U.S. patent application number 10/032366 was filed with the patent office on 2002-11-21 for human retinoid binding protein.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Bandman, Olga, Guegler, Karl J., Shah, Purvi.
Application Number | 20020173002 10/032366 |
Document ID | / |
Family ID | 25410401 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020173002 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
November 21, 2002 |
Human retinoid binding protein
Abstract
The invention provides a human retinoid binding protein (RBPhu)
and polynucleotides which identify and encode RBPhu. The invention
also provides expression vectors, host cells, agonists, antibodies
and antagonists. The invention also provides methods for treating
disorders associated with expression of RBPhu.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Guegler, Karl J.; (Menlo Park, CA)
; Shah, Purvi; (Sunnyvale, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
25410401 |
Appl. No.: |
10/032366 |
Filed: |
December 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10032366 |
Dec 21, 2001 |
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09382079 |
Aug 24, 1999 |
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09382079 |
Aug 24, 1999 |
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08899031 |
Jul 22, 1997 |
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6046027 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4702 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.2 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C07K 014/705 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising the amino acid sequence of SEQ ID NO:1,
b) a polypeptide comprising a naturally occurring 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
the amino acid sequence of SEQ ID NO:1, and d) an immunogenic
fragment of a polypeptide having the 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 the 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 RBPhu, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional RBPhu, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional RBPhu, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of RBPhu 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 RBPhu in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of RBPhu 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 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 the 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. 09/382,079, filed Aug. 24, 1999, entitled
HUMAN RETINOID BINDING PROTEIN, which is a divisional application
of U.S. application Ser. No. 08/899,031, filed Jul. 22, 1997, and
issued on Apr. 4, 2000 as U.S. Pat. No. 6,046,027, entitled HUMAN
RETINOID BINDING PROTEIN. Both of these applications are hereby
expressly incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a human retinoid binding protein and to the use of
these sequences in the diagnosis, prevention, and treatment of
cancer, inflammation, and disorders associated with retinoid
metabolism.
BACKGROUND OF THE INVENTION
[0003] Vitamin A (retinol) is a fat-soluble vitamin found mainly in
fish liver oils, liver, egg yolk, butter, and cream. Green leafy
and yellow vegetables contain beta-carotene and other provitamin
carotenoids which are converted to retinol in the mucosal cells of
the small intestine. Retinol cannot be synthesized in vivo and must
be obtained from the diet. Retinol is metabolized into the
biologically active derivative retinoic acid (RA) in a variety of
cells. The 11-cis isomer of retinal (vitamin A.sub.1 aldehyde),
combined with a protein moiety, forms the prosthetic group of
photoreceptor pigments in the retina that are involved in night,
day, and color vision. Retinol, RA, and other retinoids also
influence epithelial cell differentiation.
[0004] A number of carrier proteins which bind retinol or other
retinoids have been identified. These carrier proteins are similar
to the fatty acid binding proteins, a family of small (14-16 kDa)
cytosolic proteins which bind long-chain fatty acids, fatty
acyl-coenzyme A (CoA) derivatives, and other hydrophobic molecules
(Veerkamp, J. H. et al. (1991) Biochim. Biophys. Acta 108:1-24).
Cellular retinol-binding proteins (CRBPs) are found within cells
which participate in vitamin A metabolism or function. CRBPs, which
are expressed in a variety of organs and tissues, deliver retinol
to specific metabolic enzymes and to specific binding sites within
the nucleus, participate in the transepithelial movement of retinol
across blood-organ barriers, and are involved in the intestinal
absorption of vitamin A.
[0005] Retinoid binding proteins appear to direct bound retinoid
molecules to specific metabolic pathways. The retinoid binding
proteins also protect the cell from the damaging effects of
unliganded retinols (such as membrane disruption) and likewise
protect structurally unstable retinols from non-enzymatic side
reactions (such as isomerization and oxidation). Retinoid binding
proteins also appear to function as sensors of retinoid
concentration and act as modulators of retinoid metabolism (Napoli,
J. L. (1996) FASEB J. 10:993-1001). Retinoid binding proteins have
been cloned and characterized from a variety of sources including
mouse and human (Smith, W. C. et al. (1991) EMBO J. 10:2223-2230;
Colantuoni, V. et al. (1985) Biochem. Biophys. Res. Commun.
130:431-439).
[0006] Retinoids have been found to be effective in suppressing
tumor development in several carcinogenesis model systems and in
human subjects (Lotan, R. (1996) FASEB J. 10:1031-1039, and
references therein). Some retinoids were found to be active in
certain animal models and not in others. The effect of retinoids
was not restricted to a specific carcinogen, but rather to the type
of tissue involved. This restriction suggests that some retinoids
exhibit tissue selectivity. Paradoxically, studies have also
demonstrated that certain retinoids which are active inhibitors of
carcinogenesis in particular tissues can act as enhancers of
carcinogenesis in the same tissue of a different strain of mouse or
in a different carcinogenesis model (Lotan, supra).
[0007] The abundance of intracellular retinoid binding proteins may
play a role in the response of various tissues to retinoids.
Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA)
causes their irreversible differentiation into extraembryonic
endoderm. Boylan, J. F. et al. (1991; J. Cell. Biol. 112:965-979)
generated stably transfected F9 stem cell lines expressing either
elevated or reduced levels of functional CRABP-I protein. CRABP-I
is a retinol binding protein which preferentially binds retinoic
acid. Cell lines expressing elevated levels of CRABP-I exhibited a
significant reduction in the expression of RA-inducible mRNAs at
low exogenous RA concentrations, but this reduction was eliminated
at higher RA concentrations. Thus, higher levels of CRABP-I reduced
the potency of RA in this differentiation system. Boylan et al.
(supra) proposed that the retinol binding protein sequesters RA
within the cell and thereby prevents RA from acting to regulate
differentiation specific gene expression, and that the level of the
binding protein may affect tissue response to RA during
development.
[0008] Retinoids affect sebaceous gland activity and exhibit
immunomodulatory and anti-inflammatory properties (Orfanos, C. E.
et al. (1997) Drugs 53:358-388). Retinoids have been used for
topical and systemic treatment of psoriasis and other
hyperkeratotic and parakeratotic skin disorders, severe acne and
acne-related dermatoses, and for therapy and/or chemoprevention of
skin cancer and other neoplasia including T-cell lymphoma (Orfanos,
et al., supra). Treatment of human pancreatic carcinoma cell lines
with retinoids resulted in growth inhibition and differentiation of
ductal, but not acinar, pancreatic tumor cells (Rosewicz, S. et al.
(1995) Gastroenterology 109:1646-1660).
[0009] Toxic side-effects associated with retinoid treatments
include changes in the skin and mucous membranes (dry skin, hair
loss, dry nose, conjunctivitis), musculoskeletal symptoms,
ophthalmological effects, changes in transarinase activity, changes
in clinical chemistry markers (increase in serum triglycerides and
decrease in high-density lipoproteins) and, rarely, central nervous
system effects. A serious toxicological aspect of retinoid
treatment is teratogenesis. Retinoid therapies are not recommended
for women of childbearing age, and conception should be prevented
for a significant period of time after stopping treatment (La
Vecchia, C. et al. (1996) IARC Sci. Publ. 139:135-142).
[0010] Inadequate intake or utilization of vitamin A can impair
dark adaptation and cause night blindness; xerosis of the
conjunctiva and cornea; xerophthalmia and keratomalacia;
keratinization of lung, GI tract, and urinary tract epithelia; and
increased susceptibility to infections. Defective taste and smell,
and anemia that may be masked by hemoconcentration, have also been
reported (Berkow, R. and Fletcher, A. J. (1992) The Merck Manual of
Diagnosis and Therapy, Merck & Co., Rahway, N.J., pp.
