U.S. patent application number 10/013742 was filed with the patent office on 2002-07-04 for ubiquitin-like conjugating protein.
This patent application is currently assigned to Incyte Pharmaceuticals, Inc.. Invention is credited to Corley, Neil C., Hillman, Jennifer L., Shah, Purvi.
Application Number | 20020086408 10/013742 |
Document ID | / |
Family ID | 25534967 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086408 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
July 4, 2002 |
Ubiquitin-like conjugating protein
Abstract
The invention provides a human ubiquitin-like conjugating
protein (UBCLE) and polynucleotides which identify and encode
UBCLE. The invention also provides expression vectors, host cells,
antibodies, agonists, and antagonists. The invention also provides
methods for treating or preventing disorders associated with
expression of UBCLE.
Inventors: |
Hillman, Jennifer L.;
(Mountain View, CA) ; Shah, Purvi; (Sunnyvale,
CA) ; Corley, Neil C.; (Mountain View, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Pharmaceuticals,
Inc.
|
Family ID: |
25534967 |
Appl. No.: |
10/013742 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10013742 |
Dec 7, 2001 |
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09328866 |
Jun 9, 1999 |
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09328866 |
Jun 9, 1999 |
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08989289 |
Dec 12, 1997 |
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Current U.S.
Class: |
435/226 ;
435/252.3; 435/320.1; 435/325; 435/69.1; 536/23.2; 800/8 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 25/00 20180101; A61P 35/00 20180101; C12N 9/93 20130101; A61P
37/00 20180101 |
Class at
Publication: |
435/226 ;
435/325; 435/320.1; 435/252.3; 435/69.1; 800/8; 536/23.2 |
International
Class: |
C12N 009/64; A01K
067/00; C07H 021/04; C12P 021/02; C12N 001/21; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide comprising an amino acid sequence
selected from the group consisting of: a) a polypeptide comprising
an amino acid sequence of SEQ ID NO:1, b) a naturally occurring
polypeptide comprising an amino acid sequence at least 90%
identical to an amino acid sequence of SEQ ID NO:1, c) a
biologically active fragment of a polypeptide having an amino acid
sequence of SEQ ID NO:1, and d) an immunogenic fragment of a
polypeptide having an amino acid sequence of SEQ ID NO:1.
2. An isolated polypeptide of claim 1, having a 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, having a 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 for 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 has the sequence
of SEQ ID NO:1.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide comprising a sequence selected from
the group consisting of: a) a polynucleotide comprising a
polynucleotide sequence of SEQ ID NO:2, b) a naturally occurring
polynucleotide comprising a polynucleotide sequence at least 90%
identical to a polynucleotide sequence of SEQ ID NO:2, c) a
polynucleotide having a sequence complementary to a polynucleotide
of a), d) a polynucleotide having a sequence 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 for 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 for 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 has an amino
acid sequence of SEQ ID NO:1.
19. A method for treating a disease or condition associated with
decreased expression of functional UBCLE, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method for 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 UBCLE, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method for 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 UBCLE, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) 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 for 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 UBCLE 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 UBCLE 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 UBCLE 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 having an 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 having an amino acid sequence of SEQ
ID NO:1.
37. An antibody produced by a method of claim 36.
38. A composition comprising the 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 having an 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 having an amino acid sequence of SEQ ID NO:1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the 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 having an 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 having an amino acid sequence of SEQ
ID NO:1 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence of SEQ ID NO:1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide having an 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. 09/328,866, filed Jun. 9, 1999, which is a
divisional application of U.S. application Ser. No. 08/989,289,
filed Dec. 12, 1997, now U.S. Pat. No. 5,968,747, each of which is
entitled UBIQUITIN-LIKE CONJUGATING PROTEIN, all of which are
hereby incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a ubiquitin-like conjugating protein and to the use of
these sequences in the diagnosis, treatment, and prevention of
cancer, and developmental, immune and neuronal disorders.
