U.S. patent application number 10/359385 was filed with the patent office on 2003-07-31 for human rna binding proteins.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Bandman, Olga, Baughn, Mariah R., Corley, Neil C., Guegler, Karl J., Lu, Dyung Aina M., Tang, Y. Tom.
Application Number | 20030143622 10/359385 |
Document ID | / |
Family ID | 22645291 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143622 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
July 31, 2003 |
Human RNA binding proteins
Abstract
The invention provides human RNA binding proteins (RNABP) and
polynucleotides which identify and encode RNABP. The invention also
provides expression vectors, host cells, antibodies, agonists, and
antagonists. The invention also provides methods for diagnosing,
treating or preventing disorders associated with expression of
RNABP.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Tang, Y. Tom; (San Jose, CA) ;
Corley, Neil C.; (Castro Valley, CA) ; Guegler, Karl
J.; (Menlo Park, CA) ; Lu, Dyung Aina M.; (San
Jose, CA) ; Baughn, Mariah R.; (San Leandro,
CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
22645291 |
Appl. No.: |
10/359385 |
Filed: |
February 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10359385 |
Feb 5, 2003 |
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09421299 |
Oct 20, 1999 |
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6524579 |
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09421299 |
Oct 20, 1999 |
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09176657 |
Oct 21, 1998 |
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6020164 |
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Current U.S.
Class: |
435/6.14 ;
435/199; 435/320.1; 435/325; 435/6.16; 435/69.1; 530/358;
536/23.2 |
Current CPC
Class: |
A61P 37/06 20180101;
C07K 14/47 20130101; C07K 14/43545 20130101; C07K 14/43581
20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/199; 435/320.1; 435/325; 530/358; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/22; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1-3, b) a polypeptide comprising
a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-3, c) a biologically active fragment of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-3, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-3.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:4-6.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID
NO:1-3.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, b) a
polynucleotide comprising a naturally occurring polynucleotide
sequence at least 85% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, c) a
polynucleotide complementary to a polynucleotide of a), d) a
polynucleotide complementary to a polynucleotide of b), and e) an
RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim i and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-3.
19. A method for treating a disease or condition associated with
decreased expression of functional RNABP, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional RNABP, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional RNABP, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A method for a diagnostic test for a condition or disease
associated with the expression of RNABP in a biological sample, the
method comprising: a) combining the biological sample with an
antibody of claim 11, under conditions suitable for the antibody to
bind the polypeptide and form an antibody:polypeptide complex, and
b) detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of RNABP in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, further comprising a label.
35. A method of diagnosing a condition or disease associated with
the expression of RNABP in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibodies from the animal, and c)
screening the isolated antibodies with the polypeptide, thereby
identifying a polyclonal antibody which specifically binds to a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-3, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibody producing cells from the
animal, c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells, d)
culturing the hybridoma cells, and e) isolating from the culture
monoclonal antibody which specifically binds to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-3.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3 in a
sample, the method comprising: a) incubating the antibody of claim
11 with the sample under conditions to allow specific binding of
the antibody and the polypeptide, and b) detecting specific
binding, wherein specific binding indicates the presence of a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3 from a
sample, the method comprising: a) incubating the antibody of claim
11 with the sample under conditions to allow specific binding of
the antibody and the polypeptide, and b) separating the antibody
from the sample and obtaining the purified polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-3.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
58. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
59. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
60. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:5.
61. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:6.
Description
[0001] This application is a DIVISIONAL of pending prior
application U.S. Ser. No. 09/421,299, filed on Oct. 20, 1999, which
in turn is a DIVISIONAL of prior application U.S. Ser. No.
09/176,657, filed on Oct. 21, 1998, now issued as U.S. Pat. No.
6,020,164 on Feb. 1, 2000, both entitled HUMAN RNA BINDING
PROTEINS.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human RNA binding proteins and to the use of these
sequences in the diagnosis, treatment, and prevention of cancer,
immune disorders, and developmental disorders.
BACKGROUND OF THE INVENTION
[0003] The translation of genetic information into protein depends
on RNA as a means for storing and decoding DNA polynucleotide
sequences. The first step in this process is the transcription of
DNA into RNA which is chemically similar to DNA and retains all the
genetic information encoded in DNA. The RNA transcript undergoes
various processing steps which include splicing and
polyadenylation. The mature RNA transcript, called messenger RNA
(mRNA), is translated into protein by the ribosomal machinery.
[0004] Nascent RNA transcripts are spliced in the nucleus by the
spliceosomal complex which catalyzes the removal of introns and the
rejoining of exons. The spliceosomal complex is comprised of five
small nuclear ribonucleoprotein particles (snRNPs) designated U1,
U2, U4, U5, and U6. Each snRNP contains a single species of RNA and
about 10 proteins. The RNA components of some snRNPs recognize and
base pair with intron consensus sequences. The protein components
mediate spliceosome assembly and the splicing reaction. snRNP
proteins and other nuclear RNA binding proteins are generally
referred to as RNPs and are characterized by an RNA recognition
motif (RRM). (Reviewed in Birney, E. et al. (1993) Nucleic Acids
Res. 21:5803-5816.) The RRM is about 80 amino acids in length and
forms four .beta.-strands and two a-helices arranged in an
.alpha./.beta. sandwich. The RRM contains a core RNP-1 octapeptide
motif along with surrounding conserved sequences. In addition to
snRNP proteins, examples of RNA-binding proteins which contain the
above motifs include heteronuclear ribonucleoproteins which
stabilize nascent RNA and factors which regulate alternative
splicing. Alternative splicing factors include developmentally
regulated proteins which have been identified in lower eukaryotes
such as Drosophila melanogaster and Caenorhabditis elegans. These
proteins play key roles in developmental processes such as pattern
formation and sex determination, respectively. (See, for example,
Hodgkin, J. et al. (1994) Development 120:3681-3689.)
[0005] Although most RNPs contain an RRM or RNP-1 motif, there are
exceptions. The A' polypeptide is a unique component of the U2
snRNP that does not contain these motifs (Sillekens, P. T. et al.
(1989) Nucleic Acids Res. 17:1893-1906). A' is 255 amino acids in
length with a predicted molecular weight of 28,444 daltons. Notable
features of A' include a leucine-rich amino-terminal half and an
extremely hydrophilic carboxy-terminal half. The latter region may
be involved in RNA binding, while the former region may mediate
protein-protein interactions.
[0006] In addition to splicing, aspects of RNA metabolism include
alteration and regulation of RNA conformation and secondary
structure. These processes are mediated by RNA helicases which
utilize energy derived from ATP hydrolysis to destabilize and
unwind RNA duplexes. The most well-characterized and ubiquitous
family of RNA helicases is the DEAD-box family, so named for the
conserved B-type ATP-binding motif which is diagnostic of proteins
in this family. Over 40 DEAD-box helicases have been identified in
organisms as diverse as bacteria, insects, yeast, amphibians,
mammals, and plants. For example, a recent addition to the DEAD-box
family is the RNA helicase encoded by the Drosophila hlc gene
(Maleszka, R. et al. (1998) Proc. Natl. Acad. Sci. USA
95:3731-3736). DEAD-box helicases function in diverse processes
such as translation initiation, splicing, ribosome assembly, and
RNA editing, transport, and stability. Some DEAD-box helicases play
tissue- and stage-specific roles in spermatogenesis and
embryogenesis. All DEAD-box helicases contain several conserved
sequence motifs spread out over about 420 amino acids. These motifs
include an A-type ATP binding motif, the DEAD-box/B-type
ATP-binding motif, a serine/arginine/threonine tripeptide of
unknown function, and a C-terminal glycine-rich motif with a
possible role in substrate binding and unwinding. In addition,
alignment of divergent DEAD-box helicase sequences has shown that
37 amino acid residues are identical among these sequences,
suggesting that conservation of these residues is important for
helicase function. (Reviewed in Linder, P. et al. (1989) Nature
337:121-122.)
[0007] Overexpression of the DEAD-box I protein (DDX1) may play a
role in the progression of neuroblastoma (Nb) and retinoblastoma
(Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem.
273:21161-21168). Nb and Rb tumor progression is promoted by the
amplification of the proto-oncogene encoding MYCN, a transcription
factor. However, amplification of both the MYCN gene and the DDX1
gene, which maps in proximity to the MYCN gene on chromosome 2, is
correlated with significantly higher rates of tumor progression.
Amplification of the DDX1 gene results in increased levels of DDX1
RNA and protein, the latter being aberrantly localized in Nb and Rb
cells. These observations suggest that DDX1 may promote or enhance
tumor progression by altering the normal secondary structure and
expression levels of RNA in cancer cells. In addition, cancer cells
that have amplified both DDX1 and MYCN genes may have a selective
advantage over cancer cells that have amplified only the MYCN
gene.
[0008] Other DEAD-box helicases have been implicated either
directly or indirectly in tumorigenesis. (Discussed in Godbout,
supra.) For example, murine p68 is mutated in ultraviolet
light-induced tumors, and human DDX6 is located at a chromosomal
breakpoint associated with B-cell lymphoma. Similarly, a chimeric
protein comprised of DDX10 and NUP98, a nucleoporin protein, may be
involved in the pathogenesis of certain myeloid malignancies.
[0009] The discovery of new human RNA binding proteins and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer, immune disorders, and
developmental disorders.
SUMMARY OF THE INVENTION
[0010] The invention features substantially purified polypeptides,
human RNA binding proteins, referred to collectively as RNABP and
individually as "RNABP-1," "RNABP-2," and "RNABP-3." In one aspect,
the invention provides a substantially purified polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 (SEQ ID
NO:1-3), and fragments thereof.
[0011] The invention further provides a substantially purified
variant having at least 90% amino acid identity to at least one of
the amino acid sequences selected from the group consisting of SEQ
ID NO:1-3 and fragments thereof. The invention also provides an
isolated and purified polynucleotide encoding the polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1-3 and fragments thereof. The invention
also includes an isolated and purified polynucleotide variant
having at least 70% polynucleotide sequence identity to the
polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3 and
fragments thereof.
[0012] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:1-3
and fragments thereof. The invention also provides an isolated and
purified polynucleotide having a sequence which is complementary to
the polynucleotide encoding the polypeptide comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:1-3
and fragments thereof.
[0013] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 (SEQ
ID NO:4-6), and fragments thereof. The invention further provides
an isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity to the polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6 and fragments
thereof. The invention also provides an isolated and purified
polynucleotide having a sequence which is complementary to the
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:4-6 and fragments thereof.
[0014] The invention also provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of (a) hybridizing the complement of the
polynucleotide sequence to at least one of the polynucleotides of
the sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide in the sample. In one aspect, the method further
comprises amplifying the polynucleotide prior to hybridization.
[0015] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3 and fragments thereof. In another
aspect, the expression vector is contained within a host cell.
[0016] The invention also provides a method for producing a
polypeptide, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide under conditions suitable for the
expression of the polypeptide; and (b) recovering the polypeptide
from the host cell culture.
[0017] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO:1-3
and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
[0018] The invention further includes a purified antibody which
binds to a polypeptide selected from the group consisting of SEQ ID
NO:1-3 and fragments thereof. The invention also provides a
purified agonist and a purified antagonist to the polypeptide.
[0019] The invention also provides a method for treating or
preventing a disorder associated with decreased expression or
activity of RNABP, the method comprising administering to a subject
in need of such treatment an effective amount of a pharmaceutical
composition comprising a substantially purified polypeptide having
the amino acid sequence selected from the group consisting of SEQ
ID NO:1-3 and fragments thereof, in conjunction with a suitable
pharmaceutical carrier.
[0020] The invention also provides a method for treating or
preventing a disorder associated with increased expression or
activity of RNABP, the method comprising administering to a subject
in need of such treatment an effective amount of an antagonist of a
polypeptide having an amino acid sequence selected from the group
consisting of SEQ ID NO:1-3 and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0021] FIGS. 1A, 1B, and 1C show the amino acid sequence alignment
between RNABP-1 (1229372; SEQ ID NO:1) and hlc-encoded RNA helicase
(GI 3378056; SEQ ID NO:7). The alignment was produced using the
multisequence alignment program of LASERGENE software (DNASTAR,
Madison Wis.).
