U.S. patent application number 10/658434 was filed with the patent office on 2004-04-22 for human smn-like protein.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Corley, Neil C., Guegler, Karl J., Tang, Y. Tom.
Application Number | 20040077007 10/658434 |
Document ID | / |
Family ID | 21842832 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077007 |
Kind Code |
A1 |
Tang, Y. Tom ; et
al. |
April 22, 2004 |
Human SMN-like protein
Abstract
The invention provides a human SMN-like protein (HSLP) and
polynucleotides which identify and encode HSLP. The invention also
provides expression vectors, host cells, antibodies, agonists, and
antagonists. The invention also provides methods for treating or
preventing disorders associated with expression of HSLP.
Inventors: |
Tang, Y. Tom; (San Jose,
CA) ; Corley, Neil C.; (Castro Valley, CA) ;
Guegler, Karl J.; (Menlo Park, CA) |
Correspondence
Address: |
INCYTE CORPORATION
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
Palo Alto
CA
|
Family ID: |
21842832 |
Appl. No.: |
10/658434 |
Filed: |
September 8, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10658434 |
Sep 8, 2003 |
|
|
|
09571078 |
May 15, 2000 |
|
|
|
6620783 |
|
|
|
|
09571078 |
May 15, 2000 |
|
|
|
09028327 |
Feb 24, 1998 |
|
|
|
6130064 |
|
|
|
|
Current U.S.
Class: |
435/6.16 ;
435/199; 435/320.1; 435/325; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; A61K 38/00 20130101; A61P 15/00 20180101; A61P
25/00 20180101; C07K 14/475 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/199; 435/320.1; 435/325; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; A61K
038/17; C07H 021/04; C12N 009/22 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO:1,
b) a polypeptide comprising a naturally occurring an amino acid
sequence at least 90% identical to the amino acid sequence of SEQ
ID NO:1, c) a biologically active fragment of a polypeptide having
an amino acid sequence of SEQ ID NO:1, and d) an immunogenic
fragment of a polypeptide having an amino acid sequence of SEQ ID
NO:1.
2. An isolated polypeptide of claim 1, comprising the amino acid
sequence of SEQ ID NO:1.
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 the
polynucleotide sequence of SEQ ID NO:2.
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 the
amino acid sequence of SEQ ID NO:1.
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 of SEQ
ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to the
polynucleotide sequence of SEQ ID NO:2, 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 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises
the amino acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional HSLP, 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)
contacting a sample comprising a polypeptide of claim 1 with 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 HSLP, 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)
contacting a sample comprising a polypeptide of claim 1 with 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 HSLP, 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, said 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 polynucleotide sequence of claim 5, the
method comprising: a) contacting a sample comprising the target
polynucleotide with, 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 screening for potential 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 indicates
potential toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of HSLP 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 HSLP 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 HSLP 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 the amino
acid sequence of SEQ ID NO:1, or an immunogenic fragment thereof,
under conditions to elicit an antibody response, b) isolating
antibodies from said animal, and c) screening the isolated
antibodies with the polypeptide, thereby identifying a polyclonal
antibody which binds specifically to a polypeptide comprising the
amino acid sequence of SEQ ID NO:1.
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 the amino acid sequence
of SEQ ID NO:1, 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 binds specifically to a
polypeptide comprising the amino acid sequence of SEQ ID NO:1.
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 monoclonal 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 the amino acid
sequence of SEQ ID NO:1 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a 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 the amino acid sequence of
SEQ ID NO:1 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a 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 the amino acid sequence of SEQ ID
NO:1.
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 polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:2.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/571,078, filed May 15, 2000, now U.S. Pat.
No. 6,620,783, issued on Sep. 16, 2003, which is a continuation in
part (CIP) application of U.S. application Ser. No. 09/028,327
filed Feb. 24, 1998, now U.S. Pat. No. 6,130,064, issued Oct. 10,
2000, the contents all of which are hereby incorporated by
reference herein.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a human SMN-like protein and to the use of these
sequences in the diagnosis, treatment, and prevention of
neurological, reproductive, and cell proliferative disorders.
BACKGROUND OF THE INVENTION
[0003] Motor neurons directly control muscle activity throughout
the body. Motor neuron fibers that extend from the spinal cord to
the muscle transmit neural impulses. Motor neuron cell bodies lie
within gray matter, the inner core of the spinal cord. They are
confined to the anterior horn, one of three distinct functional
regions of gray matter. The motor neuron cell bodies receive
signals primarily from neurons contained in the other two regions
of gray matter. These neurons transmit signals from the brain and
other regions of the spinal cord.
[0004] Spinal muscular atrophy (SMA) is a fatal neurodegenerative
disorder that specifically affects motor neurons of the anterior
horn. Extensive loss of these neurons results in progressive muscle
weakness and paralysis leading to muscular atrophy. SMA is an
autosomal recessive disorder that occurs once in every 6000 live
births and has a carrier frequency of 1 in 40. Cystic fibrosis is
the only fatal autosomal recessive disorder that occurs with
greater frequency. SMA afflicts children, and three types of SMA
have been classified based on age of onset and clinical course of
the disease. Type I, also called infantile SMA or Werdnig-Hoffman
disease, is the most severe form with onset before six months of
age and death from respiratory failure by two years of age. Type
II, also called chronic childhood SMA or intermediate SMA, presents
at around 18 months of age and progresses slowly. Afflicted
children cannot walk unaided but survive beyond four years of age.
Type II, also called Wohlfart-Kugelberg-Welander disease, is the
mildest form with onset ranging from two years of age to
adolescence and variable degrees of muscular weakness (Lefebvre et
al. (1995) Cell 80:155-165).
[0005] SMA is caused by lesions in the survival motor neuron (smn)
gene on chromosome 5q13 (Burglen et al. (1996) Genomics
32:479-482). The normal chromosome 5 contains a duplication of the
smn locus, resulting in a telomere proximal smn gene (smn.sup.T)
and a centromere proximal smn gene (smn.sup.C). The two genes are
nearly identical in nucleotide sequence, and both encode a
294-amino acid protein of 38 kilodaltons. However, the smn.sup.C
RNA transcript can be alternatively spliced, and the resultant
protein is truncated at the C-terminus. The function of this
alternative protein product is unknown. Molecular genetic analysis
indicates that in over 98% of patients with SMA, sma.sup.T is
completely or partially deleted. In the remaining 2%, smn.sup.T
contains point mutations or alterations in splice site consensus
sequences. In addition, the severity of the lesion in smn.sup.T is
correlated with the clinical severity of SMA. These data indicate
that smn.sup.T, and not smn.sup.c, plays a critical role in the
determination of SMA (Lefebvre, supra). However, some studies
indicate that the activity of smn.sup.c may modulate the clinical
severity of SMA as previously established by defects in smn.sup.T
(Coovert et al. (1997) Hum Mol Genet 6:1205-1214). In general,
detection of lesions in smn.sup.T may provide the basis for
definitive prenatal and childhood diagnosis of SMA.