BACKGROUND OF THE INVENTION
[0003] The ubiquitin conjugation system (UCS) plays a major role in
the degradation of cellular proteins in eukaroytic cells and in
some bacterial cells. UCS mediates the elimination of abnormal
proteins and regulates the half-lives of other important regulatory
proteins that control gene transcription and cell cycle
progression. The UCS is reported to degrade mitotic cyclic kinases,
oncoproteins, tumor suppressors, viral proteins, transcriptional
regulators, and receptors associated with signal transduction
(Venna, R. et al. (1997) Science 278:455-460; Ciechanover, A.
(1994) Cell 79:13-21).
[0004] Several steps are involved in ubiquitin (Ub) conjugation and
protein degradation (Jentsch, S. (1992) Annu. Rev. Genet.
26:179-207). First, Ub, a small, heat stable protein, is activated
by a ubiquitin-activating enzyme (E1). This ATP dependent
activation involves binding of the C-terminus of Ub to the thiol
group of a cysteine residue of El. Activated Ub is subsequently
transferred to one of several Ub-conjugating enzymes (E2). Each E2
has a recognition subunit which allows it to interact with proteins
carrying a particular degradation signal. E2 links the Ub molecule
through its C-terminal glycine to an internal lysine of the target
protein. Different ubiquitin-dependent proteolytic pathways employ
structurally similar, but distinct, E2s, and in some instances,
accessory factors known as ubiquitin-ligases or E3s, are required
to work in conjunction with E2s for recognition of certain
substrates (Jensen, J. P. et al. (1995) J. Biol. Chem.
270:30408-30414). More than one Ub molecule may be needed to
ubiquinate a target protein which is subsequently recognized and
degraded by a proteasome. After degradation, Ub is released and
reutilized.
[0005] Prior to activation, Ub is usually expressed as a fusion
protein composed of an N-terminal ubiquitin and a C-terminal
extension protein (CEP) or as a polyubiquitin protein with Ub
monomers attached head to tail. CEPs have characteristics of a
variety of regulatory proteins; most are highly basic, contain up
to 30% lysine and arginine residues, and have nucleic acid-binding
domains (Monia, B. P. et al. (1989) J. Biol. Chem. 264:4093-4103).
The fusion protein is an important intermediate which appears to
mediate co-regulation of the cell's translational and protein
degradation activities, as well as localization of the inactive
enzyme to specific cellular sites. Once delivered, C-terminal
hydrolases cleave the fusion protein to release a functional Ub
(Monia et al., supra).
[0006] The E2s are important for substrate specificity in several
UCS pathways. All E2s have a conserved ubiquitin conjugation (UBC)
domain of approximately 16 kDa, at least 35% identity with each
other, and contain a centrally located cysteine residue which is
necessary for ubiquitin-enzyme thiolester formation (Jentsch,
supra). A highly conserved proline-rich element is located
N-terminal to the active cysteine residue. Structural variations
outside of this conserved domain are used to separate the E2
enzymes into classes. The E2s of class 1 (E2-1) consist of the
conserved UBC domain and include yeast E2-1 and UBCs 4, 5, and 7.
These E2s are thought to require E3 to carry out their activities
(Jentsch, supra). UBC7 has been shown to recognize ubiquitin as a
substrate and to form polyubiquitin chains in vitro (van Nocker, S.
et al. (1996) J. Biol. Chem. 271:12150-58). E2s of class 2 (E2-2)
have various unrelated C-terminal extensions that contribute to
substrate specificity and cellular localization. The yeast E2-2
enzymes, UBC2 and UBC3, have highly acidic C-terminal extensions
that promote interactions with basic substrates such as histones.
Yeast UBC6 has a hydrophobic signal-anchor sequence that localizes
the protein to the endoplasmic reticulum.
[0007] Abnormal activities of the UCS are implicated in a number of
diseases and disorders. These include, e.g., cachexia (Llovera, M.
et al. (1995) Int. J. Cancer 61:138-141), degradation of the
tumor-suppressor protein, p53 (Ciechanover, supra), and
neurodegeneration such as observed in Alzheimer's disease (Gregori,
L. et al. (1994) Biochem. Biophys. Res. Commun. 203:1731-1738).
Since ubiquitin conjugation is a rate-limiting step in antigen
presentation, the ubiquitin degradation pathway may also have a
critical role in the immune response (Grant E. P. et al. (1995) J.