959-960).
[0011] Primary vitamin A deficiency is usually caused by prolonged
dietary deprivation. It is endemic in areas such as southern and
eastern Asia where rice, devoid of carotene, is the staple.
Secondary deficiency may be due to inadequate conversion of
carotene, or to interference with absorption, storage, or transport
of vitamin A. Interference with absorption or storage is likely in
celiac disease, sprue, cystic fibrosis, operations on the pancreas,
duodenal bypass, congenital partial obstruction of the jejunum,
obstruction of the bile ducts, giardiasis, and cirrhosis of the
liver. Vitamin A deficiency is common in protein-energy
malnutrition not only because the diet is deficient, but also
because vitamin A storage and transport are defective. Liver stores
are depleted in deficiency before plasma levels begin to fall,
followed by retinal dysfunction, and finally by epithelial
structural changes.
[0012] The discovery of a new human retinoid binding protein and
the polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer, inflammation, and disorders
associated with retinoid metabolism.
SUMMARY OF THE INVENTION
[0013] The invention features a substantially purified polypeptide,
human retinoid binding protein (RBPhu), having the amino acid
sequence shown in SEQ ID NO:1, or fragments thereof.
[0014] The invention further provides an isolated and substantially
purified polynucleotide sequence encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or fragments
thereof and a composition comprising said polynucleotide sequence.
The invention also provides a polynucleotide sequence which
hybridizes under stringent conditions to the polynucleotide
sequence encoding the amino acid sequence SEQ ID NO:1, or fragments
of said polynucleotide sequence. The invention further provides a
polynucleotide sequence comprising the complement of the
polynucleotide sequence encoding the amino acid sequence of SEQ ID
NO:1, or fragments or variants of said polynucleotide sequence.
[0015] The invention also provides an isolated and purified
sequence comprising SEQ ID NO:2 or variants thereof. In addition,
the invention provides a polynucleotide sequence which hybridizes
under stringent conditions to the polynucleotide sequence of SEQ ID
NO:2. The invention also provides a polynucleotide sequence
comprising the complement of SEQ ID NO:2, or fragments or variants
thereof.
[0016] The present invention further provides an expression vector
containing at least a fragment of any of the claimed polynucleotide
sequences. In yet another aspect, the expression vector containing
the polynucleotide sequence is contained within a host cell.
[0017] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment thereof, the method comprising the steps of: a) culturing
the host cell containing an expression vector containing at least a
fragment of the polynucleotide sequence encoding RBPhu under
conditions suitable for the expression of the polypeptide; and b)
recovering the polypeptide from the host cell culture.
[0018] The invention also provides a pharmaceutical composition
comprising a substantially purified RBPhu having the amino acid
sequence of SEQ ID NO:1 in conjunction with a suitable
pharmaceutical carrier.
[0019] The invention also provides a purified antagonist of the
polypeptide of SEQ ID NO:1. In one aspect the invention provides a
purified antibody which binds to a polypeptide comprising the amino
acid sequence of SEQ ID NO:1.
[0020] Still further, the invention provides a purified agonist of
the polypeptide of SEQ ID NO:1.
[0021] The invention also provides a method for modulating free
intracellular retinoid concentrations comprising administering to a
subject in need of such treatment an effective amount of a
pharmaceutical composition comprising purified RBPhu.
[0022] The invention also provides a method for treating or
preventing cancer comprising administering to a subject in need of
such treatment an effective amount of an antagonist to RBPhu.
[0023] The invention also provides a method for treating or
preventing inflammation comprising administering to a subject in
need of such treatment an effective amount of an antagonist to
RBPhu.
[0024] The invention also provides a method for treating or
preventing a disorder associated with retinoid metabolism
comprising administering to a subject in need of such treatment an
effective amount of an antagonist to RBPhu.
[0025] The invention also provides a method for detecting a
polynucleotide which encodes RBPhu in a biological sample
comprising the steps of: a) hybridizing the complement of the
polynucleotide sequence which encodes SEQ ID NO:1 to nucleic acid
material of a biological sample, thereby forming a hybridization
complex; and b) detecting the hybridization complex, wherein the
presence of the complex correlates with the presence of a
polynucleotide encoding RBPhu in the biological sample. In one
aspect the nucleic acid material of the biological sample is
amplified by the polymerase chain reaction prior to
hybridization.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID
NO:1) and nucleic acid sequence (SEQ ID NO:2) of RBPhu. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering Co. Ltd. San Bruno, Calif.).
[0027] FIG. 2 shows the amino acid sequence alignments among RBPhu
(367304; SEQ ID NO:1), mouse CRBP1 (GI 50548; SEQ ID NO:3) and
human retinol binding protein (GI 190948; SEQ ID NO:4), produced
using the multisequence alignment program of DNASTAR software
(DNASTAR Inc, Madison Wis.).
[0028] FIGS. 3A and 3B show the hydrophobicity plots for RBPhu (SEQ
ID NO:1) and CRBP1 from mouse (SEQ ID NO:3), respectively; the
positive X axis reflects amino acid position, and the negative Y
axis reflects hydrophobicity (MACDNASIS PRO software).
DESCRIPTION OF THE INVENTION
[0029] Before the present proteins, nucleotide sequences, 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.
[0030] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0031] 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 mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, and methodologies which are reported in
the publications 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.
[0032] Definitions
[0033] RBPhu, as used herein, refers to the amino acid sequences of
substantially purified RBPhu obtained from any species,
particularly mammalian, including bovine, ovine, porcine, murine,
equine, and preferably human, from any source whether natural,
synthetic, semi-synthetic, or recombinant.
[0034] The term "agonist", as used herein, refers to a molecule
which, when bound to RBPhu, increases or prolongs the duration of
the effect of RBPhu. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of RBPhu.
[0035] An "allele" or "allelic sequence", as used herein, is an
alternative form of the gene encoding RBPhu. Alleles may result
from at least one mutation in the nucleic acid sequence and may
result in if altered mRNAs or 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.
[0036] "Altered" nucleic acid sequences encoding RBPhu as used
herein include those with deletions, insertions, or substitutions
of different nucleotides resulting in a polynucleotide that encodes
the same or a functionally equivalent RBPhu. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding RBPhu, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding RBPhu.
The encoded protein may also be "altered" and contain deletions,
insertions, or substitutions of amino acid residues which produce a
silent change and result in a functionally equivalent RBPhu.
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 RBPhu 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.
[0037] "Amino acid sequence" as used herein refers to an
oligopeptide, peptide, polypeptide, or protein sequence, and
fragment thereof, and to naturally occurring or synthetic
molecules. Fragments of RBPhu are preferably about 5 to about 15
amino acids in length and retain the biological activity or the
immunological activity of RBPhu. Where "amino acid sequence" is
recited herein to refer to an amino acid sequence of a naturally
occurring protein molecule, amino acid sequence, and like terms,
are not meant to limit the amino acid sequence to the complete,
native amino acid sequence associated with the recited protein
molecule.
[0038] "Amplification" as used herein refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0039] The term "antagonist" as used herein, refers to a molecule
which, when bound to RBPhu, decreases the amount or the duration of
the effect of the biological or immunological activity of RBPhu.
Antagonists may include proteins, nucleic acids, carbohydrates,
antibodies or any other molecules which decrease the effect of
RBPhu.