[0022] FIG. 2 shows the amino acid sequence alignment between amino
acids 110 to 198 of RNABP-2 (1250374; SEQ ID NO:2) and amino acids
152 to 238 of sex determination-associated RNP (GI 608464; SEQ ID
NO:8).
[0023] FIGS. 3A and 3B show the amino acid sequence alignment
between RNABP-3 (1710966; SEQ ID NO:3) and U2 snRNP A' (61 37547;
SEQ ID. NO:9).
[0024] Table 1 shows the programs, their descriptions, references,
and threshold parameters used to analyze RNABP.
DESCRIPTION OF THE INVENTION
[0025] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present
invention, the preferred machines, materials and methods are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, protocols,
reagents and vectors which are reported in the publications and
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
[0028] Definitions
[0029] "RNABP" refers to the amino acid sequences of substantially
purified RNABP obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
preferably the human species, from any source, whether natural,
synthetic, semi-synthetic, or recombinant.
[0030] The term "agonist" refers to a molecule which, when bound to
RNABP, increases or prolongs the duration of the effect of RNABP.
Agonists may include proteins, nucleic acids, carbohydrates, or any
other molecules which bind to and modulate the effect of RNABP.
[0031] An "allelic variant" is an alternative form of the gene
encoding RNABP. Allelic variants may result from at least one
mutation in the nucleic acid sequence and may result in altered
mRNAs or in polypeptides whose structure or function may or may not
be altered. Any given natural or recombinant gene may have none,
one, or many allelic forms. Common mutational changes which give
rise to allelic variants are generally ascribed to natural
deletions, additions, or substitutions of nucleotides. Each of
these types of changes may occur alone, or in combination with the
others, one or more times in a given sequence.
[0032] "Altered" nucleic acid sequences encoding RNABP include
those sequences with deletions, insertions, or substitutions of
different nucleotides, resulting in a polynucleotide the same as
RNABP or a polypeptide with at least one functional characteristic
of RNABP. Included within this definition are polymorphisms which
may or may not be readily detectable using a particular
oligonucleotide probe of the polynucleotide encoding RNABP, and
improper or unexpected hybridization to allelic variants, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding RNABP. The encoded protein may
also be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent RNABP. Deliberate amino
acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues, as long as the
biological or immunological activity of RNABP is retained. For
example, negatively charged amino acids may include aspartic acid
and glutamic acid, positively charged amino acids may include
lysine and arginine, and amino acids with uncharged polar head
groups having similar hydrophilicity values may include leucine,
isoleucine, and valine; glycine and alanine; asparagine and
glutamine; serine and threonine; and phenylalanine and
tyrosine.
[0033] The terms "amino acid" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. In this context, "fragments," "immunogenic fragments,"
or "antigenic fragments" refer to fragments of RNABP which are
preferably at least 5 to about 15 amino acids in length, most
preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of RNABP. Where
"amino acid sequence" is recited to refer to an amino acid sequence
of a naturally occurring protein molecule, "amino acid sequence"
and like terms are not meant to limit the amino acid sequence to
the complete native amino acid sequence associated with the recited
protein molecule.
[0034] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art.
[0035] The term "antagonist" refers to a molecule which, when bound
to RNABP, decreases the amount or the duration of the effect of the
biological or immunological activity of RNABP. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of RNABP.
[0036] The term "antibody" refers to intact molecules as well as to
fragments thereof, such as Fab, F(ab').sub.2, and Fv fragments,
which are capable of binding the epitopic determinant. Antibodies
that bind RNABP polypeptides can be prepared using intact
polypeptides or using fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can
be derived from the translation of RNA, or synthesized chemically,
and can be conjugated to a carrier protein if desired. Commonly
used carriers that are chemically coupled to peptides include
bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin
(KLH). The coupled peptide is then used to immunize the animal.
[0037] The term "antigenic determinant" refers to that fragment of
a molecule (i.e., an epitope) that makes contact with a particular
antibody. When a protein or a fragment of a protein is used to
immunize a host animal, numerous regions of the protein may induce
the production of antibodies which bind specifically to antigenic
determinants (given regions or three-dimensional structures on the
protein). An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
[0038] The term "antisense" refers to any composition containing a
nucleic acid sequence which is complementary to the "sense" strand
of a specific nucleic acid sequence. Antisense molecules may be
produced by any method including synthesis or transcription. Once
introduced into a cell, the complementary nucleotides combine with
natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0039] The term "biologically active," refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" refers to
the capability of the natural, recombinant, or synthetic RNABP, or
of any oligopeptide thereof, to induce a specific immune response
in appropriate animals or cells and to bind with specific
antibodies.
[0040] The terms "complementary" or "complementarity" refer to the
natural binding of polynucleotides by base pairing. For example,
the sequence "5' A-G-T 3'" bonds to the complementary sequence "3'
T-C-A 5'." Complementarity between two single-stranded molecules
may be "partial," such that only some of the nucleic acids bind, or
it may be "complete," such that total complementarity exists
between the single stranded molecules. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of the hybridization between
the nucleic acid strands. This is of particular importance in
amplification reactions, which depend upon binding between nucleic
acids strands, and in the design and use of peptide nucleic acid
(PNA) molecules.
[0041] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding RNABP or fragments of RNABP may be employed as
hybridization probes. The probes may be stored in freeze-dried form
and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.). "Consensus
sequence" refers to a nucleic acid sequence which has been
resequenced to resolve uncalled bases, extended using an XL-PCR kit
(Perkin-Elmer, Norwalk Conn.) in the 5' and/or the 3' direction,
and resequenced, or which has been assembled from the overlapping
sequences of more than one Incyte Clone using a computer program
for fragment assembly, such as the GELVIEW fragment assembly system
(GCG, Madison Wis.). Some sequences have been both extended and
assembled to produce the consensus sequence.
[0042] The term "correlates with expression of a polynucleotide"
indicates that the detection of the presence of nucleic acids, the
same or related to a nucleic acid sequence encoding RNABP, by
northern analysis is indicative of the presence of nucleic acids
encoding RNABP in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding RNABP.
[0043] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides.
[0044] The term "derivative" refers to the chemical modification of
a polypeptide sequence, or a polynucleotide sequence. Chemical
modifications of a polynucleotide sequence can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A derivative polynucleotide encodes a polypeptide which retains at
least one biological or immunological function of the natural
molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0045] The term "similarity" refers to a degree of complementarity.
There may be partial similarity or complete similarity. The word
"identity" may substitute for the word "similarity." A partially
complementary sequence that at least partially inhibits an
identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially similar." The inhibition of
hybridization of the completely complementary sequence to the
target sequence may be examined using a hybridization assay
(Southern or northern blot, solution hybridization, and the like)
under conditions of reduced stringency. A substantially similar
sequence or hybridization probe will compete for and inhibit the
binding of a completely similar (identical) sequence to the target
sequence under conditions of reduced stringency. This is not to say
that conditions of reduced stringency are such that non-specific
binding is permitted, as reduced stringency conditions require that
the binding of two sequences to one another be a specific (i.e., a
selective) interaction. The absence of non-specific binding may be
tested by the use of a second target sequence which lacks even a
partial degree of complementarity (e.g., less than about 30%
similarity or identity). In the absence of non-specific binding,
the substantially similar sequence or probe will not hybridize to
the second non-complementary target sequence.
[0046] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Madison Wis.) which creates alignments between two or
more sequences according to methods selected by the user, e.g., the
clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988)
Gene 73:237-244.) The clustal algorithm groups sequences into
clusters by examining the distances between all pairs. The clusters
are aligned pairwise and then in groups. The percentage similarity
between two amino acid sequences, e.g., sequence A and sequence B,
is calculated by dividing the length of sequence A, minus the
number of gap residues in sequence A, minus the number of gap
residues in sequence B, into the sum of the residue matches between
sequence A and sequence B, times one hundred. Gaps of low or of no
similarity between the two amino acid sequences are not included in
determining percentage similarity. Percent identity between nucleic
acid sequences can also be counted or calculated by other methods
known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J.
(1990) Methods Enzymol. 183:626-645.) Identity between sequences
can also be determined by other methods known in the art, e.g., by
varying hybridization conditions.
[0047] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size, and which contain all of the elements required for
stable mitotic chromosome segregation and maintenance.
[0048] The term "humanized antibody" refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0049] "Hybridization" refers to any process by which a strand of
nucleic acid binds with a complementary strand through base
pairing.
[0050] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., Cot or Rot analysis) or formed
between one nucleic acid sequence present in solution and another
nucleic acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0051] The words "insertion" or "addition" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively, to the
sequence found in the naturally occurring molecule.
[0052] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0053] The term "microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0054] The terms "element" or "array element" in a microarray
context, refer to hybridizable polynucleotides arranged on the
surface of a substrate.
[0055] The term "modulate" refers to a change in the activity of
RNABP. For example, modulation may cause an increase or a decrease
in protein activity, binding characteristics, or any other
biological, functional, or immunological properties of RNABP.
[0056] The phrases "nucleic acid" or "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer to DNA or RNA of genomic or
synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which, when translated, would produce polypeptides retaining some
functional characteristic, e.g., antigenicity, or structural domain
characteristic, e.g., ATP-binding site, of the full-length
polypeptide.
[0057] The terms "operably associated" or "operably linked" refer
to functionally related nucleic acid sequences. A promoter is
operably associated or operably linked with a coding sequence if
the promoter controls the translation of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in the same reading frame, certain genetic
elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the polypeptide but still bind to operator
sequences that control expression of the polypeptide.
[0058] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to 60 nucleotides, preferably about
15 to 30 nucleotides, and most preferably about 20 to 25
nucleotides, which can be used in PCR amplification or in a
hybridization assay or microarray. "Oligonucleotide" is
substantially equivalent to the terms "amplimer," "primer,"
"oligomer," and "probe," as these terms are commonly defined in the
art. "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0059] The term "sample" is used in its broadest sense. A sample
suspected of containing nucleic acids encoding RNABP, or fragments
thereof, or RNABP itself, may comprise a bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a
cell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a
substrate; a tissue; a tissue print; etc.
[0060] The terms "specific binding" or "specifically binding" refer
to that interaction between a protein or peptide and an agonist, an
antibody, or an antagonist. The interaction is dependent upon the
presence of a particular structure of the protein, e.g., the
antigenic determinant or epitope, recognized by the binding
molecule. For example, if an antibody is specific for epitope "A,"
the presence of a polypeptide containing the epitope A, or the
presence of free unlabeled A, in a reaction containing free labeled
A and the antibody will reduce the amount of labeled A that binds
to the antibody.
[0061] The term "stringent conditions" refers to conditions which
permit hybridization between polynucleotides and the claimed
polynucleotides. Stringent conditions can be defined by salt
concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0062] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least about
60% free, preferably about 75% free, and most preferably about 90%
free from other components with which they are naturally
associated.
[0063] A "substitution" refers to the replacement of one or more
amino acids or nucleotides by different amino acids or nucleotides,
respectively.
[0064] "Substrate" refers to any suitable rigid or semi-rigid
support including membranes, filters, chips, slides, wafers,
fibers, magnetic or nonmagnetic beads, gels, tubing, plates,
polymers, microparticles and capillaries. The substrate can have a
variety of surface forms, such as wells, trenches, pins, channels
and pores, to which polynucleotides or polypeptides are bound.
[0065] "Transformation" describes a process by which exogenous DNA
enters and changes a recipient cell. Transformation may occur under
natural or artificial conditions according to various methods well
known in the art, and may rely on any known method for the
insertion of foreign nucleic acid sequences into a prokaryotic or
eukaryotic host cell. The method for transformation is selected
based on the type of host cell being transformed and may include,
but is not limited to, viral infection, electroporation, heat
shock, lipofection, and particle bombardment. The term
"transformed" cells includes stably transformed cells in which the
inserted DNA is capable of replication either as an autonomously
replicating plasmid or as part of the host chromosome, as well as
transiently transformed cells which express the inserted DNA or RNA
for limited periods of time.