Immunol. 155:3750-3758).
[0008] The discovery of new ubiquitin-conjugating-like protein and
the polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
treatment, and prevention of cancer, and developmental, immune and
neuronal disorders.
SUMMARY OF THE INVENTION
[0009] The invention features a substantially purified polypeptide,
ubiquitin-like conjugating protein (UBCLE), comprising the amino
acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0010] The invention further provides a substantially purified
variant of UBCLE having at least 90% amino acid identity to the
amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide
sequence encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention
also includes an isolated and purified polynucleotide variant
having at least 90% polynucleotide identity to the polynucleotide
sequence encoding the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
[0011] Additionally, the invention provides a composition
comprising a polynucleotide sequence encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1. The invention further provides an isolated and
purified polynucleotide sequence which hybridizes under stringent
conditions to the polynucleotide sequence encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO:1 or a fragment of
SEQ ID NO:1, as well as an isolated and purified polynucleotide
sequence which is complementary to the polynucleotide sequence
encoding the polypeptide comprising the amino acid sequence of SEQ
ID NO:1 or a fragment of SEQ ID NO:1.
[0012] The invention also provides an isolated and purified
polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQ
ID NO:2, and an isolated and purified polynucleotide variant having
at least 90% polynucleotide identity to the polynucleotide sequence
comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention
also provides an isolated and purified polynucleotide sequence
which is complementary to the polynucleotide sequence comprising
SEQ ID NO:2 or a fragment of SEQ ID NO:2.
[0013] The invention further provides an expression vector
containing at least a fragment of the polynucleotide sequence
encoding the polypeptide comprising the amino acid sequence of SEQ
ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the
expression vector is contained within a host cell.
[0014] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a
fragment of SEQ ID NO:1, the method comprising the steps of: (a)
culturing the host cell containing an expression vector containing
at least a fragment of a polynucleotide sequence encoding UBCLE
under conditions suitable for the expression of the polypeptide;
and (b) recovering the polypeptide from the host cell culture.
[0015] The invention also provides a pharmaceutical composition
comprising a substantially purified UBCLE having the amino acid
sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction
with a suitable pharmaceutical carrier.
[0016] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence of SEQ ID
NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist
and a purified antagonist of the polypeptide.
[0017] The invention also provides a method for treating or
preventing a cancer, the method comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of UBCLE.
[0018] The invention also provides a method for treating or
preventing a developmental disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of UBCLE.
[0019] The invention also provides a method for treating or
preventing an immune disorder, the method comprising administering
to a subject in need of such treatment an effective amount of an
antagonist of UBCLE.
[0020] The invention also provides a method for treating or
preventing a neuronal disorder, the method comprising administering
to a subject in need of such treatment an effective amount of an
antagonist of UBCLE.
[0021] The invention also provides a method for detecting a
polynucleotide encoding UBCLE in a biological sample containing
nucleic acids, the method comprising the steps of:
[0022] (a) hybridizing the complement of the polynucleotide
sequence encoding the polypeptide comprising SEQ ID NO:1 or a
fragment of SEQ ID NO:1 to at least one of the nucleic acids of the
biological sample, thereby forming a hybridization complex; and (b)
detecting the hybridization complex, wherein the presence of the
hybridization complex correlates with the presence of a
polynucleotide encoding UBCLE in the biological sample. In one
aspect, the nucleic acids of the biological sample are amplified by
the polymerase chain reaction prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID
NO:1) and nucleic acid sequence (SEQ ID NO:2) of UBCLE. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering Co. Ltd., San Bruno, Calif.).
[0024] FIG. 2 shows the amino acid sequence alignments between
UBCLE (2501808; SEQ ID NO:1) and UBCH5C (GI 1145691; SEQ ID NO:3),
produced using the multisequence alignment program of DNASTAR
software (DNASTAR Inc, Madison Wis.).