[0006] Quantitative western analysis shows that the protein, SMN,
is normally expressed at high levels not only in the spinal cord,
but also in the kidney, liver, and brain. Intermediate SMN levels
are detected in skeletal and cardiac muscle, and low levels are
detected in primary fibroblasts and lymphoblasts. The role, if any,
for SMN outside of the spinal cord is unclear, as the pathology of
SMA is specific to motor neuron muscle control (Coovert, supra). At
the cellular level, immunocytochemistry demonstrates that SMN is
localized to both the cytoplasm and the nucleus. SMN is diffusely
distributed throughout the cytoplasm, while nuclear SMN is
concentrated at discrete foci. These foci, called gems, are novel
structures that are intimately associated with coiled bodies.
Coiled bodies are subnuclear structures involved in RNA processing
and metabolism. An in vivo screen for SMN-interacting polypeptides
identified fibrillarin, a known component of coiled bodies, and the
RGG RNA-binding motif of hnRNP U, a nuclear protein involved in RNA
processing. These data suggest that the molecular basis of SMA may
involve defects in RNA processing in motor neurons (Liu and
Dreyfuss (1996) EMBO J 15:3555-3565).
[0007] The mouse homolog of smn has been cloned and localized to
chromosome 13 in a region syntenic to that of human chromosome
5q13. Unlike human smn, mouse smn (msmn) is a single-copy gene,
suggesting that duplication of the human locus is a recent
evolutionary event. msmn encodes a 288-amino acid protein that
shares 82% amino acid identity with human smn. Northern analysis
shows that msmn RNA is widely expressed in various tissues,
including heart, brain, kidney and testis (Viollet et al. (1997)
Genomics 40:185-188). Homozygous deletion of msmn is lethal during
the morula (16-64 cell) stage of embryogenesis. This phenotype is
much more severe than that of SMA in humans, suggesting that
differences in gene copy number may influence the severity of the
SMA phenotype. In humans, smn.sup.c may partially compensate for
deletion of smn.sup.T to delay disease onset and prolong survival
(Schrank et al. (1997) Proc Natl Acad Sci 94:9920-9925).
[0008] The discovery of a new human SMN-like protein and the
polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
treatment, and prevention of neurological, reproductive, and cell
proliferative disorders.
SUMMARY OF THE INVENTION
[0009] The invention is based on the discovery of a human SMN-like
protein, HSLP, which shows homology to mouse and human SMN, a
protein involved in motor neuron survival. The invention features a
substantially purified protein comprising the amino acid sequence
of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0010] The invention further provides a substantially purified
variant having at least 90% amino acid sequence identity to the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide
encoding the protein comprising the sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1. The invention also includes an isolated
and purified polynucleotide variant having at least 90%
polynucleotide sequence identity to the polynucleotide encoding the
protein comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
[0011] The invention further provides an isolated and purified
polynucleotide which hybridizes under stringent conditions to the
polynucleotide encoding the protein comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an
isolated and purified polynucleotide which is complementary to the
polynucleotide encoding the protein comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0012] The invention also provides an isolated and purified
polynucleotide comprising the polynucleotide sequence of SEQ ID
NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified
polynucleotide variant having at least 90% polynucleotide sequence
identity to the polynucleotide comprising the polynucleotide
sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention
also provides an isolated and purified polynucleotide having a
sequence complementary to the polynucleotide comprising the
polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID
NO:2. The invention also provides a polynucleotide fragment
comprising nucleotides 712-747 for detecting the presence or
expression of an identical endogenous gene.
[0013] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
protein comprising the sequence of SEQ ID NO:1 or a fragment of SEQ
ID NO:1. In another aspect, the expression vector is contained
within a host cell.
[0014] The invention also provides a method for producing a protein
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1, the method comprising the steps of: (a) culturing the
host cell containing an expression vector containing at least a
fragment of a polynucleotide encoding the protein comprising the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1
under conditions suitable for the expression of the protein; and
(b) recovering the protein from the host cell culture.
[0015] The invention also provides a pharmaceutical composition
comprising a substantially purified protein having the sequence of
SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a
suitable pharmaceutical carrier.
[0016] The invention further includes a purified antibody which
binds to a protein comprising the sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, as well as a purified agonist and a
purified antagonist of the protein.
[0017] The invention also provides a method for treating or
preventing a neurological disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified protein having the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
[0018] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified protein having the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1.
[0019] The invention also provides a method for treating or
preventing a cell proliferative disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of the protein having the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0020] The invention also provides a method for detecting a
polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a
biological sample containing nucleic acids, the method comprising
the steps of: (a) hybridizing the complement of the polynucleotide
encoding the protein comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic
acids of the biological 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 encoding the protein comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the
biological sample. In one aspect, the nucleic acids of the
biological sample are amplified by the polymerase chain reaction
prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the amino acid
sequence (SEQ ID NO:1) and nucleic acid sequence (SEQ ID NO:2) of
HSLP. The alignment was produced using LASERGENE software (Hitachi
Software Engineering, South San Francisco Calif.).
[0022] FIGS. 2A and 2B show the amino acid sequence alignments
among HSLP (3769729; SEQ ID NO:1), mouse SMN (GI 1857114; SEQ ID
NO:3), and human SMN (GI 1314346; SEQ ID NO:4), produced using the
multisequence alignment program of LASERGENE software (DNASTAR,
Madison Wis.).
DESCRIPTION OF THE INVENTION
[0023] Before the present proteins, polynucleotides, and methods
are described, it is understood that this invention is not limited
to the particular methodology, protocols, cell lines, vectors, and
reagents 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.
[0024] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural reference unless the
context clearly dictates otherwise. For example, a reference to "a
host cell" includes a plurality of such host cells, and a reference
to "an antibody" encompasses one or more antibodies and equivalents
thereof known to those skilled in the art.
[0025] 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 methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications and patents mentioned herein are
incorporated by reference herein and are cited for the purpose of
describing and disclosing the cell lines, vectors, and
methodologies 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.
[0026] Definitions
[0027] "HSLP" refers to a protein comprising the amino acid
sequence of an SMN-like protein obtained from any species including
bovine, ovine, porcine, murine, equine, and preferably the human
species, from any source, whether natural, synthetic,
semi-synthetic, or recombinant.
[0028] "Agonist" refers to a molecule which, when bound to HSLP,
increases or prolongs the duration of the effect of HSLP. Agonists
may include proteins, nucleic acids, carbohydrates, or any other
molecules which bind to and modulate the effect of HSLP.
[0029] An "allele" is an alternative form of the gene encoding
HSLP. Alleles 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 alleles 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.
[0030] "Altered" nucleic acid sequences encoding HSLP include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polynucleotide the same HSLP or a
polypeptide with at least one functional characteristic of HSLP.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding HSLP, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding HSLP.
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 HSLP. 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 HSLP 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.
[0031] "Amino acid sequence" refers 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 HSLP which are preferably about 5 to about 15
amino acids in length and which retain some biological activity or
immunological activity of HSLP. The amino acid sequence is not
limited to the complete native amino acid sequence associated with
the recited protein molecule.
[0032] "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.