[0066] A "variant" of RNABP polypeptides refers to an amino acid
sequence that is altered by one or more amino acid residues. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software (DNASTAR).
[0067] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to RNABP. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
[0068] The Invention
[0069] The invention is based on the discovery of new human RNA
binding proteins (RNABP), the polynucleotides encoding RNABP, and
the use of these compositions for the diagnosis, treatment, or
prevention of cancer, immune disorders, and developmental
disorders.
[0070] Nucleic acids encoding the RNABP-1 of the present invention
were identified in Incyte Clone 1229372H1 from the brain tumor cDNA
library (BRAITUT01) using a computer search for nucleotide and/or
amino acid sequence alignments. A consensus sequence, SEQ ID NO:4,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 1229372H1, 1229372R6, 746935R1, and
746935F1 (BRAITUT01), 2556653H1 (THYMNOT03), 5191358H1 (OVARDIT06),
4982390H1 (HELATXT05), 995084R6 (KIDNTUT01), and 2062042R6
(OVARNOT03).
[0071] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1. RNABP-1 is 547
amino acids in length and has one potential N-glycosylation site at
N117; two potential cAMP- and cGMP-dependent protein kinase
phosphorylation sites at T75 and S515; six potential casein kinase
II phosphorylation sites at S119, T199, T240, T315, S339, and T462;
and ten potential protein kinase C phosphorylation sites at S55,
S126, S153, T217, S271, T376, S457, S517, S532, and T543. PFAM
analysis indicates that the regions of RNABP-1 from G13 to K253 and
from Y272 to N380 are similar to protein domains found in members
of the DEAD-box family of ATP-dependent helicases. Within these
regions, BLOCKS, MOTIFS, and PROFILESCAN identify several sequence
signatures that are characteristic of DEAD-box family members.
These signatures include an A-type ATP binding site from A51 to
T58, a DEAD-box motif from L163 to F176, and a putative
substrate-binding/unwinding domain from G344 to F388. Amino acids
surrounding these signatures are also highly conserved among
RNABP-1 and DEAD-box family members. In addition, 32 out of the 37
functionally significant amino acids identified by Linder et al.
(supra) are conserved in RNABP-1, including the
serine/arginine/threonine tripeptide. As shown in FIGS. 1A, 1B, and
1C, RNABP-1 has chemical and structural similarity with hlc-encoded
RNA helicase (GI 3378056; SEQ ID NO:7). In particular, RNABP-1 and
hlc-encoded RNA helicase share 44% identity. Fragments of SEQ ID
NO:4 from about nucleotide 404 to about nucleotide 433 and from
about nucleotide 1412 to about nucleotide 1441 are useful in
hybridization or amplification technologies to identify SEQ ID NO:4
and to distinguish between SEQ ID NO:4 and a related sequence.
Northern analysis shows the expression of this sequence in various
libraries, at least 62% of which are associated with cancerous or
proliferating tissue and at least 32% of which are associated with
the immune response or trauma. In particular, 22% of the libraries
expressing RNABP-1 are derived from reproductive tissue, 18% are
derived from gastrointestinal tissue, and 18% are derived from
neural tissue. For example, some of the libraries expressing
RNABP-1 are derived from white blood cells, fetal intestine, and
brain and prostate tumor tissue.
[0072] Nucleic acids encoding the RNABP-2 of the present invention
were identified in Incyte Clone 1250374H1 from the fetal lung cDNA
library (LUNGFET03) using a computer search for nucleotide and/or
amino acid sequence alignments. A consensus sequence, SEQ ID NO:5,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 1250374H1 and 1250374T6 (LUNGFET03),
1573760T6 (LNODNOT03), 960131R6 (BRSTTUT03), and 2743653H1
(BRSTTUT14).
[0073] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:2. RNABP-2 is 366
amino acids in length and has three potential N-glycosylation sites
at N58, N80, and N184; two potential casein kinase II
phosphorylation sites at T16 and T173; and four potential protein
kinase C phosphorylation sites at T109, T186, T191, and S238. PFAM
analysis indicates that the region of RNABP-2 from L113 to V182 is
similar to the RRM domain. Within this region, MOTIFS, BLOCKS, and
PROFILESCAN identify an RNP-1 signature from K150 to F157 along
with surrounding conserved amino acid sequences. RNABP-2 has
chemical and structural similarity with sex-determination
associated RNP (GI 608464; SEQ ID NO:8). As shown in FIG. 2, the
region of RNABP-2 from P110 to P198 shares 72% identity with the
region of sex-determination-associated RNP from P152 to P238. A
fragment of SEQ ID NO:5 from about nucleotide 198 to about
nucleotide 227 is useful in hybridization or amplification
technologies to identify SEQ ID NO:5 and to distinguish between SEQ
ID NO:5 and a related sequence. Northern analysis shows the
expression of this sequence in various libraries, at least 63% of
which are associated with cancerous or proliferating tissue and at
least 35% of which are associated with the immune response or
trauma. In particular, 25% of the libraries expressing RNABP-2 are
derived from reproductive tissue and 20% are derived from neural
tissue. For example, some of the libraries expressing RNABP-2 are
derived from breast tumor, fetal brain, and synovial tissue.
[0074] Nucleic acids encoding the RNABP-3 of the present invention
were identified in Incyte Clone 1710966H1 from the prostate cDNA
library (PROSNOT16) using a computer search for nucleotide and/or
amino acid sequence alignments. A consensus sequence, SEQ ID NO:6,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 1710966H1 and 1710966F6 (PROSNOT16)
and 1298650F6 (BRSTNOT07).
[0075] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:3. RNABP-3 is 226
amino acids in length and has five potential casein kinase II
phosphorylation sites at S54, T88, T152, S174, and S180 and one
potential tyrosine kinase phosphorylation site at Y132. PRINTS
analysis indicates the presence of multiple leucine-rich repeats
within the region from G30 to R153. As shown in FIGS. 3A and 3B,
RNABP-3 has chemical and structural similarity with U2 snRNP A' (GI
37547; SEQ ID NO:9). In particular, RNABP-3 and U2 snRNP A' share
19% identity. RNABP-3 and U2 snRNP A' are similar in length with
226 and 255 amino acids, respectively. In addition, hydropathy
plots indicate that the carboxy-terminal half of RNABP-3, like that
of U2 snRNP A', is hydrophilic. Likewise, the N-terminal half of
RNABP-3 is leucine-rich as discussed above. A fragment of SEQ ID
NO:6 from about nucleotide 64 to about nucleotide 93 is useful in
hybridization or amplification technologies to identify SEQ ID NO:6
and to distinguish between SEQ ID NO:6 and a related sequence.
Northern analysis shows the expression of this sequence in various
libraries, at least 63% of which are associated with cancerous or
proliferating tissue and at least 21% of which are associated with
the immune response or trauma. In particular, 26% of the libraries
expressing RNABP-3 are derived from reproductive tissue and 21% are
derived from cardiovascular tissue. For example, some of the
libraries expressing RNABP-3 are derived from lung tumor, breast
tumor, and heart atrium tissue.
[0076] The invention also encompasses RNABP variants. A preferred
RNABP variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the RNABP amino acid sequence, and which
contains at least one functional or structural characteristic of
RNABP.
[0077] The invention also encompasses polynucleotides which encode
RNABP. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO:4-6, which encodes RNABP.
[0078] The invention also encompasses a variant of a polynucleotide
sequence encoding RNABP. In particular, such a variant
polynucleotide sequence will have at least about 70%, more
preferably at least about 85%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding RNABP. A particular aspect of the invention encompasses a
variant of a polynucleotide sequence comprising a sequence selected
from the group consisting of SEQ ID NO:4-6 which has at least about
70%, more preferably at least about 85%, and most preferably at
least about 95% polynucleotide sequence identity to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:4-6. Any
one of the polynucleotide variants described above can encode an
amino acid sequence which contains at least one functional or
structural characteristic of RNABP.
[0079] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding RNABP, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring RNABP, and all such
variations are to be considered as being specifically
disclosed.
[0080] Although nucleotide sequences which encode RNABP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring RNABP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding RNABP or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. Codons may be selected to
increase the rate at which expression of the peptide occurs in a
particular prokaryotic or eukaryotic host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding RNABP and its derivatives without altering the encoded
amino acid sequences include the production of RNA transcripts
having more desirable properties, such as a greater half-life, than
transcripts produced from the naturally occurring sequence.
[0081] The invention also encompasses production of DNA sequences
which encode RNABP and RNABP derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding RNABP or any fragment thereof.
[0082] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:4-6 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will
ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide, and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0083] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0084] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE DNA polymerase (US Biochemical, Cleveland
Ohio), Taq polymerase (Perkin-Elmer), thermostable T7 polymerase
(Amersham Pharmacia Biotech, Piscataway N.J.), or combinations of
polymerases and proofreading exonucleases such as those found in
the ELONGASE amplification system (Life Technologies, Gaithersburg
Md.). Preferably, sequence preparation is automated with machines
such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno
Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and the
ABI CATALYST 800 system (Perkin-Elmer). Sequencing is then carried
out using either the ABI 373 or the the 377 DNA sequencing systems
(Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system
(Molecular Dynamics, Sunnyvale Calif.). The resulting sequences are
analyzed using a variety of algorithms which are well known in the
art. (See, e.g., Ausubel, F. M. (1997) Short Protocols in Molecular
Biology, John Wiley & Sons, New York N.Y., unit 7.7; Meyers, R.
A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York
N.Y., pp. 856-853.)
[0085] The nucleic acid sequences encoding RNABP may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based methods known in the art to detect upstream sequences,
such as promoters and regulatory elements. For example, one method
which may be employed, restriction-site PCR, uses universal and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences, Plymouth
Minn.) or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the template at temperatures of about 68.degree.
C. to 72.degree. C.
[0086] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are preferable for situations
in which an oligo d(T) library does not yield a full-length cDNA.
Genomic libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0087] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. Output/light intensity may be converted
to electrical signal using appropriate software (e.g., GENOTYPER
and SEQUENCE NAVIGATOR software, Perkin-Elmer), and the entire
process from loading of samples to computer analysis and electronic
data display may be computer controlled. Capillary electrophoresis
is especially preferable for sequencing small DNA fragments which
may be present in limited amounts in a particular sample.
[0088] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode RNABP may be cloned in
recombinant DNA molecules that direct expression of RNABP, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced and used to express
RNABP.
[0089] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter RNABP-encoding sequences for a variety of purposes including,
but not limited to, modification of the cloning, processing, and/or
expression of the gene product. DNA shuffling by random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used to engineer the nucleotide sequences.
For example, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0090] In another embodiment, sequences encoding RNABP may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, RNABP itself or a fragment
thereof may be synthesized using chemical methods. For example,
peptide synthesis can be performed using various solid-phase
techniques. (See, e.g., Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI
431A peptide synthesizer (Perkin-Elmer). Additionally, the amino
acid sequence of RNABP, or any part thereof, may be altered during
direct synthesis and/or combined with sequences from other
proteins, or any part thereof, to produce a variant
polypeptide.
[0091] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York
N.Y.)
[0092] In order to express a biologically active RNABP, the
nucleotide sequences encoding RNABP or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding RNABP. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding RNABP.
Such signals include the ATG initiation codon and adjacent
sequences, e.g., the Kozak sequence. In cases where sequences
encoding RNABP and its initiation codon and upstream regulatory
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. Exogenous translational elements and initiation codons
may be of various origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
enhancers appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.) Methods which are well known to those skilled in the
art may be used to construct expression vectors containing
sequences encoding RNABP and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. (See, e.g., Sambrook, J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,
Plainview N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995)
Current Protocols in Molecular Biology, John Wiley & Sons, New
York N.Y., ch. 9, 13, and 16.)