DESCRIPTION OF THE INVENTION
[0025] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular 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.
[0026] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any 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 cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
night be used in connection with the invention. Nothing herein is
to be construed as an admission that the invention is not entitled
to antedate such disclosure by virtue of prior invention.
[0028] Definitions
[0029] "UBCLE," as used herein, refers to the amino acid sequences
of substantially purified UBCLE obtained from any species,
particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and preferably the human species, from any source,
whether natural, synthetic, semi-synthetic, or recombinant.
[0030] The term "agonist," as used herein, refers to a molecule
which, when bound to UBCLE, increases or prolongs the duration of
the effect of UBCLE. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of UBCLE.
[0031] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding UBCLE.
Alleles may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes which give rise to alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0032] "Altered" nucleic acid sequences encoding UBCLE, as
described herein, include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the same UBCLE or a polypeptide with at least one
functional characteristic of UBCLE. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
UBCLE, and improper or unexpected hybridization to alleles, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding UBCLE. The encoded protein may
also be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent UBCLE. 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 UBCLE is retained. For
example, negatively charged amino acids may include aspartic acid
and glutamic acid, positively charged amino acids may include
lysine and arginine, and amino acids with uncharged polar head
groups having similar hydrophilicity values may include leucine,
isoleucine, and valine; glycine and alanine; asparagine and
glutamine; serine and threonine; and phenylalanine and
tyrosine.
[0033] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments" refers to
fragments of UBCLE which are preferably about 5 to about 15 amino
acids in length and which retain some biological activity or
immunological activity of UBCLE. 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.
[0034] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y.)
[0035] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to UBCLE, decreases the amount or the
duration of the effect of the biological or immunological activity
of UBCLE. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of UBCLE.
[0036] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind UBCLE polypeptides can
be prepared using intact polypeptides or using fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0037] The term "antigenic determinant," 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 a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0038] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
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
may be produced by any method including synthesis or transcription.
Once introduced into a cell, the complementary nucleotides combine
with natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0039] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
UBCLE, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0040] The terms "complementary" or "complementarity," 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," such that only some of the nucleic acids bind, or it
may be "complete," such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands. This is of particular importance in amplification
reactions, which depend upon binding between nucleic acids strands,
and in the design and use of peptide nucleic acid (PNA)
molecules.
[0041] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation or an aqueous solution. Compositions
comprising polynucleotide sequences encoding UBCLE or fragments of
UBCLE 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.).
[0042] The phrase "consensus sequence," as used herein, refers to a
nucleic acid sequence which has been resequenced to resolve
uncalled bases, extended using XL-PCR kit (Perkin Elmer, Norwalk,
Conn.) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from the overlapping sequences of more than one
Incyte Clone using a computer program for fragment assembly, such
as the GELVIEW fragment assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0043] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding UBCLE, by northern analysis is indicative of the presence
of nucleic acids encoding UBCLE in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
UBCLE.
[0044] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0045] The term "derivative," as used herein, refers to the
chemical modification of UBCLE, of a polynucleotide sequence
encoding UBCLE, or of a polynucleotide sequence complementary to a
polynucleotide sequence encoding UBCLE. Chemical modifications of a
polynucleotide sequence can include, for example, replacement of
hydrogen by an alkyl, acyl, or amino group. A derivative
polynucleotide encodes a polypeptide which retains at least one
biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains at least one
biological or immunological function of the polypeptide from which
it was derived.
[0046] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. 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
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0047] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 10 K to 10 M in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355.)
[0048] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability.
[0049] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0050] As used herein, 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 bases. A hybridization complex may be formed in
solution (e.g., C.sub.0t or R.sub.0t analysis) or formed between
one nucleic acid sequence present in solution and another nucleic
acid sequence immobilized on a solid support (e.g., paper,
membranes, filters, chips, pins or glass slides, or any other
appropriate substrate to which cells or their nucleic acids have
been fixed).