[0033] "Antagonist" refers to a molecule which, when bound to HSLP,
decreases the amount or the duration of the effect of the
biological or immunological activity of HSLP. Antagonists may
include proteins, nucleic acids, carbohydrates, antibodies, or any
other molecules which decrease the effect of HSLP.
[0034] "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 HSLP 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.
[0035] "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.
[0036] "Antisense" refers to any composition containing a nucleic
acid sequence which is complementary to a specific nucleic acid
sequence. "Antisense strand" is used in reference to a nucleic acid
strand that is complementary to the "sense" strand. 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.
[0037] "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 HSLP, or of
any oligopeptide thereof, to induce a specific immune response in
appropriate animals or cells and to bind with specific
antibodies.
[0038] "Complementary" or "complementarity" refer to the natural
binding of polynucleotides under permissive salt and temperature
conditions by base pairing. For example, the sequence "A-G-T" binds
to the complementary sequence "T-C-A". 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.
[0039] 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, an aqueous solution, or a sterile composition.
Compositions comprising polynucleotide sequences encoding HSLP or
fragments of HSLP 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., SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, and the like).
[0040] "Consensus sequence" refers to a nucleic acid sequence which
has been resequenced to resolve uncalled bases, extended using
XL-PCR kit (PE Biosystems, Foster City Calif.) 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 (Computer Genetics Group (GCG), Madison,
Wis.). Some sequences have been both extended and assembled to
produce the consensus sequence.
[0041] The phrase "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 HSLP, by
northern analysis is indicative of the presence of nucleic acids
encoding HSLP in a sample, and thereby correlates with expression
of the transcript from the polynucleotide encoding HSLP.
[0042] 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.
[0043] "Derivative" refers to the chemical modification of HSLP, of
a polynucleotide sequence encoding HSLP, or of a polynucleotide
sequence complementary to a polynucleotide sequence encoding HSLP.
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.
[0044] "Homology" refers to a degree of complementarity. A
partially complementary sequence that at least partially inhibits
an identical sequence from hybridizing to a target nucleic acid is
referred to as "substantially homologous." 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.
[0045] "Hybridization stringency" is determined by G+C content of
the probe, salt concentration, and temperature. In particular,
stringency can be increased by reducing the concentration of salt
or raising the hybridization temperature. In solutions used for
some substrate based hybridizations, additions of an organic
solvent such as formamide allows the reaction to occur at a lower
temperature. Hybridization can be performed at low stringency with
buffers, such as 5.times.SSC with 1% sodium dodecyl sulfate (SDS)
at 60.degree. C., which permits the formation of a hybridization
complex between nucleotide sequences that contain some mismatches.
Subsequent washes are performed at higher stringency with buffers
such as 0.2.times.SSC with 0.1% SDS at either 45.degree. C. (medium
stringency) or 68.degree. C. (high stringency). At high stringency,
hybridization complexes will remain stable only where the nucleic
acid sequences are completely complementary. In some membrane-based
hybridizations, perferably 35% or most preferably 50%, formamide
can be added to the hybridization solution to reduce the
temperature at which hybridization is performed, and background
signals can be reduced by the use of other detergents such as
Sarkosyl or Triton X-100 and a blocking agent such as salmon sperm
DNA. Selection of components and conditions for hybridization are
well known to those skilled in the art.
[0046] "Percent identity" refers 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.). This program can create alignments between two or
more sequences according to different methods, e.g., the clustal
method. (See, e.g., Higgins and 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 homology between the two
amino acid sequences are not included in determining percentage
similarity.
[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] "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. "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., C.sub.0t or R.sub.0t analysis) or formed
between one nucleic acid sequence present in solution and another
nucleic acid sequence immobilized on a substrate (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).
[0050] "Insertion" and "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.
[0051] "Immune response" refers to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, and the like. 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.
[0052] "Microarray" refers to an arrangement of distinct
polynucleotides on a substrate.
[0053] "Element" and "array element" refer to a hybridizable
polynucleotide arrayed on the surface of a microarray.
[0054] "Modulate" refers to a change in the activity of HSLP. For
example, modulation may cause an increase or a decrease in protein
activity, binding characteristics, or any other biological,
functional, or immunological properties of HSLP.
[0055] "Nucleic acid sequence" refers to an oligonucleotide,
nucleotide, polynucleotide, or any fragment thereof, to DNA or RNA
of genomic or synthetic origin (cDNA) 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" refer to those
nucleic acid sequences which are greater than about 60 nucleotides
in length, and most preferably are at least about 100 nucleotides,
at least about 1000 nucleotides, or at least about 10,000
nucleotides in length.
[0056] 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 transcription of the encoded polypeptide.
While operably associated or operably linked nucleic acid sequences
can be contiguous and in reading frame, certain genetic elements,
e.g., repressor genes, are not contiguously linked to the encoded
polypeptide but still bind to operator sequences that control
expression of the polypeptide.
[0057] "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.
[0058] "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 and 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 HSLP, or fragments
thereof, or HSLP 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
solid support; a tissue; a tissue print; a fingerprint, and the
like.
[0060] "Specific binding" refers 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] "Stringent conditions" refers to conditions which permit
hybridization between polynucleotide sequences and the claimed
polynucleotide sequences. Suitably stringent conditions can be
defined by GC content of the polynucleotide sequence, salt
concentration in the prehybridization and hybridization solutions,
and hybridization temperature. These conditions are well known in
the art as is the fact that stringency can be increased by reducing
the concentration of salt, increasing the concentration of
formamide, or raising the hybridization temperature.
[0062] "Substantially purified" refers to nucleic acid or amino
acid sequences that are removed from their natural environment and
are 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] "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.
[0065] "Variant" refers to an amino acid sequence that is altered
by one or more amino acids. The variant may have "conservative"
changes, wherein a substituted amino acid has similar structural or
chemical properties (e.g., replacement of leucine with isoleucine)
or "nonconservative" changes wherein the substituted amino acid is
structurally or chemically different (e.g., replacement of glycine
with tryptophan). 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).
[0066] The Invention
[0067] The invention is based on the discovery of a new human
SMN-like protein (HSLP), the polynucleotides encoding HSLP, and the
use of these compositions for the diagnosis, treatment, or
prevention of neurological, reproductive, and cell proliferative
disorders.
[0068] Nucleic acids encoding the HSLP of the present invention
were first identified in Incyte Clone 3769729 from the breast
tissue cDNA library (BRSTNOT24) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:2, was
derived from the following overlapping and/or extended sequences:
Incyte Clones 3769729 (BRSTNOT24), 637394 (NEUTGMT01), 2207558
(SINTFET03), 1643342 (HEARFET01), and 1272275 (TESTTUT02). A
fragment of SEQ ID NO:2 from about nucleotide 712 to about
nucleotide 747 is useful for distinguishing nucleotide sequences
encoding HSLP from those encoding other known SMN-like proteins.
Northern analysis shows the expression of this sequence in various
libraries, at least 67% of which are associated with cell
proliferation. In particular, 38% of libraries expressing HSLP are
derived from reproductive tissue.