[0093] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding RNABP. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0094] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding RNABP. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding RNABP can be achieved using a multifunctional E. coli
vector such as the PBLUESCRIPT plasmid (Stratagene, La Jolla
Calif.) or PSPORT1 plasmid (Life Technologies). Ligation of
sequences encoding RNABP into the vector's multiple cloning site
disrupts the lacZ gene, allowing a colorimetric screening procedure
for identification of transformed bacteria containing recombinant
molecules. In addition, these vectors may be useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of nested deletions in the cloned sequence.
(See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509.) When large quantities of RNABP are needed, e.g.,
for the production of antibodies, vectors which direct high level
expression of RNABP may be used. For example, vectors containing
the strong, inducible T5 or T7 bacteriophage promoter may be
used.
[0095] Yeast expression systems may be used for production of
RNABP. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra; Grant et al. (1987) Methods Enzymol.
153:516-54; and Scorer, C. A. et al. (1994) Bio/Technology
12:181-184.)
[0096] Plant systems may also be used for expression of RNABP.
Transcription of sequences encoding RNABP may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV used alone or in
combination with the omega leader sequence from TMV (Takamatsu, N.
(1987) EMBO J. 6:307-311). Alternatively, plant promoters such as
the small subunit of RUBISCO or heat shock promoters may be used.
(See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie,
R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991)
Results Probl. Cell Differ. 17:85-105.) These constructs can be
introduced into plant cells by direct DNA transformation or
pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0097] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding RNABP may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain infective virus which expresses RNABP in host cells. (See,
e.g., Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci.
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0098] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes. (See, e.g., Harrington, J. J. et al. (1997)
Nat. Genet. 15:345-355.)
[0099] For long term production of recombinant proteins in
mammalian systems, stable expression of RNABP in cell lines is
preferred. For example, sequences encoding RNABP can be transformed
into cell lines using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowed to
grow for about 1 to 2 days in enriched media before being switched
to selective media. The purpose of the selectable marker is to
confer resistance to a selective agent, and its presence allows
growth and recovery of cells which successfully express the
introduced sequences. Resistant clones of stably transformed cells
may be propagated using tissue culture techniques appropriate to
the cell type.
[0100] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase and adenine
phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.-
cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides,
neomycin and G-418; and a/s and pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al. (1981) J. Mol. Biol.
150:1-14.) Additional selectable genes have been described, e.g.,
trpB and hisD, which alter cellular requirements for metabolites.
(See, e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
Acad. Sci. 85:8047-8051.) Visible markers, e.g., anthocyanins,
green fluorescent proteins (GFP; Clontech), .beta.-glucuronidase
and its substrate .beta.-glucuronide, or luciferase and its
substrate luciferin may be used. These markers can be used not only
to identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system. (See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol.
55:121-131.)
[0101] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding RNABP is inserted within a marker gene
sequence, transformed cells containing sequences encoding RNABP can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding RNABP under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0102] In general, host cells that contain the nucleic acid
sequence encoding RNABP and that express RNABP may be identified by
a variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR amplification, and protein bioassay or
immunoassay techniques which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein sequences.
[0103] Immunological methods for detecting and measuring the
expression of RNABP using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
RNABP is preferred, but a competitive binding assay may be
employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St Paul Minn., Sect. IV; Coligan, J. E. et al.
(1997) Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0104] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding RNABP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding RNABP, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Amersham Pharmacia Biotech, Promega (Madison Wis.), and
US Biochemical. Suitable reporter molecules or labels which may be
used for ease of detection include radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0105] Host cells transformed with nucleotide sequences encoding
RNABP may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode RNABP may be designed to
contain signal sequences which direct secretion of RNABP through a
prokaryotic or eukaryotic cell membrane.
[0106] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and W138), are available from the American Type
Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0107] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding RNABP may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric RNABP protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of RNABP activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG
epitope (Sigma-Aldrich, St. Louis Mo.), c-myc, and hemagglutinin
(HA). GST, MBP, Trx, CBP, and 6-His enable purification of their
cognate fusion proteins on immobilized glutathione, maltose,
phenylarsine oxide, calmodulin, and metal-chelate resins,
respectively. The FLAG epitope (Sigma-Aldrich), c-myc, and
hemagglutinin (HA) enable immunoaffinity purification of fusion
proteins using commercially available monoclonal and polyclonal
antibodies that specifically recognize these epitope tags. A fusion
protein may also be engineered to contain a proteolytic cleavage
site located between the RNABP encoding sequence and the
heterologous protein sequence, so that RNABP may be cleaved away
from the heterologous moiety following purification. Methods for
fusion protein expression and purification are discussed in Ausubel
(1995, supra, ch. 10). A variety of commercially available kits may
also be used to facilitate expression and purification of fusion
proteins.
[0108] In a further embodiment of the invention, synthesis of
radiolabeled RNABP may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T?, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, preferably .sup.35S-methionine.
[0109] Fragments of RNABP may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra, pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the ABI
431A peptide synthesizer (Perkin-Elmer). Various fragments of RNABP
may be synthesized separately and then combined to produce the full
length molecule.
[0110] Therapeutics
[0111] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of RNABP and RNA
binding proteins such as DEAD-box RNA helicases and RNPs. In
addition, the expression of RNABP is closely associated with cancer
and proliferating tissue and the immune response. Therefore, RNABP
appears to play a role in cancer, immune disorders, and
developmental disorders. In the treatment of cancer, immune
disorders, and developmental disorders associated with increased
RNABP expression or activity, it is desirable to decrease the
expression or activity of RNABP. In the treatment of the above
conditions associated with decreased RNABP expression or activity,
it is desirable to increase the expression or activity of
RNABP.
[0112] Therefore, in one embodiment, RNABP or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of RNABP. Examples of such disorders include, but are not limited
to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
and uterus; immune disorders such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, trauma, X-linked agammaglobinemia of Bruton,
common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic
hypoplasia), thymic dysplasia, isolated IgA deficiency, severe
combined immunodeficiency disease (SCID), immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome),
Chediak-Higashi syndrome, chronic granulomatous diseases,
hereditary angioneurotic edema, and immunodeficiency associated
with Cushing's disease; and developmental disorders such as renal
tubular acidosis, anemia, Cushing's syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and
cerebral palsy, spina bifida, anencephaly, craniorachischisis,
congenital glaucoma, cataract, sensorineural hearing loss, and any
disorder associated with cell growth and differentiation,
embryogenesis, and morphogenesis involving any tissue, organ, or
system of a subject, e.g., the brain, adrenal gland, kidney,
skeletal or reproductive system.
[0113] In another embodiment, a vector capable of expressing RNABP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a disorder associated with decreased
expression or activity of RNABP including, but not limited to,
those described above.
[0114] In a further embodiment, a pharmaceutical composition
comprising a substantially purified RNABP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a disorder associated with decreased expression or
activity of RNABP including, but not limited to, those provided
above.
[0115] In still another embodiment, an agonist which modulates the
activity of RNABP may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of RNABP including, but not limited to, those listed above.
[0116] In a further embodiment, an antagonist of RNABP may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of RNABP. Examples of such
disorders include, but are not limited to, those cancers, immune
disorders, and developmental disorders described above. In one
aspect, an antibody which specifically binds RNABP may be used
directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express RNABP.
[0117] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding RNABP may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of RNABP including, but not
limited to, those described above.
[0118] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0119] An antagonist of RNABP may be produced using methods which
are generally known in the art. In particular, purified RNABP may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
RNABP. Antibodies to RNABP may also be generated using methods that
are well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab fragments, and fragments produced by a Fab
expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are especially preferred for therapeutic
use.
[0120] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with RNABP or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0121] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to RNABP have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of RNABP amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0122] Monoclonal antibodies to RNABP may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0123] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and Takeda, S. et al. (1985) Nature 314:452-454.) Alternatively,
techniques described for the production of single chain antibodies
may be adapted, using methods known in the art, to produce
RNABP-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0124] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al.
(1991) Nature 349:293-299.)
[0125] Antibody fragments which contain specific binding sites for
RNABP may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0126] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between RNABP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering RNABP
epitopes is preferred, but a competitive binding assay may also be
employed (Pound, supra).
[0127] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for RNABP. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
RNABP-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple RNABP epitopes,
represents the average affinity, or avidity, of the antibodies for
RNABP. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular RNABP epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
RNABP-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of RNABP, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington, D.C.; Liddell, J. E. and Cryer, A. (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0128] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is preferred for use in procedures requiring precipitation of
RNABP-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0129] In another embodiment of the invention, the polynucleotides
encoding RNABP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding RNABP may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding RNABP. Thus, complementary molecules or
fragments may be used to modulate RNABP activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding RNABP.
[0130] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding RNABP. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
[0131] Genes encoding RNABP can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding RNABP. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector, and may last even longer if appropriate replication
elements are part of the vector system.
[0132] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding RNABP. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J. E. et
al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0133] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding RNABP.
[0134] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides, corresponding to the region of the target
gene containing the cleavage site, may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0135] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding RNABP. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell lines, cells, or tissues.
[0136] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule, or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional nucleosides such as inosine, queosine, and
wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine, cytosine, guanine, thymine, and uracil
which are not as easily recognized by endogenous endonucleases.
[0137] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0138] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0139] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of RNABP, antibodies to RNABP, and
mimetics, agonists, antagonists, or inhibitors of RNABP. The
compositions may be administered alone or in combination with at
least one other agent, such as a stabilizing compound, which may be
administered in any sterile, biocompatible pharmaceutical carrier
including, but not limited to, saline, buffered saline, dextrose,
and water. The compositions may be administered to a patient alone,
or in combination with other agents, drugs, or hormones.
[0140] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0141] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing, Easton
Pa.).
[0142] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0143] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0144] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0145] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0146] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0147] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0148] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0149] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0. 1% to 2% sucrose, and 2% to 7% mannitol, at a pH
range of 4.5 to 5.5, that is combined with buffer prior to use.
[0150] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of RNABP, such
labeling would include amount, frequency, and method of
administration.
[0151] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0152] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, for example RNABP or fragments thereof,
antibodies of RNABP, and agonists, antagonists or inhibitors of
RNABP, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LD.sub.50/ED.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0155] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0156] Diagnostics
[0157] In another embodiment, antibodies which specifically bind
RNABP may be used for the diagnosis of disorders characterized by
expression of RNABP, or in assays to monitor patients being treated
with RNABP or agonists, antagonists, or inhibitors of RNABP.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for RNABP include methods which utilize the antibody and a label to
detect RNABP in human body fluids or in extracts of cells or
tissues. The antibodies may be used with or without modification,
and may be labeled by covalent or non-covalent attachment of a
reporter molecule. A wide variety of reporter molecules, several of
which are described above, are known in the art and may be
used.
[0158] A variety of protocols for measuring RNABP, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of RNABP expression.
Normal or standard values for RNABP expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to RNABP under conditions
suitable for complex formation. The amount of standard complex
formation may be quantitated by various methods, preferably by
photometric means. Quantities of RNABP expressed in subject,
control, and disease samples from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0159] In another embodiment of the invention, the polynucleotides
encoding RNABP may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of RNABP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of RNABP, and to
monitor regulation of RNABP levels during therapeutic
intervention.
[0160] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding RNABP or closely related molecules may be used
to identify nucleic acid sequences which encode RNABP. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding RNABP, allelic variants, or related
sequences.
[0161] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the RNABP encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequence of SEQ ID NO:4-6 or from genomic
sequences including promoters, enhancers, and introns of the RNABP
gene.
[0162] Means for producing specific hybridization probes for DNAs
encoding RNABP include the cloning of polynucleotide sequences
encoding RNABP or RNABP derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0163] Polynucleotide sequences encoding RNABP may be used for the
diagnosis of disorders associated with expression of RNABP.