[0040] As used herein, the term "antibody" refers to intact
molecules as well as fragments thereof, such as Fab, F(ab').sub.2,
and Fv, which are capable of binding the epitopic determinant.
Antibodies that bind RBPhu polypeptides can be prepared using
intact polypeptides or fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal 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 and thyroglobulin,
keyhole limpet hemocyanin. The coupled peptide is then used to
immunize the animal (e.g., a mouse, a rat, or a rabbit).
[0041] The term "antigenic determinant", as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or 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
a given region or three-dimensional structure on the protein; these
regions or structures are referred to as antigenic determinants. An
antigenic determinant may compete with the intact antigen (i.e.,
the immunogen used to elicit the immune response) for binding to an
antibody.
[0042] The term "antisense", as used herein, refers to any
composition containing nucleotide sequences which are complementary
to a specific DNA or RNA sequence. The term "antisense strand" is
used in reference to a nucleic acid strand that is complementary to
the "sense" strand. Antisense molecules include peptide nucleic
acids and 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 block either transcription or translation. The
designation "negative" is sometimes used in reference to the
antisense strand, and "positive" is sometimes used in reference to
the sense strand.
[0043] The term "biologically active", as used herein, 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
RBPhu, or any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0044] The terms "complementary" or "complementarity", as used
herein, 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", in which only some of the nucleic acids bind, or it
may be complete when 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 hybridization between 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
PNA molecules.
[0045] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise a dry
formulation or an aqueous solution. Compositions comprising
polynucleotide sequences encoding RBPhu (SEQ ID NO:1) or fragments
thereof (e.g., SEQ ID NO:2 and fragments thereof) 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, etc.).
[0046] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, has
been extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5'
and/or the 3' direction and resequenced, or has been assembled from
the overlapping sequences of more than one Incyte Clone using a
computer program for fragment assembly (e.g., GELVIEW fragment
assembly system, GCG, Madison, Wis.). Some sequences have been both
extended and assembled to produce the consensus sequence.
[0047] The term "correlates with expression of a polynucleotide",
as used herein, indicates that the detection of the presence of
ribonucleic acid that is similar to SEQ ID NO:2 by northern
analysis is indicative of the presence of mRNA encoding RBPhu in a
sample and thereby correlates with expression of the transcript
from the polynucleotide encoding the protein.
[0048] A "deletion", as used herein, refers to a change in the
amino acid or nucleotide sequence and results in the absence of one
or more amino acid residues or nucleotides.
[0049] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding or complementary
to RBPhu or the encoded RBPhu. Such modifications include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A nucleic acid derivative encodes a polypeptide which retains the
biological or immunological function of the natural molecule. A
derivative polypeptide is one which is modified by glycosylation,
pegylation, or any similar process which retains the biological or
immunological function of the polypeptide from which it was
derived.
[0050] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to using the functional term
"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 low
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
sequence which lacks even a partial degree of complementarity
(e.g., less than about 30% identity). In the absence of
non-specific binding, the probe will not hybridize to the second
non-complementary target sequence.
[0051] Human artificial chromosomes (HACs) are linear
microchromosomes which may contain DNA sequences of 10K to 10M in
size and contain all of the elements required for stable mitotic
chromosome segregation and maintenance (Harrington, J. J. et al.
(1997) Nat. Genet. 15:345-355).
[0052] The term "humanized antibody", as used herein, refers to
antibody molecules in which amino acids have been replaced in the
non-antigen binding regions in order to more closely resemble a
human antibody, while still retaining the original binding
ability.
[0053] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0054] The term "hybridization complex", as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen bonds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., paper, membranes,
filters, chips, pins or glass slides, or any other appropriate
substrate to which cells or their nucleic acids have been
fixed).
[0055] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, as compared to the naturally occurring molecule.
[0056] "Microarray" refers to an array of distinct polynucleotides
or oligonucleotides synthesized on a substrate, such as paper,
nylon or other type of membrane, filter, chip, glass slide, or any
other suitable solid support.
[0057] The term "modulate", as used herein, refers to a change in
the activity of RBPhu. For example, modulation may cause an
increase or a decrease in protein activity, binding
characteristics, or any other biological, functional or
immunological properties of RBPhu.
[0058] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments
thereof, and to DNA or RNA of genomic or synthetic origin which may
be single- or double-stranded, and represent the sense or antisense
strand. "Fragments" are those nucleic acid sequences which are
greater than 60 nucleotides than in length, and most preferably
includes fragments that are at least 100 nucleotides or at least
1000 nucleotides, and at least 10,000 nucleotides in length.
[0059] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to about 60 nucleotides, preferably
about 15 to 30 nucleotides, and more preferably about 20 to 25
nucleotides, which can be used in PCR amplification or a
hybridization assay, or a microarray. As used herein,
oligonucleotide is substantially equivalent to the terms
"amplimers", "primers", "oligomers", and "probes", as commonly
defined in the art.
[0060] "Peptide nucleic acid", PNA as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least five nucleotides in length linked to a
peptide backbone of amino acid residues which ends in lysine. The
terminal lysine confers solubility to the composition. PNAs may be
pegylated to extend their lifespan in the cell where they
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer
Drug Des. 8:53-63).
[0061] The term "portion", as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from five amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of SEQ ID NO:1" encompasses the full-length RBPhu and
fragments thereof.
[0062] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding RBPhu, or fragments thereof, or RBPhu 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; and the like.
[0063] The terms "specific binding" or "specifically binding", as
used herein, refers to that interaction between a protein or
peptide and an agonist, an antibody and an antagonist. The
interaction is dependent upon the presence of a particular
structure (i.e., the antigenic determinant or epitope) of the
protein recognized by the binding molecule. For example, if an
antibody is specific for epitope "A", the presence of a protein
containing epitope A (or free, unlabeled A) in a reaction
containing labeled "A" and the antibody will reduce the amount of
labeled A bound to the antibody.
[0064] The terms "stringent conditions" or "stringency", as used
herein, refer to the conditions for hybridization as defined by the
nucleic acid, salt, and temperature. These conditions are well
known in the art and may be altered in order to identify or detect
identical or related polynucleotide sequences. Numerous equivalent
conditions comprising either low or high stringency depend on
factors such as the length and nature of the sequence (DNA, RNA,
base composition), nature of the target (DNA, RNA, base
composition), milieu (in solution or immobilized on a solid
substrate), concentration of salts and other components (e.g.,
formamide, dextran sulfate and/or polyethylene glycol), and
temperature of the reactions (within a range from about 5.degree.
C. below the melting temperature of the probe to about 20.degree.
C. to 25.degree. C. below the melting temperature). One or more
factors be may be varied to generate conditions of either low or
high stringency different from, but equivalent to, the above listed
conditions.
[0065] The term "substantially purified", as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0066] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0067] "Transformation", as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method 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. Such "transformed" cells
include 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. They also include cells
which transiently express the inserted DNA or RNA for limited
periods of time.
[0068] A "variant" of RBPhu, as used herein, refers to an amino
acid sequence that is altered by w 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. More rarely, a variant may
have "nonconservative" changes, e.g., replacement of a glycine with
a tryptophan. Analogous minor variations may also include amino
acid deletions or insertions, or both. Guidance in determining
which amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0069] The Invention
[0070] The invention is based on the discovery of a new human
retinoid binding protein (hereinafter referred to as "RBPhu"), the
polynucleotides encoding RBPhu, and the use of these compositions
for the diagnosis, prevention, or treatment of cancer,
inflammation, and disorders associated with retinoid
metabolism.
