNA fragment at
68.degree. for 2 hours. The membranes were washed twice with 0.1%
SSC/0.1% SDS for 30 minutes and subjected to auto-radiography.
Southern blot analysis was carried out in
1.times.pre-hybridization/hybridization buffer (BRL, Gaithersburg,
Md.) with a .sup.32P-labeled 430 bp C-terminal restriction enzyme
PstI/PvuII fragment or 1.7 kb full length cDNA probe at 60.degree.
C. for 18 hours. The membranes were washed twice with 0.1% SSC/0.1%
SDS at 60.degree. C. for 30 minutes, and subjected to
autoradiography.
Example 4
Expression and Purification of the Cloned RNase Protein
[0040] The cDNA fragment coding the full RNase H protein sequence
was amplified by PCR using 2 primers, one of which contains
restriction enzyme NdeI site adapter and six histidine (his-tag)
codons and 22 bp protein N terminal coding sequence. The fragment
was cloned into expression vector pET17b (Novagen, Madison, Wis.)
and confirmed by DNA sequencing. The plasmid was transfected into
E.coli BL21(DE3) (Novagen, Madison, Wis.). The bacteria were grown
in M9ZB medium at 32EC and harvested when the OD.sub.600 of the
culture reached 0.8, in accordance with procedures described by
Ausubel et al., Current Protocols in Molecular Biology, Wiley and
Sons, New York, N.Y., 1988. Cells were lysed in 8M urea solution
and recombinant protein was partially purified with Ni-NTA agarose
(Qiagen, Germany). Further purification was performed with C4
reverse phase chromatography (Beckman, System Gold, Fullerton,
Calif.) with 0.1% TFA water and 0.1% TFA acetonitrile gradient of
0% to 80% in 40 minutes as described by Deutscher, M. P., Guide to
Protein Purification, Methods in Enzymology 182, Academic Press,
New York, N.Y., 1990. The recombinant proteins and control samples
were collected, lyophilized and subjected to 12% SDS-PAGE as
described by Ausubel et al. (1988) Current Protocols in Molecular
Biology, Wiley and Sons, New York, N.Y. The purified protein and
control samples were resuspended in 6 M urea solution containing 20
mM Tris HCl, pH 7.4, 400 mM NaCl, 20% glycerol, 0.2 mM PMSF, 5 mM
DTT, 10 .mu.g/ml aprotinin and leupeptin, and refolded by dialysis
with decreasing urea concentration from 6 M to 0.5 M as well as DTT
concentration from 5 mM to 0.5 mM as described by Deutscher, M. P.,
Guide to Protein Purification, Methods in Enzymology 182, Academic
Press, New York, N.Y., 1990. The refolded proteins were
concentrated (10 fold) by Centricon (Amicon, Danvers, Mass.) and
subjected to RNase H activity assay.
Example 5
RNase H Activity Assay
[0041] .sup.32P-end-labeled 17-mer RNA, GGGCGCCGTCGGTGTGG (SEQ ID
NO: 12) described by Lima, W. F. and Crooke, S. T., Biochemistry,
1997 36, 390-398, was gel-purified as described by Ausubel et al.,
Current Protocols in Molecular Biology, Wiley and Sons, New York,
N.Y., 1988 and annealed with a tenfold excess of its complementary
17-mer oligodeoxynucleotide or a 5-base DNA gapmer, i.e., a 17mer
oligonucleotide which has a central portion of 5 deoxynucleotides
(the "gap") flanked on both sides by 6 2N-methoxynucleotides.
Annealing was done in 10 mM Tris HCl, pH 8.0, 10 mM MgCl, 50 mM KCl
and 0.1 mM DTT to form one of three different substrates: (a)
single strand (ss) RNA probe, (b) full RNA/DNA duplex and (c)
RNA/DNA gapmer duplex. Each of these substrates was incubated with
protein samples at 37.degree. C. for 5 minutes to 2 hours at the
same conditions used in the annealing procedure and the reactions
were terminated by adding EDTA in accordance with procedures
described by Lima, W. F. and Crooke, S. T., Biochemistry, 1997, 36,
390-398. The reaction mixtures were precipitated with TCA
centrifugation and the supernatant was measured by liquid
scintillation counting (Beckman LS6000IC, Fullerton, Calif.). An
aliquot of the reaction mixture was also subjected to denaturing (8
M urea) acrylamide gel electrophoresis in accordance with
procedures described by Lima, W. F. and Crooke, S. T.,
Biochemistry, 1997, 36, 390-398 and Ausubel et al., Current
Protocols in Molecular Biology, Wiley and Sons, New York, N.Y.,
1988.