[0051] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0052] The term "microarray," as used herein, refers to an array of
distinct polynucleotides or oligonucleotides arrayed on a
substrate, such as paper, nylon or any other type of membrane,
filter, chip, glass slide, or any other suitable solid support.
[0053] The term "modulate," as it appears herein, refers to a
change in the activity of UBCLE. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of UBCLE.
[0054] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, to DNA or RNA of genomic
or synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0055] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimers," "primers," "oligomers," and "probes," as
these terms are commonly defined in the art.
[0056] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (Nielsen, P. E. et al. (1993) Anticancer Drug
Des. 8:53-63.
[0057] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding UBCLE, or fragments thereof, or UBCLE 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.
[0058] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein recognized by the binding molecule (i.e.,
the antigenic determinant or epitope). For example, if an antibody
is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0059] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are well known in the art. In particular, stringency can be
increased by reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature.
[0060] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and 200 .mu.g/ml sheared and denatured
salmon sperm DNA. Hybridization could occur under reduced
stringency conditions as described above, but in 35% formamide at a
reduced temperature of 35.degree. C. The temperature range
corresponding to a particular level of stringency can be further
narrowed by calculating the purine to pyrimidine ratio of the
nucleic acid of interest and adjusting the temperature accordingly.
Variations on the above ranges and conditions are well known in the
art.
[0061] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0062] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0063] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, and refers to cells which transiently
express the inserted DNA or RNA for limited periods of time.
[0064] A "variant" of UBCLE, as used herein, refers to an amino
acid sequence that is altered by one or more amino acids. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties (e.g.,
replacement of leucine with isoleucine). More rarely, a variant may
have "nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
[0065] The Invention
[0066] The invention is based on the discovery of a new human
ubiquitin-like conjugating protein (UBCLE), the polynucleotides
encoding UBCLE, and the use of these compositions for the
diagnosis, treatment, or prevention of cancer, and developmental,
immune, and neuronal disorders.
[0067] Nucleic acids encoding the UBCLE of the present invention
were first identified in Incyte Clone 2501808 from the adrenal
tumor cDNA library (ADRETUT05) 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 2501808 (ADRETUT05), 291812 (TMLR3DT01),
and 743853 (BRAITUT01).
[0068] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1. UBCLE is 198
amino acids in length and has a potential ubiquitin conjugating
enzyme active site between residues Y.sub.75 and V.sub.89, a
potential N-glycosylation site at residue N.sub.176, a potential
cAMP phosphorylation site at residue R.sub.181, a potential casein
kinase II phosphorylation site at residue T.sub.44, and two
potential protein kinase C phosphorylation sites at residues
T.sub.147 and T.sub.178. As shown in FIG. 2, UBCLE has chemical and
structural homology with UBCH5C(GI 1145691; SEQ ID NO:3). In
particular, UBCLE and UBCH5C share 32% sequence identity. Northern
analysis shows the expression of this sequence in various cDNA
libraries, at least 52% of which are immortalized or cancerous, at
least 39% of which involve cell proliferation. Of particular note
is the expression in nervous system tissues.
[0069] The invention also encompasses UBCLE variants. A preferred
UBCLE variant is one having at least about 80%, more preferably at
least about 90%, and most preferably at least about 95% amino acid
sequence identity to the UBCLE amino acid sequence.
[0070] The invention also encompasses polynucleotides which encode
UBCLE. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2,
which encodes an UBCLE.
[0071] The invention also encompasses a variant of a polynucleotide
sequence encoding UBCLE. In particular, such a variant
polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding UBCLE. A particular aspect of the invention encompasses a
variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2.
[0072] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding UBCLE, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring UBCLE, and all such variations are
to be considered as being specifically disclosed.
[0073] Although nucleotide sequences which encode UBCLE and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring UBCLE under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding UBCLE 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 UBCLE 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.
[0074] The invention also encompasses production of DNA sequences
which encode UBCLE and UBCLE derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding UBCLE or any fragment
thereof.