[0071] Nucleic acids encoding the RBPhu of the present invention
were first identified in Incyte Clone 367304 from the rheumatoid
arthritis synovial tissue cDNA library (SYNORAT01) 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 nucleic acid sequences: Incyte Clones 1516168 (PANCTUT01),
1216161 (BRSTTUT01), and 367304 (SYNORAT01).
[0072] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A, 1B, and 1C. RBPhu is 135 amino acids in length and, as
shown in FIG. 2, has chemical and structural homology with CRBP1
from mouse (GI 50548; SEQ ID NO:3) and retinol binding protein from
human (GI 190948; SEQ ID NO:4). In particular, RBPhu and mouse
CRBP1 share 55% amino acid sequence identity; RBPhu and human
retinol binding protein also share 55% identity. As illustrated by
FIGS. 3A and 3B, RBPhu and mouse CRBP1 have rather similar
hydrophobicity plots. Northern analysis shows the expression of
RBPhu in various libraries, including cancers of the brain,
bladder, liver, and pancreas; rheumatoid arthritic synovium,
splenomegalic spleen, and inflamed adenoid. Of particular note is
that expression of RBPhu is found almost exclusively in tissues
associated with inflammation, cancer, or both. The sequence
similarity of RBPhu with the intracellular CRBPs and its tissue
distribution suggest that RBPhu is a new member of the CRBP
family.
[0073] The invention also encompasses RBPhu variants. A preferred
RBPhu variant is one having at least 80%, and more preferably at
least 90%, amino acid sequence identity to the RBPhu amino acid
sequence (SEQ ID NO:1) and which retains at least one biological,
immunological or other functional characteristic or activity of
RBPhu. A most preferred RBPhu variant is one having at least 95%
amino acid sequence identity to SEQ ID NO:1.
[0074] The invention also encompasses polynucleotides which encode
RBPhu. Accordingly, any nucleic acid sequence which encodes the
amino acid sequence of RBPhu can be used to produce recombinant
molecules which express RBPhu. In a particular embodiment, the
invention encompasses the polynucleotide comprising the nucleic
acid sequence of SEQ ID NO:2 as shown in FIGS. 1A, 1B, and 1C.
[0075] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding RBPhu, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and
every possible variation of nucleotide 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 nucleotide sequence of naturally
occurring RBPhu, and all such variations are to be considered as
being specifically disclosed.
[0076] Although nucleotide sequences which encode RBPhu and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring RBPhu under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding RBPhu 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 RBPhu 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.
[0077] The invention also encompasses production of DNA sequences,
or fragments thereof, which encode RBPhu and its derivatives,
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 RBPhu or any fragment
thereof.
[0078] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NO:2, under
various conditions of stringency as taught in Wahl, G. M. and S. L.
Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.
(1987; Methods Enzymol. 152:507-511).
[0079] Methods for DNA sequencing which 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, SEQUENASE (US
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE amplification system marketed by
Gibco/BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the MICRO LAB 2200 (Hamilton, Reno, Nev.),
Peltier thermal cycler (PTC200; M J Research, Watertown, Mass.) and
the ABI CATALYST and 373 and 377 DNA sequencers (Perkin Elmer).
[0080] The nucleic acid sequences encoding RBPhu 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 (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA
is first amplified in the presence of primer to a linker sequence
and a primer specific to the known region. The amplified sequences
are then 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.
[0081] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using commercially available software such as OLIGO 4.06 primer
analysis software (National Biosciences Inc., Plymouth, Minn.), or
another appropriate program, to be 22-30 nucleotides in length, to
have a GC content of 50% or more, and to anneal to the target
sequence at temperatures about 68.degree.-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.
[0082] 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 (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may also be
used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR.
[0083] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PROMOTERFINDER libraries to walk genomic DNA (Clontech, Palo Alto,
Calif.). This process avoids the need to screen libraries and is
useful in finding intron/exon junctions.
[0084] 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
contain 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.
[0085] 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 detection of the emitted
wavelengths by a charge coupled devise camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer) and
the entire process from loading of samples to computer analysis and
electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0086] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode RBPhu may be used in
recombinant DNA molecules to direct expression of RBPhu, 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 RBPhu.
[0087] As will be understood by those of skill in the art, it may
be advantageous to produce RBPhu-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.
[0088] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter RBPhu 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.
[0089] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding RBPhu may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of RBPhu activity, it
may be useful to encode a chimeric RBPhu 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 RBPhu encoding sequence and the heterologous protein sequence,
so that RBPhu may be cleaved and purified away from the
heterologous moiety.
[0090] In another embodiment, sequences encoding RBPhu may be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers, M. H. et al. (1980) Nucleic Acids Symp.
Ser. 7:215-223, Horn, T. 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 RBPhu, or
a fragment thereof. For example, peptide synthesis can be performed
using various solid-phase techniques (Roberge, J. Y. et al. (1995)
Science 269:202-204) and automated synthesis may be achieved, for
example, using the ABI 431A peptide synthesizer (Perkin Elmer).
[0091] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
W H Freeman and Co., New York, N.Y.). The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequence of RBPhu, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with sequences from other proteins, or any
part thereof, to produce a variant polypeptide.
[0092] In order to express a biologically active RBPhu, the
nucleotide sequences encoding RBPhu or functional equivalents, 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.
[0093] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding RBPhu and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0094] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding RBPhu. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0095] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--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 such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1
plasmid (Gibco BRL) and the like 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 RBPhu, vectors based on SV40 or EBV may be used with an
appropriate selectable marker.
[0096] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for RBPhu. For example,
when large quantities of RBPhu 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, the multifunctional E. coli
cloning and expression vectors such as BLUESCRIPT (Stratagene), in
which the sequence encoding RBPhu 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; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509); and the like. PGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides 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 polypeptide of interest
can be released from the GST moiety at will.
[0097] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Bitter, G. A. et al. (1987) Methods
Enzymol. 153:516-544.
[0098] In cases where plant expression vectors are used, the
expression of sequences encoding RBPhu 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, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196.
[0099] An insect system may also be used to express RBPhu. 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 RBPhu 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 RBPhu 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 RBPhu may be expressed (Engelhard, E. K. et al. (1994) Proc.
Natl. Acad. Sci. USA 91:3224-3227).
[0100] 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 RBPhu 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 RBPhu in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. USA 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.
[0101] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of 6 to 10M are constructed and delivered via
conventional delivery methods (liposomes, polycationic amino
polymers, or vesicles) for therapeutic purposes.
[0102] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding RBPhu. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding RBPhu, 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 which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0103] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
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 (ATCC; Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0104] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express RBPhu may 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 1-2 days in an enriched
media before to they are 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.
[0105] 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 (Wigler, M. et al. (1977)
Cell 11:223-232) and adenine phosphoribosyltransferase (Lowy, I. et
al. (1980) Cell 22:817-823) genes which can be employed in tk.sup.-
and apr.sup.- cells, respectively. Also, antimetabolite, antibiotic
or herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14) and als
and pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
USA 85:8047-8051). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0106] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding RBPhu is inserted within a marker gene sequence,
transformed cells containing sequences encoding RBPhu can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding RBPhu
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.
[0107] Alternatively, host cells which contain the nucleic acid
sequence encoding RBPhu and express RBPhu 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.
[0108] The presence of polynucleotide sequences encoding RBPhu can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
RBPhu. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding RBPhu
to detect transformants containing DNA or RNA encoding RBPhu.
[0109] A variety of protocols for detecting and measuring the
expression of RBPhu, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on RBPhu is preferred, but
a competitive binding assay may be employed. These and other assays
are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St. Paul,
Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0110] 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 RBPhu include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding RBPhu, 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 (Pharmacia & Upjohn
(Kalamazoo, Mich.); Promega (Madison, Wis.); and U.S. Biochemical
Corp. (Cleveland, Ohio) ). 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.