Sequence CWU 1
1
12 1 286 PRT Homo sapiens 1 Met Ser Trp Leu Leu Phe Leu Ala His Arg
Val Ala Leu Ala Ala Leu 1 5 10 15 Pro Cys Arg Arg Gly Ser Arg Gly
Phe Gly Met Phe Tyr Ala Val Arg 20 25 30 Arg Gly Arg Lys Thr Gly
Val Phe Leu Thr Trp Asn Glu Cys Arg Ala 35 40 45 Gln Val Asp Arg
Phe Pro Ala Ala Arg Phe Lys Lys Phe Ala Thr Glu 50 55 60 Asp Glu
Ala Trp Ala Phe Val Arg Lys Ser Ala Ser Pro Glu Val Ser 65 70 75 80
Glu Gly His Glu Asn Gln His Gly Gln Glu Ser Glu Ala Lys Pro Gly 85
90 95 Lys Arg Leu Arg Glu Pro Leu Asp Gly Asp Gly His Glu Ser Ala
Gln 100 105 110 Pro Tyr Ala Lys His Met Lys Pro Ser Val Glu Pro Ala
Pro Pro Val 115 120 125 Ser Arg Asp Thr Phe Ser Tyr Met Gly Asp Phe
Val Val Val Tyr Thr 130 135 140 Asp Gly Cys Cys Ser Ser Asn Gly Arg
Arg Lys Pro Arg Ala Gly Ile 145 150 155 160 Gly Val Tyr Trp Gly Pro
Gly His Pro Leu Asn Val Gly Ile Arg Leu 165 170 175 Pro Gly Arg Gln
Thr Asn Gln Arg Ala Glu Ile His Ala Ala Cys Lys 180 185 190 Ala Ile
Glu Gln Ala Lys Thr Gln Asn Ile Asn Lys Leu Val Leu Tyr 195 200 205
Thr Asp Ser Met Phe Thr Ile Asn Gly Ile Thr Asn Trp Val Gln Gly 210
215 220 Trp Lys Lys Asn Gly Trp Lys Thr Ser Ala Gly Lys Glu Val Ile
Asn 225 230 235 240 Lys Glu Asp Phe Val Ala Leu Glu Arg Leu Thr Gln
Gly Met Asp Ile 245 250 255 Gln Trp Met His Val Pro Gly His Ser Gly
Phe Ile Gly Asn Glu Glu 260 265 270 Ala Asp Arg Leu Ala Arg Glu Gly
Ala Lys Gln Ser Glu Asp 275 280 285 2 293 PRT Gallus sp. 2 Met Leu
Arg Trp Leu Val Ala Leu Leu Ser His Ser Cys Phe Val Ser 1 5 10 15
Lys Gly Gly Gly Met Phe Tyr Ala Val Arg Lys Gly Arg Gln Thr Gly 20
25 30 Val Tyr Arg Thr Trp Ala Glu Cys Gln Gln Gln Val Asn Arg Phe
Pro 35 40 45 Ser Ala Ser Phe Lys Lys Phe Ala Thr Glu Lys Glu Ala
Trp Ala Phe 50 55 60 Val Gly Ala Gly Pro Pro Asp Gly Gln Gln Ser
Ala Pro Ala Glu Thr 65 70 75 80 His Gly Ala Ser Ala Val Ala Gln Glu
Asn Ala Ser His Arg Glu Glu 85 90 95 Pro Glu Thr Asp Val Leu Cys
Cys Asn Ala Cys Lys Arg Arg Tyr Glu 100 105 110 Gln Ser Thr Asn Glu
Glu His Thr Val Arg Arg Ala Lys His Asp Glu 115 120 125 Glu Gln Ser
Thr Pro Val Val Ser Glu Ala Lys Phe Ser Tyr Met Gly 130 135 140 Glu
Phe Ala Val Val Tyr Thr Asp Gly Cys Cys Ser Gly Asn Gly Arg 145 150
155 160 Asn Arg Ala Arg Ala Gly Ile Gly Val Tyr Trp Gly Pro Gly His
Pro 165 170 175 Leu Asn Ile Ser Glu Arg Leu Pro Gly Arg Gln Thr Asn
Gln Arg Ala 180 185 190 Glu Ile His Ala Ala Cys Lys Ala Ile Glu Gln