Examples of such disorders include, but are not limited to, cancers
such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
immune disorders such as acquired immunodeficiency syndrome (AIDS),
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, trauma, X-linked agammaglobinemia of Bruton,
common variable immunodeficiency (CVI), DiGeorge's syndrome (thymic
hypoplasia), thymic dysplasia, isolated IgA deficiency, severe
combined immunodeficiency disease (SCID), immunodeficiency with
thrombocytopenia and eczema (Wiskott-Aldrich syndrome),
Chediak-Higashi syndrome, chronic granulomatous diseases,
hereditary angioneurotic edema, and immunodeficiency associated
with Cushing's disease; and developmental disorders such as renal
tubular acidosis, anemia, Cushing's syndrome, achondroplastic
dwarfism, Duchenne and Becker muscular dystrophy, epilepsy, gonadal
dysgenesis, WAGR syndrome (Wilms' tumor, aniridia, genitourinary
abnormalities, and mental retardation), Smith-Magenis syndrome,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and
cerebral palsy, spina bifida, anencephaly, craniorachischisis,
congenital glaucoma, cataract, sensorineural hearing loss, and any
disorder associated with cell growth and differentiation,
embryogenesis, and morphogenesis involving any tissue, organ, or
system of a subject, e.g., the brain, adrenal gland, kidney,
skeletal or reproductive system. The polynucleotide sequences
encoding RNABP may be used in Southern or northern analysis, dot
blot, or other membrane-based technologies; in PCR technologies; in
dipstick, pin, and multiformat ELISA-like assays; and in
microarrays utilizing fluids or tissues from patients to detect
altered RNABP expression. Such qualitative or quantitative methods
are well known in the art.
[0164] In a particular aspect, the nucleotide sequences encoding
RNABP may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding RNABP may be labeled by standard
methods and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantitated and compared with a standard value. If the
amount of signal in the patient sample is significantly altered in
comparison to a control sample then the presence of altered levels
of nucleotide sequences encoding RNABP in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0165] In order to provide a basis for the diagnosis of a disorder
associated with expression of RNABP, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding RNABP, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0166] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0167] With respect to cancer, the presence of an abnormal amount
of transcript (either under- or overexpressed) in biopsied tissue
from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting
the disease prior to the appearance of actual clinical symptoms. A
more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the cancer.
[0168] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding RNABP may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding RNABP, or a fragment of a
polynucleotide complementary to the polynucleotide encoding RNABP,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0169] Methods which may also be used to quantitate the expression
of RNABP include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid
quantitation.
[0170] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0171] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0172] In another embodiment of the invention, nucleic acid
sequences encoding RNABP may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Harrington, J. J. et al. (1997) Nat. Genet.
15:345-355; Price, C. M. (1993) Blood Rev. 7:127-134; and Trask, B.
J. (1991) Trends Genet. 7:149-154.) Fluorescent in situ
hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g.,
Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.)
Examples of genetic map data can be found in various scientific
journals or at the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the location of the gene encoding RNABP on a
physical chromosomal map and a specific disorder, or a
predisposition to a specific disorder, may help define the region
of DNA associated with that disorder. The nucleotide sequences of
the invention may be used to detect differences in gene sequences
among normal, carrier, and affected individuals.
[0173] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms by
physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, e.g., ataxia-telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence of
the subject invention may also be used to detect differences in the
chromosomal location due to translocation, inversion, etc., among
normal, carrier, or affected individuals.
[0174] In another embodiment of the invention, RNABP, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between RNABP and the agent being tested may be
measured.
[0175] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test
compounds are reacted with RNABP, or fragments thereof, and washed.
Bound RNABP is then detected by methods well known in the art.
Purified RNABP can also be coated directly onto plates for use in
the aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0176] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding RNABP specifically compete with a test compound for binding
RNABP. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with RNABP.
[0177] In additional embodiments, the nucleotide sequences which
encode RNABP may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0178] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0179] I. Construction of cDNA Libraries
[0180] BRAITUT01
[0181] The BRAITUT01 cDNA library was constructed using RNA
isolated from brain tumor tissue removed from a 50-year-old
Caucasian female during a frontal lobectomy. Pathology indicated
recurrent grade 3 oligoastrocytoma with focal necrosis and
extensive calcification. Patient history included speech
disturbance, epilepsy, and radiation treatment. Family history
included a brain tumor.
[0182] The frozen tissue was homogenized and lysed in guanidinium
isothiocyanate solution using a POLYTRON-PT 3000 homogenizer
(Brinkmann Instruments, Westbury N.Y.). RNA was isolated as per
Stratagene's RNA isolation protocol. RNA was extracted once with
acid phenol, precipitated with sodium acetate and ethanol,
resuspended in RNase-free water, and treated with DNase. Poly (A+)
RNA was isolated using the OLIGOTEX mRNA purification kit (QIAGEN,
Chatsworth Calif.).
[0183] Poly(A+) RNA was used for cDNA synthesis and construction of
the cDNA library according to the recommended protocols in the
SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were
fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech), and those cDNAs exceeding 400 bp were ligated into the
PSPORT plasmid (Life Technologies). Recombinant plasmids were
transformed into DH5a competent cells (Life Technologies).
[0184] LUNGFET03, PROSNOT16
[0185] The LUNGFET03 cDNA library was constructed using RNA
isolated from lung tissue removed from a Caucasian female fetus who
died at 20 weeks' gestation. The PROSNOT16 cDNA library was
constructed using RNA isolated from diseased prostate tissue
removed from a 68-year-old Caucasian male during a radical
prostatectomy. Pathology indicated adenofibromatous hyperplasia.
Pathology for the associated tumor tissue indicated adenocarcinoma
(Gleason grade 3-4). The patient presented with elevated prostate
specific antigen (PSA). The patient was concurrently diagnosed with
myasthenia gravis. Patient history included osteoarthritis and type
II diabetes. Family history included benign hypertension, acute
myocardial infarction, hyperlipidemia, and arteriosclerotic
coronary artery disease.
[0186] Frozen tissue from each of the above sources was homogenized
and lysed in guanidinium isothiocyanate solution using a POLYTRON
PT-3000 homogenizer (Brinkmann Instruments). The lysate was
centrifuged over a CsCl cushion to isolate RNA. The RNA was
extracted with acid phenol, precipitated with sodium acetate and
ethanol, resuspended in RNase-free water, and treated with DNase.
The RNA was re-extracted with acid phenol and reprecipitated as
described above. Poly(A+) RNA was isolated using the OLIGOTEX mRNA
purification kit (QIAGEN).
[0187] Poly(A+) RNA was used for cDNA synthesis and construction of
the cDNA library according to the recommended protocols in the
SUPERSCRIPT plasmid system (Life Technologies). The cDNAs were
fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia
Biotech), and those cDNAs exceeding 400 bp were ligated into pINCY
(Incyte Pharmaceuticals, Palo Alto Calif.). Recombinant plasmids
were transformed into DH5a competent cells (Life Technologies).
[0188] II. Isolation of cDNA Clones
[0189] For each of the above cDNA libraries, plasmid DNA was
released from host cells and purified using the R. E. A. L. PREP 96
plasmid kit (QIAGEN). The recommended protocol was employed except
for the following changes: 1) the bacteria were cultured in 1 ml of
sterile Terrific Broth (Life Technologies) with carbenicillin at 25
mg/l and glycerol at 0.4%; 2) after the cultures were incubated for
19 hours, the cells were lysed with 0.3 ml of lysis buffer; and 3)
following isopropanol precipitation, the plasmid DNA pellets were
each resuspended in 0.1 ml of distilled water. The DNA samples were
stored at 4.degree. C.
[0190] III. Sequencing and Analysis
[0191] The cDNAs were prepared for sequencing using the ABI
CATALYST 800 (Perkin-Elmer) or the HYDRA microdispenser (Robbins
Scientific) or MICROLAB 2200 (Hamilton) systems in combination with
the PTC-200 thermal cyclers (MJ Research). The cDNAs were sequenced
using the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer)
and standard ABI protocols, base calling software, and kits. In one
alternative, cDNAs were sequenced using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were amplified and sequenced using the ABI PRISM BIGDYE
terminator cycle sequencing ready reaction kit (Perkin-Elmer). In
yet another alternative, cDNAs were sequenced using solutions and
dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs
were determined using standard methods (reviewed in Ausubel, 1997,
supra, unit 7.7). Some of the cDNA sequences were selected for
extension using the techniques disclosed in Example V.
[0192] The polynucleotide sequences derived from cDNA, extension,
and shotgun sequencing were assembled and analyzed using a
combination of software programs which utilize algorithms well
known to those skilled in the art. Table 1 summarizes the software
programs, descriptions, references, and threshold parameters used.
The first column of Table 1 shows the tools, programs, and
algorithms used, the second column provides a brief description
thereof, the third column presents the references which are
incorporated by reference herein, and the fourth column presents,
where applicable, the scores, probability values, and other
parameters used to evaluate the strength of a match between two
sequences (the higher the probability the greater the homology).
Sequences were analyzed using MACDNASIS PRO software (Hitachi
Software Engineering, South San Francisco Calif.) and LASERGENE
software (DNASTAR).
[0193] The polynucleotide sequences were validated by removing
vector, linker, and polyA sequences and by masking ambiguous bases,
using algorithms and programs based on BLAST, dynamic programing,
and dinucleotide nearest neighbor analysis. The sequences were then
queried against a selection of public databases such as GenBank
primate, rodent, mammalian, vertebrate, and eukaryote databases,
and BLOCKS to acquire annotation, using programs based on BLAST,
FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide sequences using programs based on Phred, Phrap, and
Consed, and were screened for open reading frames using programs
based on GeneMark, BLAST, and FASTA. The full length polynucleotide
sequences were translated to derive the corresponding full length
amino acid sequences, and these full length sequences were
subsequently analyzed by querying against databases such as the
GenBank databases (described above), SwissProt, BLOCKS, PRINTS,
Prosite, and Hidden Markov Model (HMM)-based protein family
databases such as PFAM. HMM is a probalistic approach which
analyzes consensus primary structures of gene families. (See, e.g.,
Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365.) The programs
described above for the assembly and analysis of full length
polynucleotide and amino acid sequences were also used to identify
polynucleotide sequence fragments from SEQ ID NO:4-6. Fragments
from about 20 to about 4000 nucleotides which are useful in
hybridization and amplification technologies were described in The
Invention section above.
[0194] IV. Northern Analysis
[0195] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; Ausubel, 1995, supra, ch. 4 and
16.)
[0196] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or similar. The basis of the search is the
product score, which is defined as: 1 % sequence identity .times. %
maximum BLAST score 100
[0197] % sequence identity x % maximum BLAST score 100
[0198] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0199] The results of northern analyses are reported as a
percentage distribution of libraries in which the transcript
encoding RNABP occurred. Analysis involved the categorization of
cDNA libraries by organ/tissue and disease. The organ/tissue
categories included cardiovascular, dermatologic, developmental,
endocrine, gastrointestinal, hematopoietic/immune, musculoskeletal,
nervous, reproductive, and urologic. The disease/condition
categories included cancer, inflammation/trauma, cell
proliferation, neurological, and pooled. For each category, the
number of libraries expressing the sequence of interest was counted
and divided by the total number of libraries across all categories.
Percentage values of tissue-specific and disease- or
condition-specific expression are reported in The Invention section
above.
[0200] V. Extension of RNABP Encoding Polynucleotides
[0201] The full length nucleic acid sequences of SEQ ID NO:4-6 were
produced by extension of an appropriate fragment of the full length
molecule using oligonucleotide primers designed from this fragment.
One primer was synthesized to initiate 5' extension of the known
fragment, and the other primer, to initiate 3' extension of the
known fragment. The initial primers were designed using OLIGO 4.06
software (National Biosciences), or another appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the target sequence at
temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0202] Selected human cDNA libraries were used to extend the
sequence. If more than one extension was necessary or desired,
additional or nested sets of primers were designed.
[0203] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
.beta.-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair PCI A and PCI B: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. In
the alternative, the parameters for primer pair T7 and SK+were as
follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C.
[0204] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1.times.TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a FLUOROSKAN II
microplate reader (Labsystems Oy, Helsinki, Finland) to measure the
fluorescence of the sample and to quantify the concentration of
DNA. A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a 1% agarose mini-gel to determine
which reactions were successful in extending the sequence.