[0111] Host cells transformed with nucleotide sequences encoding
RBPhu 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 RBPhu may be designed to
contain signal Is sequences which direct secretion of RBPhu through
a prokaryotic or eukaryotic cell membrane. Other constructions may
be used to join sequences encoding RBPhu to nucleotide sequence
encoding a polypeptide 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 FLAGS
extension/affinity purification system (Immunex Corp., 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 RBPhu may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing RBPhu and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilized metal ion affinity
chromatography) as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3: 263-281) while the enterokinase cleavage site provides a
means for purifying RBPhu from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453).
[0112] In addition to recombinant production, fragments of RBPhu
may be produced by direct peptide synthesis using solid-phase
techniques (Merrifield, J. (1963) J. Am. Chem. Soc. 85:2149-2154).
Protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer).
Various fragments of RBPhu may be chemically synthesized separately
and combined using chemical methods to produce the full length
molecule.
[0113] Therapeutics
[0114] Chemical and structural homology exist among RBPhu, CRBP1
from mouse (GI 50548) and retinol binding protein from human (GI
190948). In addition, RBPhu is expressed in tumors and inflamed
tissues. Therefore, RBPhu appears to play a role in cancer,
inflammation, and disorders associated with retinoid
metabolism.
[0115] In some instances, excessive levels of retinoids in the cell
may be cytotoxic or produce unwanted side-effects. It may be
desirable to modulate the intracellular concentrations of retinoids
by administering RBPhu. Therefore, in one embodiment, RBPhu or a
fragment or derivative thereof may be administered to a subject to
modulate free intracellular retinoid concentrations. Such
modulation may be useful to counteract excessive dosages of
retinoid therapeutics and would be particularly useful in
pregnancy.
[0116] In another embodiment, a vector capable of expressing RBPhu,
or a fragment or a derivative thereof, may also be administered to
a subject to modulate free intracellular retinoid
concentrations.
[0117] In still another embodiment, an agonist which modulates the
activity of RBPhu may also be administered to a subject to modulate
free intracellular retinoid concentrations.
[0118] Expression of RBPhu appears to be associated with cancer.
Therefore, in one embodiment, an antagonist of RBPhu may be
administered to a subject to prevent or treat cancer. Such cancers
may include, but are not limited to, adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, and
particularly cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds RBPhu 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
RBPhu.
[0119] In another embodiment, a vector expressing the complement of
the polynucleotide encoding RBPhu may be administered to a subject
to treat or prevent cancer including, but not limited to, those
cancers described above.
[0120] Expression of RBPhu appears to be associated with
inflammation. Therefore, in one embodiment, an antagonist of RBPhu
may be administered to a subject to prevent or treat inflammation
of any type and, in particular, that which results from a
particular disorder. Such disorders of inflammation include, but
are not limited to, AIDS, Addison's disease, adult respiratory
distress syndrome, allergies, anemia, asthma, atherosclerosis,
bronchitis, cholecystitis, Crohn's disease, ulcerative colitis,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
erythema nodosum, atrophic gastritis, glomerulonephritis, gout,
Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, splenomegaly, and autoimmune thyroiditis;
complications of cancer, hemodialysis, extracorporeal circulation;
viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections and trauma.
[0121] In another embodiment, a vector expressing the complement of
the polynucleotide encoding RBPhu may be administered to a subject
to treat or prevent inflammation of any type including, but not
limited to, the disorders of inflammation described above.
[0122] In another embodiment, an antagonist of RBPhu may be
administered to a subject to prevent or treat a disorder associated
with retinoid metabolism. Such disorders include, but are not
limited to, skin diseases such as severe acne and acne-related
dermatoses, dernatitis, eczema, ichthyosis, keratosis, psoriasis,
scleroderma, and skin atrophy; night blindness; xerosis of the
conjunctiva and cornea; xerophthalmia and keratomalacia;
keratinization of lung, GI tract, and urinary tract epithelia; and
abnormalities in retinoid absorption or storage associated with
disorders such as celiac disease, sprue, cystic fibrosis,
operations on the pancreas, duodenal bypass, congenital partial
obstruction of the jejunum, obstruction of the bile ducts,
giardiasis, cirrhosis of the liver, and malnutrition.
[0123] In another embodiment, a vector expressing the complement of
the polynucleotide encoding RBPhu may be administered to a subject
to treat or prevent a disorder associated with retinoid metabolism
including, but not limited to, the disorders described above.
[0124] 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.
[0125] An antagonist of RBPhu may be produced using methods which
are generally known in the art. In particular, purified RBPhu may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
RBPhu.
[0126] Antibodies to RBPhu may be generated using methods that are
well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, single chain, 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.
[0127] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with RBPhu or 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, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
[0128] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to RBPhu have an amino acid
sequence consisting of at least five amino acids and more
preferably at least 10 amino acids. It is also preferable that they
are identical to a portion of the amino acid sequence of the
natural protein, and they may contain the entire amino acid
sequence of a small, naturally occurring molecule. Short stretches
of RBPhu amino acids may be fused with those of another protein
such as keyhole limpet hemocyanin and antibody produced against the
chimeric molecule.
[0129] Monoclonal antibodies to RBPhu 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 (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
USA 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0130] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.
S. et al. (1984) Nature 312:604-608; Takeda, S. et al. (1985)
Nature 314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce RBPhu-specific single chain
antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be generated by chain shuffling from
random combinatorial immunoglobulin libraries (Burton, D. R. (1991)
Proc. Natl. Acad. Sci. USA 88:11120-11123).
[0131] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0132] Antibody fragments which contain specific binding sites for
RBPhu may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be 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 (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0133] 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 RBPhu and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering RBPhu
epitopes is preferred, but a competitive binding assay may also be
employed (Maddox, supra).
[0134] In another embodiment of the invention, the polynucleotides
encoding RBPhu, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding RBPhu 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 RBPhu. Thus, complementary molecules or
fragments may be used to modulate RBPhu 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 RBPhu.
[0135] Expression vectors derived from retro viruses, adenovirus,
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 sequence which is complementary to the
polynucleotides of the gene encoding RBPhu. These techniques are
described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0136] Genes encoding RBPhu can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide or fragment thereof which encodes RBPhu. 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 even longer if appropriate replication elements are part
of the vector system.
[0137] 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 RB Phu (signal sequence, promoters,
enhancers, and introns). Oligonucleotides derived from the
transcription initiation site, e.g., between 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 (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The complementary sequence or antisense molecule
may also be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0138] 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 nibozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples which may be used include engineered hammerhead
motif ribozyme molecules that can specifically and efficiently
catalyze endonucleolytic cleavage of sequences encoding RBPhu.
[0139] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include 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.
[0140] 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 RBPhu. 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.
[0141] 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.
[0142] 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 polycationic amino polymers (Goldman, C.
K. et al. (1997) Nat. Biotechnol. 15:462-466; incorporated herein
by reference) may be achieved using methods which are well known in
the art.
[0143] 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.
[0144] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of RBPhu, antibodies to RBPhu, mimetics, agonists,
antagonists, or inhibitors of RBPhu. The compositions may be
administered alone or in combination with at least one other agent,
such as 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.
[0145] 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.
[0146] 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 Co.,
Easton, Pa.).
[0147] 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.
[0148] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or 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,
alginic acid, or a salt thereof, such as sodium alginate.
[0149] 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.