Ala Lys Ser Gln Asn 195 200 205 Ile Lys Lys Leu Ile Ile Tyr Thr Asp
Ser Lys Phe Thr Ile Asn Gly 210 215 220 Ile Thr Ser Trp Val Glu Asn
Trp Lys Thr Asn Gly Trp Arg Thr Ser 225 230 235 240 Ser Gly Gly Ser
Val Ile Asn Lys Glu Asp Phe Gln Lys Leu Asp Ser 245 250 255 Leu Ser
Lys Gly Ile Glu Ile Gln Trp Met His Ile Pro Gly His Ala 260 265 270
Gly Phe Gln Gly Asn Glu Glu Ala Asp Arg Leu Ala Arg Glu Gly Ala 275
280 285 Ser Lys Gln Lys Leu 290 3 348 PRT Saccharomyces sp. 3 Met
Ala Arg Gln Gly Asn Phe Tyr Ala Val Arg Lys Gly Arg Glu Thr 1 5 10
15 Gly Ile Tyr Asn Thr Trp Asn Glu Cys Lys Asn Gln Val Asp Gly Tyr
20 25 30 Gly Gly Ala Ile Tyr Lys Lys Phe Asn Ser Tyr Glu Gln Ala
Lys Ser 35 40 45 Phe Leu Gly Gln Pro Asn Thr Thr Ser Asn Tyr Gly
Ser Ser Thr His 50 55 60 Ala Gly Gly Gln Val Ser Lys Pro His Thr
Thr Gln Lys Arg Val His 65 70 75 80 Arg Arg Asn Arg Pro Leu His Tyr
Ser Ser Leu Thr Ser Ser Ser Ala 85 90 95 Cys Ser Ser Leu Ser Ser
Ala Asn Thr Asn Thr Phe Tyr Ser Val Lys 100 105 110 Ser Asn Val Pro
Asn Ile Glu Ser Lys Ile Phe Asn Asn Trp Lys Asp 115 120 125 Cys Gln
Ala Tyr Val Lys His Lys Arg Gly Ile Thr Phe Lys Lys Phe 130 135 140
Glu Asp Gln Leu Ala Ala Glu Asn Phe Ile Ser Gly Met Ser Ala His 145
150 155 160 Asp Tyr Lys Leu Met Asn Ile Ser Lys Glu Ser Phe Glu Ser
Lys Tyr 165 170 175 Lys Leu Ser Ser Asn Thr Met Tyr Asn Lys Ser Met
Asn Val Tyr Cys 180 185 190 Asp Gly Ser Ser Phe Gly Asn Gly Thr Ser
Ser Ser Arg Ala Gly Tyr 195 200 205 Gly Ala Tyr Phe Glu Gly Ala Pro
Glu Glu Asn Ile Ser Glu Pro Leu 210 215 220 Leu Ser Gly Ala Gln Thr
Asn Asn Arg Ala Glu Ile Glu Ala Val Ser 225 230 235 240 Glu Ala Leu
Lys Lys Ile Trp Glu Lys Leu Thr Asn Glu Lys Glu Lys 245 250 255 Val
Asn Tyr Gln Ile Lys Thr Asp Ser Glu Tyr Val Thr Lys Leu Leu 260 265
270 Asn Asp Arg Tyr Met Thr Tyr Asp Asn Lys Lys Leu Glu Gly Leu Pro
275 280 285 Asn Ser Asp Leu Ile Val Pro Leu Val Gln Arg Phe Val Lys
Val Lys 290 295 300 Lys Tyr Tyr Glu Leu Asn Lys Glu Cys Phe Lys Asn
Asn Gly Lys Phe 305 310 315 320 Gln Ile Glu Trp Val Lys Gly His Asp
Gly Asp Pro Gly Asn Glu Met 325 330 335 Ala Asp Phe Leu Ala Lys Lys
Gly Ala Ser Arg Arg 340 345 4 155 PRT Escherichia coli 4 Met Leu
Lys Gln Val Glu Ile Phe Thr Asp Gly Ser Cys Leu Gly Asn 1 5 10 15
Pro Gly Pro Gly Gly Tyr Gly Ala Ile Leu Arg Tyr Arg Gly Arg Glu 20