[0069] In one embodiment, the invention encompasses a protein
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A-1F. HSLP is 238 amino acids in length and has a potential
N-glycosylation site at N.sub.101; a potential casein kinase II
phosphorylation site at S.sub.141; and five potential protein
kinase C phosphorylation sites at S.sub.11, S.sub.72, S.sub.141,
T.sub.206, and T.sub.227. As shown in FIGS. 2A and 2B, HSLP has
chemical and structural homology with SMN from mouse (GI 1857114)
and from human (GI 1314346). In particular, HSLP and mouse SMN
share 18% identity, and HSLP and human SMN share 17% identity. In
addition, the regions of HSLP from W.sub.73 to D.sub.98, and from
G.sub.110 to E.sub.127 are highly conserved among SMN proteins from
three divergent mammalian species: human, mouse, and dog. For
example, these two regions of HSLP share 73% and 47% identity,
respectively, with the homologous regions of mouse and human SMN.
HSLP is similar in size to mouse and human SMN which are 288 and
294 amino acids in length, respectively.
[0070] The invention also encompasses HSLP variants. A preferred
HSLP 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 HSLP amino acid sequence, and which
contains at least one functional or structural characteristic of
HSLP.
[0071] The invention also encompasses polynucleotides with a
deletion, insertion, or substitution but which encodes HSLP or at
least one functional domain of HSLP. The protein produced by an
altered
[0072] The invention also encompasses a variant of a polynucleotide
sequence encoding HSLP. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding HSLP. A particular aspect of the invention encompasses a
variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2. Any one of the
polynucleotide variants described above can encode an amino acid
sequence which contains at least one functional or structural
characteristic of HSLP.
[0073] 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 HSLP, some bearing minimal
homology 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 HSLP, and all such variations are
to be considered as being specifically disclosed.
[0074] Although nucleotide sequences which encode HSLP and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HSLP under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HSLP or its derivatives
possessing a substantially different codon usage. 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 HSLP 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.
[0075] The invention also encompasses production of DNA sequences
which encode HSLP and HSLP 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 that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding HSLP or any fragment
thereof.
[0076] 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:2, or a fragment of SEQ ID NO:2, under various conditions of
stringency. (See, e.g., Wahl and Berger (1987) Methods Enzymol
152:399-407, and Kimmel (1987) Methods Enzymol 152:507-511.)
[0077] Methods for DNA sequencing are well known and generally
available 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, T7 SEQUENASE DNA
polymerase, Taq DNA polymerase, and THERMOSEQUENASE DNA polymerase
(Amersham Pharmacia Biotech (APB), Picataway N.J.), or combinations
of polymerases and proofreading exonucleases such as those found in
the ELONGASE amplification system (Life Technologies, Gaithersburg
Md.). Preferably, the process is automated with machines such as
the MICROLAB 2200 system (Hamilton, Reno Nev.), DNA ENGINE thermal
cycler (MJ Research, Watertown Mass.), and the ABI CATALYST thermal
cycler and ABI PRISM 373 and 377 DNA sequencing systems (PE
Biosytems).
[0078] The nucleic acid sequences encoding HSLP may be extended
utilizing a partial nucleotide sequence and employing various
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 primers to
retrieve unknown sequence adjacent to a known locus. (See, e.g.,
Sarkar (1993) PCR Methods Applic 2:318-322.) In particular, genomic
DNA is first amplified in the presence of a primer which is
complementary to a linker sequence within the vector and a primer
specific to a region of the nucleotide sequenc. The amplified
sequences are subjected to a second round of PCR with the same
linker primer and another specific primer internal to the first
one. Products of each round of PCR are transcribed with an
appropriate RNA polymerase and sequenced using reverse
transcriptase.
[0079] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (See, e.g.,
Triglia et al. (1988) Nucleic Acids Res 16:8186.) The primers may
be designed using commercially available software such as OLIGO
4.06 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 target
sequence at temperatures of about 68.degree. C. to 72.degree. C.
The method uses several restriction enzymes to generate a suitable
fragment in the known region of a gene. The fragment is then
circularized by intramolecular ligation and used as a PCR
template.
[0080] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (See, e.g.,
Lagerstrom et al. (1991) PCR Methods Applic 1:111-119.) In this
method, multiple restriction enzyme digestions and ligations may be
used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR. Other
methods which may be used to retrieve unknown sequences are known
in the art. (See, e.g., Parker et al. (1991) Nucleic Acids Res
19:3055-3060.) Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries (Clontech, Palo Alto Calif.) to walk
genomic DNA. This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0081] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially 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.
[0082] 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 fluorescent dyes (one for each
nucleotide) which are laser activated, 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, PE
Biosystems), 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
the sequencing of small pieces of DNA which might be present in
limited amounts in a particular sample.
[0083] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HSLP may be used in
recombinant DNA molecules to direct expression of HSLP, 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 these sequences
may be used to clone and express HSLP.
[0084] As will be understood by those of skill in the art, it may
be advantageous to produce HSLP-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0085] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HSLP-encoding sequences for a variety of reasons including,
but not limited to, alterations which modify 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, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0086] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HSLP may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of HSLP activity, it may
be useful to encode a chimeric HSLP protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the HSLP
encoding sequence and the heterologous protein sequence, so that
HSLP may be cleaved and purified away from the heterologous
moiety.
[0087] In another embodiment, sequences encoding HSLP may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers et al. (1980) Nucleic Acids Symp.
Ser. 7:215-223, and Horn et al. (1980) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, the protein itself may be produced using
chemical methods to synthesize the amino acid sequence of HSLP, or
a fragment thereof. For example, peptide synthesis can be performed
using various solid-phase techniques. (See, e.g., Roberge et a.
(1995) Science 269:202-204.) Automated synthesis maybe achieved
using the ABI 431A peptide synthesizer (PE Biosystems).
Additionally, the amino acid sequence of HSLP, 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 protein.
[0088] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez and
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 (1983) Proteins, Structures and
Molecular Properties, W H Freeman, New York N.Y.)
[0089] In order to express a biologically active HSLP, the
nucleotide sequences encoding HSLP or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0090] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HSLP 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 et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; and Ausubel et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0091] A variety of expression vector/host systems and control
elements may be utilized to contain and express sequences encoding
HSLP. The "control elements" or "regulatory sequences" are those
non-translated regions, e.g., enhancers, promoters, and 5' and
3'untranslated regions, of the vector and polynucleotide sequences
encoding HSLP which interact with host cellular proteins to carry
out transcription and translation. Such elements may vary in their
strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used. For example, when cloning in bacterial systems, inducible
promoters, e.g., hybrid lacZ promoter of the PBLUESCRIPT phagemid
(Stratagene, La Jolla Calif.) or PSPORT1 plasmid (Life
Technologies), may be used. The baculovirus polyhedrin promoter may
be used in insect cells. Promoters or enhancers derived from the
genomes of plant cells (e.g., heat shock, RUBISCO, and storage
protein genes) or from plant viruses (e.g., viral promoters or
leader sequences) may be cloned into the vector. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are preferable. If it is necessary to generate a cell line that
contains multiple copies of the sequence encoding HSLP, vectors
based on SV40 or EBV may be used with an appropriate selectable
marker.