[0150] 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 a
filler 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.
[0151] 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 or triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0152] 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.
[0153] 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.
[0154] 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, succinic
acids, etc. 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: 1-50 mM histidine,
0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5,
that is combined with buffer prior to use.
[0155] 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 RBPhu, such
labeling would include amount, frequency, and method of
administration.
[0156] 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.
[0157] 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, usually mice, rabbits, dogs,
or pigs. The 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.
[0158] A therapeutically effective dose refers to that amount of
active ingredient, for example RBPhu or fragments thereof,
antibodies of RBPhu, agonists, antagonists or inhibitors of RBPhu,
which ameliorates the symptoms or condition. Therapeutic efficacy
and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio
between therapeutic and toxic effects is the therapeutic index, and
it can be expressed as the ratio, LD50/ED50. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is
used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0159] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires 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, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0160] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
[0161] Diagnostics
[0162] In another embodiment, antibodies which specifically bind
RBPhu may be used for the diagnosis of conditions or diseases
characterized by expression of RBPhu, or in assays to monitor
patients being treated with RBPhu, agonists, antagonists or
inhibitors. The antibodies useful for fir diagnostic purposes may
be prepared in the same manner as those described above for
therapeutics. Diagnostic assays for RBPhu include methods which
utilize the antibody and a label to detect RBPhu in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0163] A variety of protocols including ELISA, RIA, and FACS for
measuring RBPhu are known in the art and provide a basis for
diagnosing altered or abnormal levels of RBPhu expression. Normal
or standard values for RBPhu expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to RBPhu under conditions
suitable for complex formation. The amount of standard complex
formation may be quantified by various methods, but preferably by
photometric, means. Quantities of RBPhu expressed in subject
samples, control and disease, from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0164] In another embodiment of the invention, the polynucleotides
encoding RBPhu 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 RBPhu may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
RBPhu, and to monitor regulation of RBPhu levels during therapeutic
intervention.
[0165] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding RBPhu or closely related molecules, may be used
to identify nucleic acid sequences which encode RBPhu. The
specificity of the probe, whether it is made from a highly specific
region, e.g., 10 unique nucleotides in the 5' regulatory region, or
a less specific region, e.g., especially in the 3' coding region,
and the stringency of the hybridization or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding RBPhu,
alleles, or related sequences.
[0166] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the RBPhu encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
derived from the nucleotide sequence of SEQ ID NO:2 or from genomic
sequence including promoter, enhancer elements, and introns of the
naturally occurring RBPhu.
[0167] Means for producing specific hybridization probes for DNAs
encoding RBPhu include the cloning of nucleic acid sequences
encoding RBPhu or RBPhu derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, 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, radionuclides
such as 32P or 35S, or enzymatic labels, such as alkaline
phosphatase coupled to the probe via avidinrbiotin coupling
systems, and the like.
[0168] Polynucleotide sequences encoding RBPhu may be used for the
diagnosis of conditions or disorders which are associated with
expression of RBPhu. Examples of such conditions or disorders
include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, and teratocarcinoma, and particularly cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
AIDS, Addison's disease, adult respiratory distress syndrome,
allergies, anemia, asthma, atherosclerosis, bronchitis,
cholecystitis, Crohn's disease, ulcerative colitis, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythema
nodosum, atrophic gastritis, glomerulonephritis, gout, Graves'
disease, hypereosinophilia, irritable bowel syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, rheumatoid arthritis, scleroderma,
Sjogren's syndrome, splenomegaly, and autoimmune thyroiditis;
complications of cancer, hemodialysis, extracorporeal circulation;
viral, bacterial, fungal, parasitic, protozoal, and helminthic
infections and trauma; severe acne and acne-related dermatoses,
dermatitis, eczema, ichthyosis, keratosis, psoriasis, scleroderma,
and skin atrophy; night blindness; xerosis of the conjunctiva and
cornea; xerophthalmia and keratomalacia; keratinization of lung, GI
tract, and urinary tract epithelia; celiac disease, sprue, cystic
fibrosis, congenital partial obstruction of the jejunum,
obstruction of the bile ducts, giardiasis, cirrhosis of the liver,
and malnutrition. The polynucleotide sequences encoding RBPhu may
be used in Southern or northern analysis, dot blot, or other
membrane-based technologies; in PCR technologies; or in dipstick,
pin, ELISA assays or microarrays utilizing fluids or tissues from
patient biopsies to detect altered RBPhu expression. Such
qualitative or quantitative methods are well known in the art.
[0169] In a particular aspect, the nucleotide sequences encoding
RBPhu may be useful in assays that detect activation or induction
of various cancers, particularly those mentioned above. The
nucleotide sequences encoding RBPhu 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 biopsied or extracted sample is
significantly altered from that of a comparable control sample, the
nucleotide sequences have hybridized with nucleotide sequences in
the sample, and the presence of altered levels of nucleotide
sequences encoding RBPhu in the sample indicates the presence of
the associated disease. Such assays may also be used to evaluate
the efficacy of a particular therapeutic treatment regimen in
animal studies, in clinical trials, or in monitoring the treatment
of an individual patient.
[0170] In order to provide a basis for the diagnosis of disease
associated with expression of RBPhu, 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,
which encodes RBPhu, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease.
[0171] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0172] 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.
[0173] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding RBPhu may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation (5'->3') and
another with antisense (340 <-5'), employed under optimized
conditions for identification of a specific gene or condition. The
same two oligomers, nested sets of oligomers, or even a degenerate
pool of oligomers may be employed under less stringent conditions
for detection and/or quantitation of closely related DNA or RNA
sequences.
[0174] Methods which may also be used to quantitate the expression
of RBPhu include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236). The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantitation.
[0175] In further embodiments, an oligonucleotide derived from any
of the polynucleotide sequences described herein may be used as a
target in a microarray. The microarray can be used to monitor the
expression level of large numbers of genes simultaneously (to
produce a transcript image), and to identify genetic variants,
mutations and polymorphisms. This information will be useful in
determining gene function, understanding the genetic basis of
disease, diagnosing disease, and in developing and monitoring the
activity of therapeutic agents (Heller, R. et al. (1997) Proc.
Natl. Acad. Sci. USA 94:2150-2155).
[0176] In one embodiment, the microarray is prepared and used
according to the methods described in PCT application WO95/11995
(Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotechnol.
14:1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci.
USA 93:10614-10619), all of which are incorporated herein in their
entirety by reference.
[0177] The microarray is preferably composed of a large number of
unique, single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length,
and most preferably about 20-25 nucleotides in length. For a
certain type of microarray, it may be preferable to use
oligonucleotides which are only 7-10 nucleotides in length. The
microarray may contain oligonucleotides which cover the known 5',
or 3', sequence, sequential oligonucleotides which cover the full
length sequence; or unique oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides
used in the microarray may be oligonucleotides that are specific to
a gene or genes of interest in which at least a fragment of the
sequence is known or that are specific to one or more unidentified
cDNAs which are common to a particular cell type, developmental or
disease state.
[0178] In order to produce oligonucleotides to a known sequence for
a microarray, the gene of interest is examined using a computer
algorithm which starts at the 5' or more preferably at the 3' end
of the nucleotide sequence. The algorithm identifies oligomers of
defined length that are unique to the gene, have a GC content
within a range suitable for hybridization, and lack predicted
secondary structure that may interfere with hybridization. In
certain situations it may be appropriate to use pairs of
oligonucleotides on a microarray. The "pairs" will be identical,
except for one nucleotide which preferably is located in the center
of the sequence. The second oligonucleotide in the pair (mismatched
by one) serves as a control. The number of oligonucleotide pairs
may range from two to one million. The oligomers are synthesized at
designated areas on a substrate using a light-directed chemical
process. The substrate may be paper, nylon or other type of
membrane, filter, chip, glass slide or any other suitable solid
support.