[0205] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into PUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with
AGARACE enzyme (Promega). Extended clones were religated using T4
ligase (New England Biolabs, Beverly Mass.) into PUC 18 vector
(Amersham Pharmacia Biotech), treated with Pfu DNA polymerase
(Stratagene) to fill-in restriction site overhangs, and transfected
into competent E. coli cells. Transformed cells were selected on
antibiotic-containing media, individual colonies were picked and
cultured overnight at 37.degree. C. in 384-well plates in LB/2x
carb liquid media.
[0206] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE terminator
cycle sequencing ready reaction kit (Perkin-Elmer).
[0207] In like manner, the nucleotide sequences of SEQ ID NO:4-6
are used to obtain 5' regulatory sequences using the procedure
above, oligonucleotides designed for such extension, and an
appropriate genomic library.
[0208] VI. Labeling and Use of Individual Hybridization Probes
[0209] Hybridization probes derived from SEQ ID NO:4-6 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [y_.sup.32P] adenosine triphosphate (Amersham Pharmacia
Biotech), and T4 polynucleotide kinase (DuPont NEN, Boston Mass.).
The labeled oligonucleotides are substantially purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column
(Amersham Pharmacia Biotech). An aliquot containing 10.sup.7 counts
per minute of the labeled probe is used in a typical membrane-based
hybridization analysis of human genomic DNA digested with one of
the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I,
or Pvu II (DuPont NEN).
[0210] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (NYTRAN PLUS, Schleicher
& Schuell, Durham N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times.saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT-AR film (Eastman Kodak,
Rochester N.Y.) is exposed to the blots, hybridization patterns are
compared visually.
[0211] VII. Microarrays
[0212] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0213] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one
of the nucleotide sequences of the present invention, or selected
at random from a cDNA library relevant to the present invention,
are arranged on an appropriate substrate, e.g., a glass slide. The
cDNA is fixed to the slide using, e.g., UV cross-linking followed
by thermal and chemical treatments and subsequent drying. (See,
e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et
al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared
and used for hybridization to the elements on the substrate. The
substrate is analyzed by procedures described above.
[0214] VIII. Complementary Polynucleotides
[0215] Sequences complementary to the RNABP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring RNABP. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 software (National Biosciences) and the
coding sequence of RNABP. To inhibit transcription, a complementary
oligonucleotide is designed from the most unique 5' sequence and
used to prevent promoter binding to the coding sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent
ribosomal binding to the RNABP-encoding transcript.
[0216] IX. Expression of RNABP
[0217] Expression and purification of RNABP is achieved using
bacterial or virus-based expression systems. For expression of
RNABP in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express RNABP upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of RNABP
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding RNABP by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0218] In most expression systems, RNABP is synthesized as a fusion
protein with, e.g., glutathione S-transferase (GST) or a peptide
epitope tag, such as the FLAG epitope (Sigma-Aldrich) or 6-His,
permitting rapid, single-step, affinity-based purification of
recombinant fusion protein from crude cell lysates. GST, a
26-kilodalton enzyme from Schistosoma japonicum, enables the
purification of fusion proteins on immobilized glutathione under
conditions that maintain protein activity and antigenicity
(Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from RNABP at specifically
engineered sites. The FLAG epitope (Sigma-Aldrich), an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak). 6-His, a stretch of six consecutive histidine residues,
enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression and purification are discussed in Ausubel (1995,
supra, ch 10 and 16). Purified RNABP obtained by these methods can
be used directly in the following activity assay.
[0219] X. Demonstration of RNABP Activity
[0220] RNABP activity is demonstrated by the formation of an
RNABP-RNA complex as detected by polyacrylamide gel mobility-shift
assay. In preparation for this assay, RNABP is expressed by
transforming a mammalian cell line such as COS7, HeLa or CHO with a
eukaryotic expression vector containing RNABP cDNA. The cells are
incubated for 48-72 hours after transformation under conditions
which allow expression and accumulation of RNABP. Extracts
containing solubilized proteins can be prepared from cells
expressing RNABP by methods well known in the art. Portions of the
extract containing RNABP are added to [.sup.32P]-labeled RNA.
Radioactive RNA can be synthesized in vitro by techniques well
known in the art. The mixtures are incubated at 25.degree. C. in
the presence of RNase inhibitors under buffered conditions for 5-10
minutes. After incubation, the samples are analyzed by
polyacrylamide gel electrophoresis followed by autoradiography. The
presence of a high molecular weight band on the autoradiogram
indicates the formation of a complex between RNABP and the
radioactive transcript. A band of significantly lower molecular
weight will be present in samples prepared using control extracts
prepared from untransformed cells. The amount of RNABP-RNA complex
can be quantified using phospho-image analysis and is proportional
to the activity of RNABP.
[0221] XI. Functional Assays
[0222] RNABP function is assessed by expressing the sequences
encoding RNABP at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include the PCMV SPORT plasmid (Life
Technologies) and the PCR3.1 plasmid (Invitrogen, Carlsbad Calif.),
both of which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, preferably of endothelial or hematopoietic origin, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP, and to
evaluate properties, for example, their apoptotic state. FCM
detects and quantifies the uptake of fluorescent molecules that
diagnose events preceding or coincident with cell death. These
events include changes in nuclear DNA content as measured by
staining of DNA with propidium iodide; changes in cell size and
granularity as measured by forward light scatter and 90 degree side
light scatter; down-regulation of DNA synthesis as measured by
decrease in bromodeoxyuridine uptake; alterations in expression of
cell surface and intracellular proteins as measured by reactivity
with specific antibodies; and alterations in plasma membrane
composition as measured by the binding of fluorescein-conjugated
Annexin V protein to the cell surface. Methods in flow cytometry
are discussed in Ormerod, M. G. (1994) Flow Cytometry, Oxford, New
York N.Y.
[0223] The influence of RNABP on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding RNABP and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding RNABP and other genes of interest can
be analyzed by northern analysis or microarray techniques.
[0224] XII. Production of RNABP Specific Antibodies
[0225] RNABP substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0226] Alternatively, the RNABP amino acid sequence is analyzed
using LASERGENE software (DNASTAR) to determine regions of high
immunogenicity, and a corresponding oligopeptide is synthesized and
used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions are well described in
the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
[0227] Typically, oligopeptides 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Perkin-Elmer)
using fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction
with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits
are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity by, for example, binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
[0228] XIII. Purification of Naturally Occurring RNABP Using
Specific Antibodies
[0229] Naturally occurring or recombinant RNABP is substantially
purified by immunoaffinity chromatography using antibodies specific
for RNABP. An immunoaffinity column is constructed by covalently
coupling anti-RNABP antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0230] Media containing RNABP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of RNABP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/RNABP binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and RNABP is collected.