[0092] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for HSLP. For example,
when large quantities of HSLP are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as PBLUESCRIPT phagemid (Stratagene),
in which the sequence encoding HSLP may be ligated into the vector
in frame with sequences for the amino-terminal Met and the
subsequent 7 residues of .beta.-galactosidase so that a hybrid
protein is produced, and pIN vectors (Van Heeke and Schuster (1989)
J Biol Chem 264:5503-5509). PGEX vectors (APB) may also be used to
express foreign proteins as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned protein of interest can be released from the GST
moiety at will.
[0093] In the yeast Saccharomvces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. (See, e.g., Ausubel,
supra; and Grant et al., (1987) Methods Enzymol 153:516-544.)
[0094] In cases where plant expression vectors are used, the
expression of sequences encoding HSLP may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi et
al. (1984) EMBO J. 3:1671-1680; Broglie et al. (1984) Science
224:838-843; and Winter et al. (1991) Results Probl Cell Differ
17:85-105). These constructs can be introduced into plant cells by
direct DNA or pathogen-mediated transformation. Such techniques are
described in a number of available reviews. (See, e.g., Hobbs or
Murry (1992) In: Yearbook of Science and Technology, McGraw Hill,
N.Y. N.Y.; pp. 191-196.)
[0095] An insect system may also be used to express HSLP. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding HSLP may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of sequences
encoding HSLP will render the polyhedrin gene inactive and produce
recombinant virus lacking coat protein. The recombinant viruses may
then be used to infect, for example, S. frugiperda cells or
Trichoplusia larvae in which HSLP may be expressed. (See, e.g.,
Engelhard et al. (1994) Proc Natl Acad Sci 91:3224-3227.)
[0096] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HSLP 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 a viable virus which is capable of expressing HSLP in
infected host cells. (See, e.g., Logan and 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.
[0097] HACs may also be employed to deliver larger fragments of DNA
than can be contained and expressed in 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.
[0098] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding HSLP. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding HSLP and its initiation codon and upstream
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 the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. 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 cell
system used. (See, e.g., Scharf et al. (1994) Results Probl Cell
Differ 20:125-162.)
[0099] 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 protein 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
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (Manassas Va.) and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0100] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing HSLP can be transformed 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
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type. The invention is
not limited by the vector, host cell or control elements
employed.
[0101] 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 genes and adenine
phosphoribosyltransferase genes, which can be employed in tk or apr
cells, respectively. (See, e.g., Wigler et al. (1977) Cell
11:223-232, and Lowy 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; npt confers resistance to the aminoglycosides
neomycin and G-418; and als and pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler et al. (1980) Proc Natl Acad Sci 77:3567-3570,
Colbere-Garapin et al. (1981) J Mol Biol 150:1-14, and Murry,
supra.) Additional selectable genes have been described, e.g.,
trpB, which allows cells to utilize indole in place of tryptophan,
or hisD, which allows cells to utilize histinol in place of
histidine. (See, e.g., Hartman and Mulligan (1988) Proc Natl Acad
Sci 85:8047-8051.) Visible markers, e.g., anthocyanins, B
glucuronidase and its substrate GUS, luciferase and its substrate
luciferin may be used. Green fluorescent proteins (GFP; Clontech)
can also 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 et al. (1995) Methods Mol Biol 55:121-131.)
[0102] 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 HSLP is inserted within a marker gene
sequence, transformed cells containing sequences encoding HSLP can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding HSLP 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.
[0103] Alternatively, host cells which contain the nucleic acid
sequence encoding HSLP and express HSLP 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 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.
[0104] The presence of polynucleotide sequences encoding HSLP can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
HSLP. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding HSLP
to detect transformants containing DNA or RNA encoding HSLP.
[0105] A variety of protocols for detecting and measuring the
expression of HSLP, using either polyclonal or monoclonal
antibodies specific for the protein, 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
HSLP is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art. (See, e.g.,
Hampton et al. (1990) Serological Methods, a Laboratory Manual, APS
Press, St Paul Minn., Section IV; and Maddox et al. (1983) J Exp
Med 158:1211-1216).
[0106] 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 HSLP include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HSLP, 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 ABP and Promega (Madison Wis.). 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.
[0107] Host cells transformed with nucleotide sequences encoding
HSLP 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 contained 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 HSLP may be designed to
contain signal sequences which direct secretion of HSLP through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding HSLP to nucleotide sequences
encoding a protein domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAG
extension/affinity purification system (Immunex, Seattle Wash.).
The inclusion of cleavable linker sequences, such as those specific
for Factor XA or enterokinase (Invitrogen, San Diego Calif.),
between the purification domain and the HSLP encoding sequence may
be used to facilitate purification. One such expression vector
provides for expression of a fusion protein containing HSLP and a
nucleic acid encoding 6 histidine residues preceding a thioredoxin
or an enterokinase cleavage site. The histidine residues facilitate
purification on immobilized metal ion affinity chromatography
(IMIAC; Porath et al. (1992) Prot Exp Purif 3:263-281). The
enterokinase cleavage site provides a means for purifying HSLP from
the fusion protein (Kroll et al. (1993) DNA Cell Biol
12:441-453).
[0108] Fragments of HSLP may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton (1984) Protein: Structures and
Molecular Properties, W H Freeman, New York N.Y., 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 (PE Biosystems). Various fragments
of HSLP may be synthesized separately and then combined to produce
the full length molecule.
[0109] Therapeutics
[0110] Chemical and structural homology exists among HSLP and SMN
from mouse (GI 1857114) and human (GI 1314346). In addition, HSLP
is expressed in reproductive and proliferating tissues. Therefore,
HSLP appears to play a role in neurological, reproductive, and cell
proliferative disorders.
[0111] Therefore, in one embodiment, HSLP or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a neurological disorder. Such disorders can include, but
are not limited to, akathesia, Alzheimer's disease, amnesia,
amyotrophic lateral sclerosis, bipolar disorder, catatonia,
cerebral neoplasms, dementia, depression, diabetic neuropathy,
Down's syndrome, tardive dyskinesia, dystonias, epilepsy,
Huntington's disease, peripheral neuropathy, multiple sclerosis,
neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia, schizophrenia, and Tourette's disorder.
[0112] In another embodiment, a vector capable of expressing HSLP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a neurological disorder including, but
not limited to, those described above.
[0113] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HSLP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a neurological disorder including, but not limited
to, those provided above.
[0114] In still another embodiment, an agonist which modulates the
activity of HSLP may be administered to a subject to treat or
prevent a neurological disorder including, but not limited to,
those listed above.
[0115] In another embodiment, HSLP or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
reproductive disorder. Such disorders can include, but are not
limited to, abnormal prolactin production, infertility, tubal
disease, ovulatory defects, endometriosis, perturbations of the
estrous and menstrual cycles, polycystic ovary syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors,
autoimmune disorders, ectopic pregnancy, teratogenesis, breast
cancer, fibrocystic breast disease, galactorrhea, abnormal
spermatogenesis, abnormal sperm physiology, testicular cancer,
prostate cancer, benign prostatic hyperplasia, prostatitis, and
gynecomastia.