[0179] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any
other number between two and one million which lends itself to the
efficient use of commercially available instrumentation.
[0180] In order to conduct sample analysis using a microarray, the
RNA or DNA from a biological sample is made into hybridization
probes. The mRNA is isolated, and cDNA is produced and used as a
template to make antisense RNA (aRNA). The aRNA is amplified in the
presence of fluorescent nucleotides, and labeled probes are
incubated with the microarray so that the probe sequences hybridize
to complementary oligonucleotides of the microarray. Incubation
conditions are adjusted so that hybridization occurs with precise
complementary matches or with various degrees of less
complementarity. After removal of nonhybridized probes, a scanner
is used to determine the levels and patterns of fluorescence. The
scanned images are examined to determine degree of complementarity
and the relative abundance of each oligonucleotide sequence on the
microarray. The biological samples may be obtained from any bodily
fluids (such as blood, urine, saliva, phlegm, gastric juices,
etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be used to measure the absence, presence, and
amount of hybridization for all of the distinct sequences
simultaneously. This data may be used for large scale correlation
studies on the sequences, mutations, variants, or polymorphisms
among samples.
[0181] In another embodiment of the invention, the nucleic acid
sequences which encode RBPhu may also be used to generate
hybridization probes which are useful for 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, such as human artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs), bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood
Rev. 7:127-134; and Trask, B. J. (1991) Trends Genet.
7:149-154.
[0182] Fluorescent in situ hybridization (FISH as described in
Verma et al. (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York, N.Y.) may be correlated with
other physical chromosome mapping techniques and genetic map data.
Examples of genetic map data can be found in various scientific
journals or at Online Mendelian Inheritance in Man (OMIM).
Correlation between the location of the gene encoding RBPhu on a
physical chromosomal map and a specific disease , or predisposition
to a specific disease, may help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of
the subject invention may be used to detect differences in gene
sequences between normal, carrier, or affected individuals.
[0183] 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, or parts
thereof, 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, for example, AT to 11q22-23 (Gatti, R. A. et al.
(1988) Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the subject invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, etc. among normal, carrier, or affected
individuals.
[0184] In another embodiment of the invention, RBPhu, 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 RBPhu and the agent being tested, may be
measured.
[0185] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
RBPhu 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 RBPhu, or
fragments thereof, and washed. Bound RBPhu is then detected by
methods well known in the art. Purified RBPhu 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.
[0186] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding RBPhu specifically compete with a test compound for binding
RBPhu. In this manner, the antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with RBPhu.
[0187] In additional embodiments, the nucleotide sequences which
encode RBPhu 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.
[0188] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0189] I SYNORAT01 cDNA Library Construction
[0190] The SYNORAT01 cDNA library was prepared from rheumatoid
synovial elbow tissue obtained from UC Davis (lot #48). The frozen
tissue was ground in a mortar and pestle and lysed immediately in a
buffer containing guanidinium isothiocyanate. The lysate was
extracted twice with phenol chloroform at pH 8.0 and centrifuged
over a CsCl cushion using a Beckman SW28 rotor in a Beckman L8-70M
ultracentrifuge (Beckman Instruments). The RNA was precipitated
using 0.3 M sodium acetate and 2.5 volumes of ethanol and
resuspended in water.
[0191] The RNA was DNase treated for 15 min at 37.degree. C. before
library construction. First strand cDNA synthesis was accomplished
using an oligo d(T) primer/linker which also contained an XhoI
restriction site. Second strand synthesis was performed using a
combination of DNA polymerase I, E. coli ligase and RNase H,
followed by the addition of an EcoRI adaptor to the blunt ended
cDNA. The EcoRI adapted, double-stranded cDNA was then digested
with XhoI restriction enzyme and fractionated on SEPHACRYL S400 to
obtain sequences which exceeded 1000 bp in size. The size selected
cDNAs were inserted into the LAMBDAZAP vector system (Stratagene);
and the vector, which contains the pBLUESCRIPT phagemid
(Stratagene), was transformed into cells of E. coli, strain
XLI-BLUEMRF (Stratagene).
[0192] The plasmid forms of individual cDNA clones were obtained by
the in vivo excision process. Enzymes from both pBLUESCRIPT and a
cotransformed f1 helper phage nicked the DNA, initiated new DNA
synthesis, and created the smaller, single-stranded circular
phagemid DNA molecules which contained the cDNA insert. The
phagemid DNA was released, purified, and used to reinfect fresh
host cells (SOLR, Stratagene). Presence of the phagemid which
carries the gene for .beta.-lactamase allowed transformed bacteria
to grow on medium containing ampicillin.
[0193] II Isolation and Sequencing of cDNA Clones
[0194] Plasmid DNA was purified using the MINIPREP kit (Catalogue
#77468, Advanced Genetic Technologies Corporation, Gaithersburg
Md.), a 96-well block kit with reagents for 960 purifications. The
recommended protocol included with the kit was employed except for
the following changes. Each of the 96 wells was filled with only 1
ml of sterile Terrific Broth (Catalog #22711, GIBCO/BRL) with
carbenicillin at 25 mg/L and glycerol at 0.4%. After the wells were
inoculated, the bacteria were cultured for 24 hours and lysed with
60 .mu.l of lysis buffer. A centrifugation step (Beckman GS-6R
@2900 rpm for 5 min; Beckman Instruments) was performed before the
contents of the block were added to the primary filter plate. The
optional step of adding isopropanol to TRIS buffer was not
routinely performed. After the last step in the protocol, samples
were transferred to a Beckman 96-well block for storage.
[0195] The cDNAs were sequenced by the method of Sanger, F. and A.
R. Coulson (1975; J. Mol. Biol. 94:441f), using a MICRO LAB 2200
(Hamilton, Reno Nev.) in combination with four Peltier thermal
cyclers (PTC200 from M J Research, Watertown Mass.) and Applied
Biosystems 377 or 373 DNA sequencing systems (Perkin Elmer), and
the reading frame was determined.
[0196] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0197] The nucleotide sequences of the Sequence Listing or amino
acid sequences deduced from them were used as query sequences
against databases such as GenBank, SwissProt, BLOCKS, and Pima II.
These databases which contain previously identified and annotated
sequences were searched for regions of homology (similarity) using
BLAST, which stands for Basic Local Alignment Search Tool
(Altschul, S. F. (1993) J. Mol. Evol. 36:290-300; Altschul et al.
(1990) J. Mol. Biol. 215:403-410).
[0198] BLAST produces alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST is 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 such as the one described in Smith, R. F.
and T. F. Smith (1992; Protein Eng. 5:35-51), incorporated herein
by reference, can be used when dealing with primary sequence
patterns and secondary structure gap penalties. As disclosed in
this application, the sequences have lengths of at least 49
nucleotides, and no more than 12% uncalled bases (where N is
recorded rather than A, C, G, or T).
[0199] The BLAST approach, as detailed in Karlin, S. and S. F.
Altschul (1993; Proc. Nat. Acad. Sci. USA 90:5873-5877) and
incorporated herein by reference, searches for matches between a
query sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-14 for peptides.
[0200] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and mammalian
sequences (mam). Deduced amino acid sequences from the same clones
are searched against GenBank functional protein databases,
mammalian (mamp), vertebrate (vrtp) and eukaryote (eukp), for
homology.