[0231] XIV. Identification of Molecules Which Interact with
RNABP
[0232] RNABP, or biologically active fragments thereof, are labeled
with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et al.
(1973) Biochem. J. 133:529.) Candidate molecules previously arrayed
in the wells of a multi-well plate are incubated with the labeled
RNABP, washed, and any wells with labeled RNABP complex are
assayed. Data obtained using different concentrations of RNABP are
used to calculate values for the number, affinity, and association
of RNABP with the candidate molecules.
[0233] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
9 1 547 PRT Homo sapiens 1229372 1 Met Glu Asp Ser Glu Ala Leu Gly
Phe Glu His Met Gly Leu Asp 1 5 10 15 Pro Arg Leu Leu Gln Ala Val
Thr Asp Leu Gly Trp Ser Arg Pro 20 25 30 Thr Leu Ile Gln Glu Lys
Ala Ile Pro Leu Ala Leu Glu Gly Lys 35 40 45 Asp Leu Leu Ala Arg
Ala Arg Thr Gly Ser Gly Lys Thr Ala Ala 50 55 60 Tyr Ala Ile Pro
Met Leu Gln Leu Leu Leu His Arg Lys Ala Thr 65 70 75 Gly Pro Val
Val Glu Gln Ala Val Arg Gly Leu Val Leu Val Pro 80 85 90 Thr Lys
Glu Leu Ala Arg Gln Ala Gln Ser Met Ile Gln Gln Leu 95 100 105 Ala
Thr Tyr Cys Ala Arg Asp Val Arg Val Ala Asn Val Ser Ala 110 115 120
Ala Glu Asp Ser Val Ser Gln Arg Ala Val Leu Met Glu Lys Pro 125 130
135 Asp Val Val Val Gly Thr Pro Ser Arg Ile Leu Ser His Leu Gln 140
145 150 Gln Asp Ser Leu Lys Leu Arg Asp Ser Leu Glu Leu Leu Val Val
155 160 165 Asp Glu Ala Asp Leu Leu Phe Ser Phe Gly Phe Glu Glu Glu
Leu 170 175 180 Lys Ser Leu Leu Cys His Leu Pro Arg Ile Tyr Gln Ala
Phe Leu 185 190 195 Met Ser Ala Thr Phe Asn Glu Asp Val Gln Ala Leu
Lys Glu Leu 200 205 210 Ile Leu His Asn Pro Val Thr Leu Lys Leu Gln
Glu Ser Gln Leu 215 220 225 Pro Gly Pro Asp Gln Leu Gln Gln Phe Gln
Val Val Cys Glu Thr 230 235 240 Glu Glu Asp Lys Phe Leu Leu Leu Tyr
Ala Leu Leu Lys Leu Ser 245 250 255 Leu Ile Arg Gly Lys Ser Leu Leu
Phe Val Asn Thr Leu Glu Arg 260 265 270 Ser Tyr Arg Leu Arg Leu Phe
Leu Glu Gln Phe Ser Ile Pro Thr 275 280 285 Cys Val Leu Asn Gly Glu
Leu Pro Leu Arg Ser Arg Cys His Ile 290 295 300 Ile Ser Gln Phe Asn
Gln Gly Phe Tyr Asp Cys Val Ile Ala Thr 305 310 315 Asp Ala Glu Val
Leu Gly Ala Pro Val Lys Gly Lys Arg Arg Gly 320 325 330 Arg Gly Pro
Lys Gly Asp Lys Ala Ser Asp Pro Glu Ala Gly Val 335 340 345 Ala Arg
Gly Ile Asp Phe His His Val Ser Ala Val Leu Asn Phe 350 355 360 Asp
Leu Pro Pro Thr Pro Glu Ala Tyr Ile His Arg Ala Gly Arg 365 370 375
Thr Ala Arg Ala Asn Asn Pro Gly Ile Val Leu Thr Phe Val Leu 380 385
390 Pro Thr Glu Gln Phe His Leu Gly Lys Ile Glu Glu Leu Leu Ser 395
400 405 Gly Glu Asn Arg Gly Pro Ile Leu Leu Pro Tyr Gln Phe Arg Met
410 415 420 Glu Glu Ile Glu Gly Phe Arg Tyr Arg Cys Arg Asp Ala Met
Arg 425 430 435 Ser Val Thr Lys Gln Ala Ile Arg Glu Ala Arg Leu Lys
Glu Ile 440 445 450 Lys Glu Glu Leu Leu His Ser Glu Lys Leu Lys Thr
Tyr Phe Glu 455 460 465 Asp Asn Pro Arg Asp Leu Gln Leu Leu Arg His
Asp Leu Pro Leu 470 475 480 His Pro Ala Val Val Lys Pro His Leu Gly
His Val Pro Asp Tyr 485 490 495 Leu Val Pro Pro Ala Leu Arg Gly Leu
Val Arg Pro His Lys Lys 500 505 510 Arg Lys Lys Leu Ser Ser Ser Cys
Arg Lys Ala Lys Arg Ala Lys 515 520 525 Ser Gln Asn Pro Leu Arg Ser
Phe Lys His Lys Gly Lys Lys Phe 530 535 540 Arg Pro Thr Ala Lys Pro
Ser 545 2 366 PRT Homo sapiens 1250374 2 Met Glu Lys Lys Lys Met
Val Thr Gln Gly Asn Gln Glu Pro Thr 1 5 10 15 Thr Thr Pro Asp Ala
Met Val Gln Pro Phe Thr Thr Ile Pro Phe 20 25 30 Pro Pro Pro Pro
Gln Asn Gly Ile Pro Thr Glu Tyr Gly Val Pro 35 40 45 His Thr Gln
Asp Tyr Ala Gly Gln Thr Gly Glu His Asn Leu Thr 50 55 60 Leu Tyr
Gly Ser Thr Gln Ala His Gly Glu Gln Ser Ser Asn Ser 65 70 75 Pro
Ser Thr Gln Asn Gly Ser Leu Thr Thr Glu Gly Gly Ala Gln 80 85 90
Thr Asp Gly Gln Gln Ser Gln Thr Gln Ser Ser Glu Asn Ser Glu 95 100
105 Ser Lys Ser Thr Pro Lys Arg Leu His Val Ser Asn Ile Pro Phe 110
115 120 Arg Phe Arg Asp Pro Asp Leu Arg Gln Met Phe Gly Gln Phe Gly
125 130 135 Lys Ile Leu Asp Val Glu Ile Ile Phe Asn Glu Arg Gly Ser
Lys 140 145 150 Gly Phe Gly Phe Val Thr Phe Glu Asn Ser Ala Asp Ala
Asp Arg 155 160 165 Ala Arg Glu Lys Leu His Gly Thr Val Val Glu Gly
Arg Lys Ile 170 175 180 Glu Val Asn Asn Ala Thr Ala Arg Val Met Thr
Asn Lys Lys Met 185 190 195 Val Thr Pro Tyr Ala Asn Gly Trp Lys Leu
Ser Pro Val Val Gly 200 205 210 Ala Val Tyr Gly Pro Glu Leu Tyr Ala
Ala Ser Ser Phe Gln Ala 215 220 225 Asp Val Ser Leu Gly Asn Asp Ala
Ala Val Pro Leu Ser Gly Arg 230 235 240 Gly Gly Ile Asn Thr Tyr Ile
Pro Leu Ile Ile Pro Gly Phe Pro 245 250 255 Tyr Pro Thr Ala Ala Thr
Thr Ala Ala Ala Phe Arg Gly Ala His 260 265 270 Leu Arg Gly Arg Gly
Arg Thr Val Tyr Gly Ala Val Arg Ala Val 275 280 285 Pro Pro Thr Ala
Ile Pro Ala Tyr Pro Gly Val Asp Met Gln Pro 290 295 300 Thr Asp Met
His Ser Leu Leu Leu Gln Pro Gln Pro Pro Leu Leu 305 310 315 Gln Pro
Leu Gln Pro Leu Thr Val Thr Val Met Ala Gly Cys Thr 320 325 330 Gln
Pro Thr Pro Thr Met Pro Leu Pro Leu Pro Leu Ala Met Glu 335 340 345
Leu Ala Leu Trp Arg Val Tyr Thr Glu Val Ala Thr Ala Asp Leu 350 355
360 Pro Pro Thr Glu Val Thr 365 3 226 PRT Homo sapiens 1710966 3
Met Ala Gly Leu Val Val Arg Gly Thr Gln Val Ser Tyr Ile Gly 1 5 10
15 Gln Asp Cys Arg Glu Ile Pro Glu His Leu Gly Arg Asp Cys Gly 20
25 30 His Phe Ala Lys Arg Leu Asp Leu Ser Phe Asn Leu Leu Arg Ser
35 40 45 Leu Glu Gly Leu Ser Ala Phe Arg Ser Leu Glu Glu Leu Ile
Leu 50 55 60 Asp Asn Asn Gln Leu Gly Asp Asp Leu Val Leu Pro Gly
Leu Pro 65 70 75 Arg Leu His Thr Leu Thr Leu Asn Lys Asn Arg Ile
Thr Asp Leu 80 85 90 Glu Asn Leu Leu Asp His Leu Ala Glu Val Thr
Pro Ala Leu Glu 95 100 105 Tyr Leu Ser Leu Leu Gly Asn Val Ala Cys
Pro Asn Glu Leu Val 110 115 120 Ser Leu Glu Lys Asp Glu Glu Asp Tyr
Lys Arg Tyr Arg Cys Phe 125 130 135 Val Leu Tyr Lys Leu Pro Asn Leu
Lys Phe Leu Asp Ala Gln Lys 140 145 150 Val Thr Arg Gln Glu Arg Glu
Glu Ala Leu Val Arg Gly Val Phe 155 160 165 Met Lys Val Val Lys Pro
Lys Ala Ser Ser Glu Asp Val Ala Ser 170 175 180 Ser Pro Glu Arg His
Tyr Thr Pro Leu Pro Ser Ala Ser Arg Glu 185 190 195 Leu Thr Ser His
Gln Gly Val Leu Gly Lys Cys Arg Tyr Val Tyr 200 205 210 Tyr Gly Lys
Asn Ser Glu Gly Asn Arg Phe Ile Arg Asp Asp Gln 215 220 225 Leu 4
2286 DNA Homo sapiens 1229372 4 ggttctcctc agcccagtct atctcagtgg
ctccattcat agggtgatgt gcccggcggg 60 acactaaccc taaccaagca
gagagacggt catgcccgtc acgacctcgg ccctcgcccc 120 ggccgaggct
tctcctgcag gtcgcgagaa tcaggtgcgt cacggcgtcc gggaacgccg 180
gaagagccag tggagcggct ctgtagtcca aagtaccccg tcgaccccag cacggccgct
240 ccaccgcctc ctactagacc cagtcctagg gactgcgcag tcgcagagct
ccgtccgagt 300 accggaagcc taggccgcca gcacttccgg gaagtgactt
cgtctccgaa gccgattggt 360 tgttgctttg ctcccgctcg cgtcggtggc
gtttttcctg cagcgcgtgc gtgctgcgct 420 actgagcagc gccatggagg
actctgaagc actgggcttc gaacacatgg gcctcgatcc 480 ccggctcctt
caggctgtca ccgatctggg ctggtcgcga cctacgctga tccaggagaa 540
ggccatccca ctggccctag aagggaagga cctcctggct cgggcccgca cgggctccgg
600 gaagacggcc gcttatgcta ttccgatgct gcagctgttg ctccatagga
aggcgacagg 660 tccggtggta gaacaggcag tgagaggcct tgttcttgtt
cctaccaagg agctggcacg 720 gcaagcacag tccatgattc agcagctggc
tacctactgt gctcgggatg tccgagtggc 780 caatgtctca gctgctgaag
actcagtctc tcagagagct gtgctgatgg agaagccaga 840 tgtggtagta
gggaccccat ctcgcatatt aagccacttg cagcaagaca gcctgaaact 900
tcgtgactcc ctggagcttt tggtggtgga cgaagctgac cttctttttt cctttggctt
960 tgaagaagag ctcaagagtc tcctctgtca cttgccccgg atttaccagg
cttttctcat 1020 gtcagctact tttaacgagg acgtacaagc actcaaggag
ctgatattac ataacccggt 1080 tacccttaag ttacaggagt cccagctgcc
tgggccagac cagttacagc agtttcaggt 1140 ggtctgtgag actgaggaag
acaaattcct cctgctgtat gccctgctca agctgtcatt 1200 gattcggggc
aagtctctgc tctttgtcaa cactctagaa cggagttacc ggctacgcct 1260
gttcttggaa cagttcagca tccccacctg tgtgctcaat ggagagcttc cactgcgctc
1320 caggtgccac atcatctcac agttcaacca aggcttctac gactgtgtca
tagcaactga 1380 tgctgaagtc ctgggggccc cagtcaaggg caagcgtcgg
ggccgagggc ccaaagggga 1440 caaggcctct gatccggaag caggtgtggc
ccggggcata gacttccacc atgtgtctgc 1500 tgtgctcaac tttgatcttc
ccccaacccc tgaggcctac atccatcgag ctggcaggac 1560 agcacgcgct
aacaacccag gcatagtctt aacctttgtg cttcccacgg agcagttcca 1620
cttaggcaag attgaggagc ttctcagtgg agagaacagg ggccccattc tgctccccta
1680 ccagttccgg atggaggaga tcgagggctt ccgctatcgc tgcagggatg
ccatgcgctc 1740 agtgactaag caggccattc gggaggcaag attgaaggag
atcaaggaag agcttctgca 1800 ttctgagaag cttaagacat actttgaaga
caaccctagg gacctccagc tgctgcggca 1860 tgacctacct ttgcaccccg
cagtggtgaa gccccacctg ggccatgttc ctgactacct 1920 ggttcctcct
gctctccgtg gcctggtgcg ccctcacaag aagcggaaga agctgtcttc 1980
ctcttgtagg aaggccaaga gagcaaagtc ccagaaccca ctgcgcagct tcaagcacaa
2040 aggaaagaaa ttcagaccca cagccaagcc ctcctgaggt tgttgggcct
ctctggagct 2100 gagcacattg tggagcacag gcttacaccc ttcgtggaca
ggcgaggctc tggtgcttac 2160 tgcacagcct gaacagacag ttctggggcc
ggcagtgctg ggccctttag ctccttggca 2220 cttccaagct ggcatcttgc