[0116] In another embodiment, a vector capable of expressing HSLP
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a reproductive disorder including, but
not limited to, those described above.
[0117] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HSLP in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a reproductive disorder including, but not limited
to, those provided above.
[0118] In still another embodiment, an agonist which modulates the
activity of HSLP may be administered to a subject to treat or
prevent a reproductive disorder including, but not limited to,
those listed above.
[0119] In a further embodiment, an antagonist of HSLP may be
administered to a subject to treat or prevent a cell proliferative
disorder. Such disorders may include, but are not limited to,
arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis,
mixed connective tissue disease, myelofibrosis, paroxysmal
nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary
thrombocythemia, and cancers including 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,
nerve, pancreas, parathyroid, penis, prostate, salivary glands,
skin, spleen, testis, thymus, thyroid, and uterus. In one aspect,
an antibody which specifically binds HSLP 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 HSLP.
[0120] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding HSLP may be administered
to a subject to treat or prevent a cell proliferative disorder
including, but not limited to, those described above.
[0121] 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.
[0122] An antagonist of HSLP may be produced using methods which
are generally known in the art. In particular, purified HSLP may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind HSLP. Antibodies
to HSLP 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.
[0123] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with HSLP 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.
[0124] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to HSLP 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 HSLP amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0125] Monoclonal antibodies to HSLP 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 et
al. (1975) Nature 256:495-497, Kozbor et al. (1985) J Immunol
Methods 81:31-42, Cote et al. (1983) Proc Natl Acad Sci
80:2026-2030, and Cole et al. (1984) Mol Cell Biol 62:109-120.)
[0126] 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 et al. (1984) Proc Natl Acad Sci 81:6851-6855,
Neuberger et al. (1984) Nature 312:604-608, and Takeda 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 HSLP-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
(1991) Proc Natl Acad Sci 88:10134-10137.)
[0127] Antibodies may also be produced by inducing n 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 et al.
(1989) Proc Natl Acad Sci 86: 3833-3837, and Winter et al. (1991)
Nature 349:293-299.)
[0128] Antibody fragments which contain specific binding sites for
HSLP 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 et al. (1989) Science
246:1275-1281.)
[0129] 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 HSLP and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering HSLP epitopes
is preferred, but a competitive binding assay may also be employed.
(Maddox, supra.)
[0130] In another embodiment of the invention, the polynucleotides
encoding HSLP, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding HSLP 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 HSLP. Thus, complementary molecules or
fragments may be used to modulate HSLP 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 HSLP.
[0131] 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 which
will express nucleic acid sequences complementary to the
polynucleotides of the gene encoding HSLP. (See, e.g., Sambrook,
supra; and Ausubel, supra.)
[0132] Genes encoding HSLP can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide, or fragment thereof, encoding HSLP. 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.
[0133] 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 HSLP. 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 et al.
(1994) In: Huber and 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.
[0134] 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 HSLP.
[0135] 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.
[0136] 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 HSLP. 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.
[0137] 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 bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0138] 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 et al. (1997) Nature Biotechnol 15:462-466.)
[0139] 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.
[0140] 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 HSLP, antibodies to HSLP, and mimetics,
agonists, antagonists, or inhibitors of HSLP. 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.
[0141] 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.
[0142] 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.).
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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 acids. 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.
[0151] 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 HSLP, such
labeling would include amount, frequency, and method of
administration.
[0152] 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.
[0153] 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.
[0154] A therapeutically effective dose refers to that amount of
active ingredient, for example HSLP or fragments thereof,
antibodies of HSLP, and agonists, antagonists or inhibitors of
HSLP, 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 ED50 (the dose therapeutically effective in 50%
of the population) or LD50 (the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
LD50/ED50 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 ED50 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.
[0155] 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.
[0156] 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 proteins will be specific to particular cells,
conditions, locations, and the like
[0157] Diagnostics
[0158] In another embodiment, antibodies which specifically bind
HSLP may be used for the diagnosis of disorders characterized by
expression of HSLP, or in assays to monitor patients being treated
with HSLP or agonists, antagonists, or inhibitors of HSLP.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as described above for therapeutics. Diagnostic assays
for HSLP include methods which utilize the antibody and a label to
detect HSLP 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.
[0159] A variety of protocols for measuring HSLP, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of HSLP expression. Normal or
standard values for HSLP expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to HSLP under conditions suitable
for complex formation. The amount of standard complex formation may
be quantitated by various methods, preferably by photometric means.
Quantities of HSLP 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.
[0160] In another embodiment of the invention, the polynucleotides
encoding HSLP 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 HSLP may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of HSLP, and to
monitor regulation of HSLP levels during therapeutic
intervention.
[0161] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HSLP or closely related molecules may be used
to identify nucleic acid sequences which encode HSLP. 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 HSLP, alleles, or related
sequences.
[0162] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the HSLP 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:2 or from genomic sequences
including promoters, enhancers, and introns of the HSLP gene.
[0163] Means for producing specific hybridization probes for DNAs
encoding HSLP include the cloning of polynucleotide sequences
encoding HSLP or HSLP 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.
[0164] Polynucleotide sequences encoding HSLP may be used for the
diagnosis of a disorder associated with expression of HSLP.
Examples of such a disorder include, but are not limited to, a
neurological disorder such as akathesia, Alzheimer's disease,
amnesia, amyotrophic lateral sclerosis, bipolar disorder,
catatonia, cerebral neoplasms, dementia, depression, diabetic
neuropathy, Down's syndrome, tardive dyskinesia, dystonias,
epilepsy, Huntington's disease, peripheral neuropathy, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid
psychoses, postherpetic neuralgia, schizophrenia, and Tourette's
disorder; a reproductive disorder such as abnormal prolactin
production, infertility, tubal disease, ovulatory defects,
endometriosis, perturbations of the estrous and menstrual cycles,
polycystic ovary syndrome, ovarian hyperstimulation syndrome,
endometrial and ovarian tumors, autoimmune disorders, ectopic
pregnancy, teratogenesis, breast cancer, fibrocystic breast
disease, galactorrhea, abnormal spermatogenesis, abnormal sperm
physiology, testicular cancer, prostate cancer, benign prostatic
hyperplasia, prostatitis, and gynecomastia; and a cell
proliferative disorder such as arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis, mixed connective tissue disease,
myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia
vera, psoriasis, primary thrombocythemia, and cancers including
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, nerve, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus.
The polynucleotide sequences encoding HSLP may be used in Southern
or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; in dipstick, pin, and ELISA-like
assays; and in microarrays utilizing fluids or tissues from
patients to detect altered HSLP expression. Such qualitative or
quantitative methods are well known in the art.
[0165] In a particular aspect, the nucleotide sequences encoding
HSLP may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding HSLP 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 HSLP 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.
[0166] In order to provide a basis for the diagnosis of a disorder
associated with expression of HSLP, 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 HSLP, 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.