[0201] IV Northern Analysis
[0202] 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
(Sambrook et al., supra).
[0203] Analogous computer techniques using BLAST (Altschul, S. F.
(1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J.
Mol. Evol. 215:403-410) are used to search for identical or related
molecules in nucleotide databases such as GenBank or the LIFESEQ
database (Incyte Pharmaceuticals). This analysis is much faster
than multiple, membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or
homologous.
[0204] The basis of the search is the product score which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0205] 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 1-2% error; and at 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.
[0206] The results of northern analysis are reported as a list of
libraries in which the transcript encoding RBPhu 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.
[0207] V Extension of RBPhu Encoding Polynucleotides
[0208] The nucleic acid sequence of the Incyte Clone 367304 was
used to design oligonucleotide primers for extending a partial
nucleotide sequence to full length. One primer was synthesized to
initiate extension in the antisense direction, and the other was
synthesized to extend sequence in the sense direction. 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
about 30 nucleotides in length, to have a GC content of 50% or
more, and to anneal to the target sequence at temperatures of about
68.degree. to about 72.degree. C. Any stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations
was avoided.
[0209] Selected human cDNA libraries (Gibco/BRL) 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.
[0210] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR was performed using the Peltier thermal cycler
(PTC200; M. J. Research, Watertown, Mass.) and the following
parameters:
1 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 step 4-6 for 15 additional
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 step 8-10 for
12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C. (and
holding)
[0211] A 5-10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) 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 (QIAGEN Inc.,
Chatsworth, Calif.), and trimmed of overhangs using Klenow enzyme
to facilitate religation and cloning.
[0212] 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 2-3 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 et al., supra). After
incubation for one hour at 37.degree. C., the E. coli mixture was
plated on Luria Bertani (LB)-agar (Sambrook et al., supra)
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 of each sample was transferred into a PCR
array.
[0213] 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:
2 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 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0214] 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.
[0215] 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.
[0216] VI Labeling and Use of Individual Hybridization Probes
[0217] 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 software (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham) and
T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). The labeled
oligonucleotides are substantially purified with SEPHADEX G-25
superfine resin column (Pharmacia & Upjohn). A aliquot
containing 10.sup.7 counts per minute of the labeled probe is used
in a typical membrane-based hybridization analysis of human genomic
DNA digested with one of the following endonucleases (Ase I, Bgl
II, Eco RI, Pst I, Xba I, or Pvu II; DuPont NEN).
[0218] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (NYTRAN PLUS,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times.saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film
(Kodak, Rochester, N.Y.) is exposed to the blots in a PHOSPHOIMAGER
cassette (Molecular Dynamics, Sunnyvale, Calif.), hybridization
patterns are compared visually.
[0219] VII Microarrays
[0220] To produce oligonucleotides for a microarray, the nucleotide
sequence described herein is examined using a computer algorithm
which starts at the 3' end of the nucleotide sequence. The
algorithm identifies oligomers of defined length that are unique to
the gene, have a GC content within a range suitable for
hybridization, and lack predicted secondary structure that would
interfere with hybridization. The algorithm identifies 20
sequence-specific oligonucleotides of 20 nucleotides in length
(20-mers). A matched set of oligonucleotides is created in which
one nucleotide in the center of each sequence is altered. This
process is repeated for each gene in the microarray, and double
sets of twenty 20 mers are synthesized and arranged on the surface
of the silicon chip using a light-directed chemical process (Chee,
M. et al., PCT/WO95/11995, incorporated herein by reference).
[0221] In the alternative, a chemical coupling procedure and an ink
jet device are used to synthesize oligomers on the surface of a
substrate (Baldeschweiler, J. D. et al., PCT/WO95/25116,
incorporated herein by reference). In another alternative, a
"gridded" array analogous to a dot (or slot) blot is used to
arrange and link cDNA fragments or oligonucleotides to the surface
of a substrate using a vacuum system, thermal, UV, mechanical or
chemical bonding procedures. An array may be produced by hand or
using available materials and machines and contain grids of 8 dots,
24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots. After
hybridization, the microarray is washed to remove nonhybridized
probes, and a scanner is used to determine the levels and patterns
of fluorescence. The scanned images are examined to determine
degree of complementarity and the relative abundance of each
oligonucleotide sequence on the microarray.
[0222] VIII Complementary Polynucleotides
[0223] Sequence complementary to the RBPhu-encoding sequence, or
any part thereof, is used to decrease or inhibit expression of
naturally occurring RBPhu. Although use of oligonucleotides
comprising from about 15 to about 30 base-pairs is described,
essentially the same procedure is used with smaller or larger
sequence fragments. Appropriate oligonucleotides are designed using
OLIGO 4.06 software and the coding sequence of RBPhu, SEQ ID NO:1.
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 RBPhu-encoding transcript.
[0224] IX Expression of RBPhu
[0225] Expression of RBPhu is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector is also used to express
RBPhu in E. coli. Upstream of the cloning site, this vector
contains a promoter for .beta.-galactosidase, followed by sequence
containing the amino-terminal Met, and the subsequent seven
residues of .beta.-galactosidase. Immediately following these eight
residues is a bacteriophage promoter useful for transcription and a
linker containing a number of unique restriction sites.
[0226] Induction of an isolated, transformed bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first eight residues of .beta.-galactosidase, about
5 to 15 residues of linker, and the full length protein. The signal
residues direct the secretion of RBPhu into the bacterial growth
media which can be used directly in the following assay for
activity.
[0227] X Demonstration of RBPhu Activity
[0228] The ligand binding activity of RBPhu is characterized with
the fluorescent probe 1-anilinonapthalene 8-sulfonic acid
(1,8-ANS). To quantitate the specificity and affinity of ligand
binding, a competition assay (as described in Kane, C. D. and
Bemlohr, D. A. (1996) Anal. Biochem. 233:197-204) monitors the
ability of various retinoids to displace bound 1,8-ANS from
Hu-RBP.
[0229] XI Production of RBPhu Specific Antibodies
[0230] RBPhu that is substantially purified using PAGE
electrophoresis (Sambrook, supra), or other purification
techniques, is used to immunize rabbits and to produce antibodies
using standard protocols. The amino acid sequence deduced from SEQ
ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc.) to
determine regions of high immunogenicity and a corresponding
oligopeptide is synthesized and used to raise antibodies by means
known to those of skill in the art. Selection of appropriate
epitopes, such as those near the C-terminus or in hydrophilic
regions, is described by Ausubel et al. (supra), and others.
[0231] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems 431A peptide synthesizer
using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,
supra). Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. The 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.
[0232] XII Purification of Naturally Occurring RBPhu Using Specific
Antibodies
[0233] Naturally occurring or recombinant RBPhu is substantially
purified by immunoaffinity chromatography using antibodies specific
for RBPhu. An immunoaffinity column is constructed by covalently
coupling RBPhu antibody to an activated chromatographic resin, such
as CNBr-activated Sepharose (Pharmacia & Upjohn). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0234] Media containing RBPhu is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of RBPhu (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/RBPhu binding (eg, a buffer of pH
2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and RBPhu is collected.
[0235] XIII Identification of Molecules Which Interact with
RBPhu
[0236] RBPhu or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent (Bolton, A. E. and W. M.
Hunter (1973) Biochem. J. 133:529-539). Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated
with the labeled RBPhu, washed and any wells with labeled RBPhu
complex are assayed. Data obtained using different concentrations
of RBPhu are used to calculate values for the number, affinity, and
association of RBPhu with the candidate molecules.
[0237] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system 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
* * * * *