25 30 Lys Thr Phe Ser Ala Gly Tyr Thr Arg Thr Thr Asn Asn Arg Met
Glu 35 40 45 Leu Met Ala Ala Ile Val Ala Leu Glu Ala Leu Lys Glu
His Cys Glu 50 55 60 Val Ile Leu Ser Thr Asp Ser Gln Tyr Val Arg
Gln Gly Ile Thr Gln 65 70 75 80 Trp Ile His Asn Trp Lys Lys Arg Gly
Trp Lys Thr Ala Asp Lys Lys 85 90 95 Pro Val Lys Asn Val Asp Leu
Trp Gln Arg Leu Asp Ala Ala Leu Gly 100 105 110 Gln His Gln Ile Lys
Trp Glu Trp Val Lys Gly His Ala Gly His Pro 115 120 125 Glu Asn Glu
Arg Cys Asp Glu Leu Ala Arg Ala Ala Ala Met Asn Pro 130 135 140 Thr
Leu Glu Asp Thr Gly Tyr Gln Val Glu Val 145 150 155 5 216 PRT Mus
musculus 5 Gly Ile Cys Gly Leu Gly Met Phe Tyr Ala Val Arg Arg Gly
Arg Arg 1 5 10 15 Pro Gly Val Phe Leu Ser Trp Ser Glu Cys Lys Ala
Gln Val Asp Arg 20 25 30 Phe Pro Ala Ala Arg Phe Lys Lys Phe Ala
Thr Glu Asp Glu Ala Trp 35 40 45 Ala Phe Val Arg Ser Ser Ser Ser
Pro Asp Gly Ser Lys Gly Gln Glu 50 55 60 Ser Ala His Glu Gln Lys
Ser Gln Ala Lys Thr Ser Lys Arg Pro Arg 65 70 75 80 Glu Pro Leu Val
Val Val Tyr Thr Asp Gly Cys Cys Ser Ser Asn Gly 85 90 95 Arg Lys
Arg Ala Arg Ala Gly Ile Gly Val Tyr Trp Gly Pro Gly His 100 105 110
Pro Leu Asn Val Arg Ile Arg Leu Pro Gly Arg Gln Thr Asn Gln Arg 115
120 125 Ala Glu Ile His Ala Ala Cys Lys Ala Val Met Gln Ala Lys Ala
Gln 130 135 140 Asn Ile Ser Lys Leu Val Leu Tyr Thr Asp Ser Met Phe
Thr Ile Asn 145 150 155 160 Gly Ile Thr Asn Trp Val Gln Gly Trp Lys
Lys Asn Gly Trp Arg Thr 165 170 175 Ser Thr Gly Lys Asp Val Ile Asn
Lys Glu Asp Phe Met Glu Leu Asp 180 185 190 Glu Leu Thr Gln Gly Met
Asp Ile Gln Trp Met His Ile Pro Gly His 195 200 205 Ser Gly Phe Val
Gly Asn Glu Glu 210 215 6 26 DNA Artificial Sequence Description of
Artificial SequenceSynthetic 6 acgctggccg ggagtcgaaa tgcttc 26 7 28
DNA Artificial Sequence Description of Artificial SequenceSynthetic
7 ctgttcctgg cccacagagt cgccttgg 28 8 29 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 8 ggtctttctg acctggaatg
agtgcagag 29 9 29 DNA Artificial Sequence Description of Artificial
SequenceSynthetic 9 cttgcctggt ttcgccctcc gattcttgt 29 10 29 DNA
Artificial Sequence Description of Artificial SequenceSynthetic 10
ttgattttca tgcccttctg aaacttccg 29 11 34 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 11 cctcatcctc
tatggcaaac ttcttaaatc tggc 34 12 17 DNA Artificial Sequence
Description of Artificial SequenceSynthetic 12 gggcgccgtc ggtgtgg
17
* * * * *