[0167] 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.
[0168] With respect to cancer, the presence of a relatively high
amount of transcript 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.
[0169] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HSLP 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 HSLP, or a fragment of a
polynucleotide complementary to the polynucleotide encoding HSLP,
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.
[0170] Methods which may also be used to quantitate the expression
of HSLP include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby et al. (1993) J
Immunol Methods 159:235-244, and Duplaa 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-like format where the
oligomer of interest is presented in various dilutions and a
spectrophotometric or colorimetric response gives rapid
quantitation.
[0171] 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.
[0172] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan et al. (1995) U.S.
Pat. No. 5,474,796; Schena et al. (1996) Proc Natl Acad Sci
93:10614-10619; Baldeschweiler et al. (1995) WO95/251116; Shalon et
al. (1995) WO95/35505; Heller et al. (1997) Proc Natl Acad Sci
94:2150-2155; and Heller et al. U.S. Pat. No. 5,605,662.)
[0173] In another embodiment of the invention, nucleic acid
sequences encoding HSLP 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., HACs, yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), bacterial P1
constructions, or single chromosome cDNA libraries. (See, e.g.,
Price (1993) Blood Rev 7:127-134, and Trask (1991) Trends Genet
7:149-154.)
[0174] 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, Molecular
Biology and Biotechnology, VCH Publishers, New York N.Y., 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
HSLP 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.
[0175] 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., AT to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti 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, and the like, among normal, is carrier,
or affected individuals.
[0176] In another embodiment of the invention, HSLP, 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 HSLP and the agent being tested may be
measured.
[0177] 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)
WO84/03564.) In this method, large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The test compounds are reacted with
HSLP, or fragments thereof, and washed. Bound HSLP is then detected
by methods well known in the art. Purified HSLP 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.
[0178] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HSLP specifically compete with a test compound for binding
HSLP. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
HSLP.
[0179] In additional embodiments, the nucleotide sequences which
encode HSLP 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.
[0180] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0181] I. BRSTNOT24 cDNA Library Construction
[0182] The BRSTNOT24 cDNA library was constructed from diseased
breast tissue removed from a 46-year-old Caucasian female during
bilateral subcutaneous mammectomy, bilateral breast augmentation,
and total breast reconstruction. Pathology indicated benign
fibrocystic disease bilaterally. The patient presented with
fibrosclerosis of the breast. Family history included breast cancer
in the mother and sibling.
[0183] The frozen tissue was homogenized and lysed in TRIZOL
reagent (1 gm tissue/10 ml 1; Life Technologies) using a POLYTRON
homogenizer (PT-3000 homogenizer; Brinkmann Instruments, Westbury
N.Y.). After brief incubation on ice, chloroform was added (1:5
v/v), and the mixture was centrifuged to separate the phases. The
aqueous phase was removed to a fresh tube, and isopropanol was
added to precipitate the RNA. The RNA was resuspended in RNase-free
water and treated with DNase. The RNA was re-extracted once with
acid phenol-chloroform and reprecipitated with sodium acetate and
ethanol. Poly(A+) RNA was isolated using the OLIGOTEX kit (Qiagen,
Chatsworth, Calif.).
[0184] Poly(A+) RNA was used to construct the BRSTNOT24 cDNA
library according to the recommended protocols in the SUPERSCRIPT
plasmid system (Life Technologies). The cDNAs were fractionated on
a SEPHAROSE CL4B column (APB), and those cDNAs exceeding 400 bp
were ligated into the pINCY plasmid (Incyte Corporation, Palo Alto
Calif.). The plasmid was subsequently transformed into DH5.alpha.
competent cells (Life Technologies).
[0185] II. Isolation and Sequencing of cDNA Clones
[0186] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96-well plasmid purification 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 (Carb) 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 was
resuspended in 0.1 ml of distilled water. The DNA was stored at
4.degree. C.
[0187] The cDNAs were prepared using a MICROLAB 2200 system
(Hamilton) in combination with DNA ENGINE thermal cyclers (M J
Research) and sequenced by the method of Sanger et al. (1975, J Mol
Biol 94:441f), using ABI PRISM 377 DNA sequencing systems (PE
Biosystems).
[0188] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0189] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (See, e.g., Altschul (1993) J Mol Evol 36:290-300,
and Altschul et al. (1990) J Mol Biol 215:403-410.)
[0190] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms could have been used when dealing with
primary sequence patterns and secondary structure gap penalties.
(See, e.g., Smith et al. (1992) Protein Engineering 5:35-51.) The
sequences disclosed in this application have lengths of at least 49
nucleotides and have no more than 12% uncalled bases (where N is
recorded rather than A, C, G, or T).
[0191] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-8 for peptides.
[0192] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam). Deduced amino acid sequences from the
same clones were then searched against GenBank functional protein
databases, mammalian (mamp), vertebrate (vrtp), and eukaryote
(eukp), for homology.
[0193] Additionally, sequences identified from cDNA libraries may
be analyzed to identify those gene sequences encoding conserved
protein motifs using an appropriate analysis program, e.g., BLOCK 2
bioanalysis program (Incyte Corporation). This motif analysis
program, based on sequence information contained in the Swiss-Prot
Database and PROSITE, is a method of determining the function of
uncharacterized proteins translated from genornic or cDNA
sequences. (See, e.g., Bairoch et al. (1997) Nucleic Acids Res
25:217-221, and Attwood et al. (1997) J Chem Inf Comput Sci
37:417-424.) PROSITE may be used to identify common functional or
structural domains in divergent proteins. The method is based on
weight matrices. Motifs identified by this method are then
calibrated against the SWISS-PROT database in order to obtain a
measure of the chance distribution of the matches.
[0194] In another alternative, Hidden Markov models (HMMs) may be
used to find protein domains, each defined by a dataset of proteins
known to have a common biological function. (See, e.g., Pearson,
and Lipman (1988) Proc Natl Acad Sci 85:2444-2448, and Smith and
Waterman (1981) J Mol Biol 147:195-197.) HMMs were initially
developed to examine speech recognition patterns, but are now being
used in a biological context to analyze protein and nucleic acid
sequences as well as to model protein structure. (See, e.g., Krogh
et al. (1994) J Mol Biol 235:1501-1531, and Collin et al. (1993)
Protein Sci 2:305-314.) HMMs have a formal probabilistic basis and
use position-specific scores for amino acids or nucleotides. The
algorithm continues to incorporate information from newly
identified sequences to increase its motif analysis
capabilities.
[0195] IV. Northern Analysis
[0196] 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; and Ausubel, supra.)
[0197] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Corporation). 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 homologous.
[0198] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0199] 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. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0200] The results of northern analysis are reported as a list of
libraries in which the transcript encoding HSLP occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0201] V. Extension of HSLP Encoding Polynucleotides
[0202] The nucleic acid sequence of Incyte Clone 3769729 was used
to design oligonucleotide primers for extending a partial
nucleotide sequence to full length. One primer was synthesized to
initiate extension of an antisense polynucleotide, and the other
was synthesized to initiate extension of a sense polynucleotide.