cccttgacaa cagaataaaa attttagctg ccccaaaaaa 2280 aaaaaa 2286 5 1506
DNA Homo sapiens 1250374 5 cggacgcgtg gctgccagag aggaagagaa
aagaaaggaa gaaacattag aaagaaaaag 60 gaaggaaaac ggtataaaga
gagatcaatt acccaccctt aaatagctag attggggggg 120 gaggggggtg
gaaaagaaag ctgtggaggt gtgccccagc acggctgctt tgaaaggttt 180
atcatctatc cgtttggttt atggagaaaa agaaaatggt aactcagggt aaccaggagc
240 cgacaacaac tcctgacgca atggttcagc cttttactac catcccattt
ccaccacctc 300 cgcagaatgg aattcccaca gagtatgggg tgccacacac
tcaagactat gccggccaga 360 ccggtgagca taacctgaca ctctacggaa
gtacgcaagc ccacggggag cagagcagca 420 actcacccag cacacaaaat
ggatctctta cgacagaagg tggagcacag acagacggcc 480 agcagtcaca
gacacaaagt agtgaaaatt cagagagtaa atctaccccg aaacggctgc 540
atgtctctaa tattcctttc cgcttccggg accctgacct ccggcagatg tttgggcagt
600 ttggcaaaat cctagatgta gaaataatct ttaatgaacg tggctctaag
ggattcgggt 660 tcgtaacttt cgagaatagt gctgatgcag acagggccag
ggagaaatta cacggcaccg 720 tggtagaggg ccgtaaaatc gaggtgaata
atgctacagc acgtgtaatg accaataaga 780 agatggtcac accatatgca
aatggttgga aattaagccc agtagttgga gctgtatatg 840 gtccggagtt
atatgcagca tccagctttc aagcagatgt gtccctaggc aatgatgcag 900
cagtgcccct atcaggaaga gggggtatca acacttacat tcctttaatc attcctggct
960 tcccttaccc tactgcagcc accacggcag ccgctttcag aggagcccat
ttgaggggca 1020 gagggcggac agtatatggt gcagtccgag cggtacctcc
aacagccatc cccgcctatc 1080 caggggtgga tatgcagcct acagatatgc
acagcctgct actgcaaccg cagccaccgc 1140 tgctgcagcc gctgcagccg
cttacagtga cggttatggc agggtgtaca cagccgaccc 1200 ctaccatgcc
cttgcccctg ccgctagcta tggagttggc gctgtggcga gtttataccg 1260
aggtggctac agccgatttg ccccctactg aagtgacgtg agacccctgc aaatgggaca
1320 gccccccagt tcatgaggcc tggctattgc aatatttact agtagaggaa
ctctatagca 1380 agatgaagag gaaaaacaaa caaacaaaca aacaaaaaca
caaaaaaaga aagaatactt 1440 ttttatacct cactatgttc tttgaatatg
tatttttcct ttaaatttct gcctttaaaa 1500 aaaaaa 1506 6 834 DNA Homo
sapiens 1710966 6 gccgcgctcc ccgctgctgc cgccgcgacc ccgcgctccg
tcccgcgcgc ccgcagcgtc 60 ctggccgcca tggccgggct cgtggtgcgt
ggaactcaag tgtcctacat aggccaggac 120 tgcagagaaa ttccagagca
ccttggcagg gactgtggac atttcgcaaa gaggcttgat 180 ctgagcttta
accttctgag gtcactggaa ggactgagcg cattcaggag cctggaggaa 240
ctcatcttgg acaacaatca gctgggggac gaccttgtgt tgccagggtt acccagactg
300 cataccttaa ccctcaacaa gaaccgaatc actgatttgg agaacctgct
ggatcacttg 360 gcagaagtga caccagctct ggagtacctc agtctgctgg
gcaacgtggc ctgtcccaac 420 gagctggtca gcctggaaaa ggatgaggaa
gactacaaga gatacagatg ctttgttctg 480 tacaagctgc ccaacttgaa
atttctggat gcccagaaag taaccagaca agaacgagag 540 gaggcgttgg
tcagaggagt cttcatgaag gtggtgaagc ccaaggcttc tagtgaggac 600
gttgccagct ccccggagcg ccactacacg cccttgcctt ctgcttccag ggaactcacc
660 agtcaccaag gtgtcctggg gaagtgtcgc tacgtttact atgggaaaaa
ctcagagggc 720 aacaggttta tccgagatga ccagctctga agccaacttc
tgtatacctt cacccatttc 780 atgaaaataa aatcaaaagg gaaatcaaaa
ataaagaaaa cgctaaaaaa aaaa 834 7 560 PRT Drosophila melanogaster
g3378056 7 Met Ser Gln Met Thr Gln Lys Thr Val Gln Phe His Glu Leu
Glu 1 5 10 15 Leu Asp Gln Arg Ile Leu Lys Ala Val Ala Gln Leu Gly
Trp Gln 20 25 30 Gln Pro Thr Leu Ile Gln Ser Thr Ala Ile Pro Leu
Leu Leu Glu 35 40 45 Gly Lys Asp Val Val Val Arg Ala Arg Thr Gly
Ser Gly Lys Thr 50 55 60 Ala Thr Tyr Ala Leu Pro Leu Ile Gln Lys
Ile Leu Asn Ser Lys 65 70 75 Leu Asn Ala Ser Glu Gln Tyr Val Ser
Ala Val Val Leu Ala Pro 80 85 90 Thr Lys Glu Leu Cys Arg Gln Ser
Arg Lys Val Ile Glu Gln Leu 95 100 105 Val Glu Ser Cys Gly Lys Val
Val Arg Val Ala Asp Ile Ala Asp 110 115 120 Ser Ser Asn Asp Thr Val
Thr Gln Arg His Ala Leu Ser Glu Ser 125 130 135 Pro Asp Ile Val Val
Ala Thr Pro Ala Asn Leu Leu Ala Tyr Ala 140 145 150 Glu Ala Gly Ser
Val Val Asp Leu Lys His Val Glu Thr Leu Val 155 160 165 Val Asp Glu
Ala Asp Leu Val Phe Ala Tyr Gly Tyr Glu Lys Asp 170 175 180 Phe Lys
Arg Leu Ile Lys His Leu Pro Pro Ile Tyr Gln Ala Val 185 190 195 Leu
Val Ser Ala Thr Leu Thr Asp Asp Val Val Arg Met Lys Gly 200 205 210
Leu Cys Leu Asn Asn Pro Val Thr Leu Lys Leu Glu Glu Pro Glu 215 220
225 Leu Val Pro Gln Asp Gln Leu Ser His Gln Arg Ile Leu Ala Glu 230
235 240 Glu Asn Asp Lys Pro Ala Ile Leu Tyr Ala Leu Leu Lys Leu Arg
245 250 255 Leu Ile Arg Gly Lys Ser Ile Ile Phe Val Asn Ser Ile Asp
Arg 260 265 270 Cys Tyr Lys Val Arg Leu Phe Leu Glu Gln Phe Gly Ile
Arg Ala 275 280 285 Cys Val Leu Asn Ser Glu Leu Pro Ala Asn Ile Arg
Ile His Thr 290 295 300 Ile Ser Gln Phe Asn Lys Gly Thr Tyr Asp Ile
Ile Ile Ala Ser 305 310 315 Asp Glu His His Met Glu Lys Pro Gly Gly
Lys Ser Ala Thr Asn 320 325 330 Arg Lys Ser Pro Arg Ser Gly Asp Met
Glu Ser Ser Ala Ser Arg 335 340 345 Gly Ile Asp Phe Gln Cys Val Asn
Asn Val Ile Asn Phe Asp Phe 350 355 360 Pro Arg Asp Val Thr Ser Tyr
Ile His Arg Ala Gly Arg Thr Ala 365 370 375 Arg Gly Asn Asn Lys Gly
Ser Val Leu Ser Phe Val Ser Met Lys 380 385 390 Glu Ser Lys Val Asn
Asp Ser Val Glu Lys Lys Leu Cys Asp Ser 395 400 405 Phe Ala Ala Gln
Glu Gly
Glu Gln Ile Ile Lys Asn Tyr Gln Phe 410 415 420 Lys Met Glu Glu Val
Glu Ser Phe Arg Tyr Arg Ala Gln Asp Cys 425 430 435 Trp Arg Ala Ala
Thr Arg Val Ala Val His Asp Thr Arg Ile Arg 440 445 450 Glu Ile Lys
Ile Glu Ile Leu Asn Cys Glu Lys Leu Lys Ala Phe 455 460 465 Phe Glu
Glu Asn Lys Arg Asp Leu Gln Ala Leu Arg His Asp Lys 470 475 480 Pro
Leu Arg Ala Ile Lys Val Gln Ser His Leu Ser Asp Met Pro 485 490 495
Glu Tyr Ile Val Pro Lys Ala Leu Lys Arg Val Val Gly Thr Ser 500 505
510 Ser Ser Pro Val Gly Ala Ser Glu Ala Lys Gln Pro Arg Gln Ser 515
520 525 Ala Ala Lys Ala Ala Phe Glu Arg Gln Val Asn Asp Pro Leu Met
530 535 540 Ala Ser Gln Val Asp Phe Gly Lys Arg Arg Pro Ala His Arg
Arg 545 550 555 Lys Lys Lys Ala Leu 560 8 415 PRT Caenorhabditis
elegans g608464 8 Met Gln Ala Leu Tyr Gln Leu Ser Ala Thr Gly Ala
Gln Gln Gln 1 5 10 15 Asn Gln Gln Ile Pro Ile Gly Leu Ser Asn Ser
Leu Leu Tyr Gln 20 25 30 Gln Leu Ala Ala His Gln Gln Ile Ala Ala
Gln Gln His Gln Gln 35 40 45 Gln Leu Ala Val Ser Ala Ala His Gln
Thr Gln Asn Asn Ile Met 50 55 60 Leu Ala Thr Ser Ala Pro Ser Leu
Ile Asn His Met Glu Asn Ser 65 70 75 Thr Asp Gly Lys Val Lys Asp
Asp Pro Asn Ser Asp Tyr Asp Leu 80 85 90 Gln Leu Ser Ile Gln Gln
Arg Leu Ala Ala Ala Ala Gln Ala Ala 95 100 105 Gln Met Gly Gln Thr
Gln Ile Gly Pro Gln Ile Val Gly Gln Gln 110 115 120 Gly Gln Pro Val
Val Ala Thr Thr Ala Gly Ser Thr Asn Gly Ser 125 130 135 Ala Ala Val
Thr Gln Pro Asp Pro Ser Thr Ser Ser Gly Pro Asp 140 145 150 Gly Pro
Lys Arg Leu His Val Ser Asn Ile Pro Phe Arg Phe Arg 155 160 165 Asp
Pro Asp Leu Lys Thr Met Phe Glu Lys Phe Gly Val Val Ser 170 175 180
Asp Val Glu Ile Ile Phe Asn Glu Arg Gly Ser Lys Gly Phe Gly 185 190
195 Phe Val Thr Met Glu Arg Pro Gln Asp Ala Glu Arg Ala Arg Gln 200
205 210 Glu Leu His Gly Ser Met Ile Glu Gly Arg Lys Ile Glu Val Asn
215 220 225 Cys Ala Thr Ala Arg Val His Ser Lys Lys Val Lys Pro Thr
Gly 230 235 240 Gly Ile Leu Asp Gln Met Asn Pro Leu Met Ala Gln Ser
Ala Leu 245 250 255 Ala Ala Gln Ala Gln Met Asn Arg Ala Leu Leu Leu
Arg Ser Pro 260 265 270 Leu Val Ala Gln Ser Leu Leu Gly Arg Gly Pro
Ala Leu Ile Pro 275 280 285 Gly Met Gln Gln Pro Ala Phe Gln Leu Gln
Ala Ala Leu Ala Gly 290 295 300 Asn Pro Leu Ala Gln Leu Gln Gly Gln
Pro Leu Leu Phe Asn Ala 305 310 315 Ala Ala Leu Gln Thr Asn Ala Leu
Gln Gln Ser Ala Phe Gly Met 320 325 330 Asp Pro Ala Ala Val Leu Ala
Ala Leu Leu Ala Asn Glu Gln Ala 335 340 345 Arg Phe Gln Leu Ala Ala
Ala Ala Ala Gln Gly Asn Glu Tyr Ile 350 355 360 Met Tyr His Gln Ala
Lys Gln Gln Glu Leu Pro Gly Arg Ile Pro 365 370 375 Ser Ser Gly Asn
Ala Ser Ala Phe Gly Glu Gln Tyr Leu Ser Asn 380 385 390 Ala Leu Ala
Thr Ala Ser Leu Pro Ser Tyr Gln Met Asn Pro Ala 395 400 405 Leu Arg
Thr Leu Asn Arg Phe Thr Pro Tyr 410 415 9 255 PRT Homo sapiens
g37547 9 Met Val Lys Leu Thr Ala Glu Leu Ile Glu Gln Ala Ala Gln
Tyr 1 5 10 15 Thr Asn Ala Val Arg Asp Arg Glu Leu Asp Leu Arg Gly
Tyr Lys 20 25 30 Ile Pro Val Ile Glu Asn Leu Gly Ala Thr Leu Asp
Gln Phe Asp 35 40 45 Ala Ile Asp Phe Ser Asp Asn Glu Ile Arg Lys
Leu Asp Gly Phe 50 55 60 Pro Leu Leu Arg Arg Leu Lys Thr Leu Leu
Val Asn Asn Asn Arg 65 70 75 Ile Cys Arg Ile Gly Glu Gly Leu Asp
Gln Ala Leu Pro Cys Leu 80 85 90 Thr Glu Leu Ile Leu Thr Asn Asn
Ser Leu Val Glu Leu Gly Asp 95 100 105 Leu Asp Pro Leu Ala Ser Leu
Lys Ser Leu Thr Tyr Leu Ser Ile 110 115 120 Leu Arg Asn Pro Val Thr
Asn Lys Lys His Tyr Arg Leu Tyr Val 125 130 135 Ile Tyr Lys Val Pro
Gln Val Arg Val Leu Asp Phe Gln Lys Val 140 145 150 Lys Leu Lys Glu
Arg Gln Glu Ala Glu Lys Met Phe Lys Gly Lys 155 160 165 Arg Gly Ala
Gln Leu Ala Lys Asp Ile Ala Arg Arg Ser Lys Thr 170 175 180 Phe Asn
Pro Gly Ala Gly Leu Pro Thr Asp Lys Lys Arg Gly Gly 185 190 195 Pro
Ser Pro Gly Asp Val Glu Ala Ile Lys Asn Ala Ile Ala Asn 200 205 210
Ala Ser Thr Leu Ala Glu Val Glu Arg Leu Lys Gly Leu Leu Gln 215 220
225 Ser Gly Gln Ile Pro Gly Arg Glu Arg Arg Ser Gly Pro Thr Asp 230
235 240 Asp Gly Glu Glu Glu Met Glu Glu Asp Thr Val Thr Asn Gly Ser
245 250 255
* * * * *