Primers were used to facilitate the extension of the known sequence
"outward" generating amplicons containing new unknown nucleotide
sequence for the region of interest. The initial primers were
designed from the cDNA using OLIGO 4.06 primer analysis 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.
[0203] Selected human cDNA libraries (Life Technologies) were used
to extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0204] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (PE Biosystems) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the DNA
ENGINE thermal cycler (M J Research), beginning with 40 pmol of
each primer and the recommended concentrations of all other
components of the kit, with the following parameters: Step 1,
94.degree. C. for 1 min (initial denaturation); Step 2, 65.degree.
C. for 1 min; Step 3, 68.degree. C. for 6 min; Step 4, 94.degree.
C. for 15 sec; Step 5, 65.degree. C. for 1 min; Step 6, 68.degree.
C. for 7 min; Step 7, Repeat steps 4 through 6 for an additional 15
cycles; Step 8, 94.degree. C. for 15 sec; Step 9, 65.degree. C. for
1 min; Step 10, 68.degree. C. for 7:15 min; Step 11, Repeat steps 8
through 10 for an additional 12 cycles; Step 12, 72.degree. C. for
8 min; and Step 13, hold at 4.degree. C.
[0205] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK DNA
purification kit (Qiagen), and trimmed of overhangs using Klenow
enzyme to facilitate religation and cloning.
[0206] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook, supra, Appendix A, p.
2). After incubation for one hour at 37.degree. C., the E. coli
mixture was plated on Luria Bertani (LB) agar (Sambrook, supra,
Appendix A, p. 1) containing 2.times.Carb. The following day,
several colonies were randomly picked from each plate and cultured
in 150 .mu.l of liquid LB/2.times.Carb medium placed in an
individual well of an appropriate commercially-available sterile
96-well microtiter plate. The following day, 5 .mu.l of each
overnight culture was transferred into a non-sterile 96-well plate
and, after dilution 1:10 with water, 5 .mu.l from each sample was
transferred into a PCR array.
[0207] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions: Step 1, 94.degree. C.
for 60 sec; Step 2, 94.degree. C. for 20 sec; Step 3, 55.degree. C.
for 30 sec; Step 4, 72.degree. C. for 90 sec; Step 5, Repeat steps
2 through 4 for an additional 29 cycles, Step 6, 72.degree. C. for
180 sec; and Step 7, hold at 4.degree. C.
[0208] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0209] In like manner, the nucleotide sequence of SEQ ID NO:2 is
used to obtain 5' regulatory sequences using the procedure above,
oligonucleotides designed for 5' extension, and an appropriate
genomic library.
[0210] VI. Labeling and Use of Individual Hybridization Probes
[0211] Hybridization probes derived from SEQ ID NO:2 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 primer analysis
software (National Biosciences) and labeled by combining 50 pmol of
each oligomer, 250 .mu.Ci of [.gamma.-.sup.32P] adenosine
triphosphate (APB), and T4 polynucleotide kinase (NEN Life Science
Products, Boston Mass.). The labeled oligonucleotides are
substantially purified using a SEPHADEX G-25 superfine resin column
(APB). 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 (NEN
Life Science Products).
[0212] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to NYTRAN PLUS nylon membranes
(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
autoradiography film (Eastman Kodak, Rochester N.Y.) is exposed to
the blots, hybridization patterns are compared.
[0213] VII. Microarrays
[0214] 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.
[0215] Full-length cDNAs 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). cDNAs 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 et al. (1995) Science 270:467-470, and Shalon 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.
[0216] VIII. Complementary Polynucleotides
[0217] Sequences complementary to the HSLP-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring HSLP. 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 primer analysis software and the coding
sequence of HSLP. 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 HSLP-encoding transcript.
[0218] IX. Expression of HSLP
[0219] Expression of HSLP is accomplished by subcloning the cDNA
into an appropriate vector and transforming the vector into host
cells. This vector contains an appropriate promoter, e.g.,
.beta.-galactosidase, upstream of the cloning site, operably
associated with the cDNA of interest. (See, e.g., Sambrook, supra,
pp. 404-433; and Rosenberg et al (1983) Methods Enzymol
101:123-138.)
[0220] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopy-ranoside using standard methods
produces a fusion protein which consists of the first 8 residues of
.beta.-galactosidase, about 5 to 15 residues of linker, and the
protein. The signal residues direct the secretion of HSLP into
bacterial growth media which can be used directly in the following
assay for activity.
[0221] X. Demonstration of HSLP Activity
[0222] An assay for HSLP activity measures its affinity for
proteins involved in RNA processing. The yeast two-hybrid system is
a sensitive, enzymatic method for detection of protein-protein
interactions in vivo. This method is used to identify
SMN-interacting proteins (Liu and Dreyfuss, supra) and is well
known by those skilled in the art. Recombinant DNA methods are used
to express HSLP, fibrillarin, and the RGG RNA-binding motif of
hnRNP U (RGG) in the yeast Saccharomyces cerevisiae. These proteins
are expressed as fusions with other protein fragments involved in
gene regulation. The interaction of HSLP with either fibrillarin or
RGG triggers the expression of a reporter gene. This gene encodes a
metabolic enzyme that generates a colored reaction product. When
plated on the appropriate substratum, the yeast will turn from
white to blue in color. The amount of reaction product can be
quantified spectrophotometrically and is proportional to the
affinity of HSLP for either fibrillarin or RGG.
[0223] XI. Production of HSLP Specific Antibodies
[0224] HSLP substantially purified using PAGE electrophoresis
(Harrington (1990) Methods Enzymol 182:488-495), or other
purification techniques, is used to immunize rabbits and to produce
antibodies using standard protocols well known in the art. The HSLP
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. Methods
for selection of appropriate epitopes, such as those near the
C-terminus or in hydrophilic regions are well described in Ausubel
(supra).
[0225] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an ABI 431A peptide synthesizer using
Fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St. Louis Mo.) by
reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester to
increase immunogenicity. (See Ausubel, supra.) Rabbits are
immunized with the oligopeptide-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide activity,
for example, by binding the peptide to plastic, blocking with 1%
BSA, reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0226] XII. Purification of Naturally Occurring HSLP Using Specific
Antibodies
[0227] Naturally occurring or recombinant HSLP is substantially
purified by immunoaffinity chromatography using antibodies specific
for HSLP. An immunoaffinity column is constructed by covalently
coupling anti-HSLP antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (APB). After the coupling, the
resin is blocked and washed according to the manufacturer's
instructions.
[0228] Media containing HSLP are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of HSLP (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/HSLP 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 HSLP is collected.
[0229] XIII. Identification of Molecules Which Interact with
HSLP
[0230] HSLP, or biologically active fragments thereof, are labeled
with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973)
Biochem J 133:529-539.) Candidate molecules previously arrayed in
the wells of a multi-well plate are incubated with the labeled
HSLP, washed, and any wells with labeled HSLP complex are assayed.
Data obtained using different concentrations of HSLP are used to
calculate values for the number, affinity, and association of HSLP
with the candidate molecules
[0231] 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
* * * * *