[0075] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:2, or a fragment of SEQ ID NO:2, under various conditions of
stringency as taught in Wahi, G. M. and S. L. Berger (1987; Methods
Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.
152:507-511).
[0076] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, 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 amplification system (GIBco/BRL (Gaithersburg,
Md.). Preferably, the process is automated with machines such as
the Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.), Peltiert thermal
cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI CATALYST
and 373 and 377 DNA SEQUENCERS (Perkin Elmer).
[0077] The nucleic acid sequences encoding UBCLE 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 a 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.
[0078] 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 about 22 to 30 nucleotides in
length, to have a GC content of about 50% or more, and to anneal to
the target sequence at temperatures of about 68.degree. C. to
72.degree. C. The method uses several restriction enzymes to
generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used
as a PCR template.
[0079] 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 be used to
place an engineered double-stranded sequence into an unknown
fragment of the DNA molecule before performing PCR. 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.
[0080] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genoric libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0081] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., SEQUENCE NAVIGATIOR and GENOTYPER, 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.
[0082] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode UBCLE may be used in
recombinant DNA molecules to direct expression of UBCLE, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced, and these sequences
may be used to clone and express UBCLE.
[0083] As will be understood by those of skill in the art, it may
be advantageous to produce UBCLE-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.
[0084] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter UBCLE 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
[0085] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding UBCLE may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of UBCLE activity, it
may be useful to encode a chimeric UBCLE 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 UBCLE encoding sequence and the heterologous protein sequence,
so that UBCLE may be cleaved and purified away from the
heterologous moiety.
[0086] In another embodiment, sequences encoding UBCLE may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See Caruthers, M. H. et al. (1980) Nucl. Acids Res.
Symp. Ser. 7:215-223, and Horn, T. et al. (1980) Nucl. Acids Res.
Symp. Ser. 7:225-232.) Alternatively, the protein itself may be
produced using chemical methods to synthesize the amino acid
sequence of UBCLE, 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 using the ABI 431A peptide synthesizer
(Perkin Elmer).
[0087] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography. (See, for
example, Creighton, T. (1983) Proteins, Structures and Molecular
Principles, WH Freeman and Co., New York, N.Y.) The composition of
the synthetic peptides may be confirmed by amino acid analysis or
by sequencing. (See, for example, the Edman degradation procedure
described in Creighton, supra.) Additionally, the amino acid
sequence of UBCLE, or any part thereof, may be altered during
direct synthesis and/or combined with sequences from other
proteins, or any part thereof, to produce a variant
polypeptide.
[0088] In order to express a biologically active UBCLE, the
nucleotide sequences encoding UBCLE or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0089] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding UBCLE 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.).
[0090] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding UBCLE. 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) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0091] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters,
and 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, La Jolla, 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 UBCLE, vectors based on SV40 or EBV may be
used with an appropriate selectable marker.
[0092] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for UBCLE. For example,
when large quantities of UBCLE are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as BLUESCRIPT (Stratagene), in which
the sequence encoding UBCLE 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.
[0093] 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 (supra) and Grant et al. (1987; Methods Enzymol.
153:516-544).
[0094] In cases where plant expression vectors are used, the
expression of sequences encoding UBCLE 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.)
[0095] An insect system may also be used to express UBCLE. 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 UBCLE 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 UBCLE 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 UBCLE may be expressed. (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227.)
[0096] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding UBCLE 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 UBCLE in
infected host cells. (Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. 81:3655-3659.) In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0097] 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 about 6 M to 10 M are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0098] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding UBCLE. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding UBCLE and its initiation codon and
upstream sequences are inserted into the appropriate expression
vector, no additional transcriptional or translational control
signals may be needed. However, in cases where only coding
sequence, or a fragment thereof, is inserted, exogenous
translational control signals including the ATG initiation codon
should be provided. Furthermore, the initiation codon should be in
the correct reading frame to ensure translation of the entire
insert. Exogenous translational elements and initiation codons may
be of various origins, both natural and synthetic. The efficiency
of expression may be enhanced by the inclusion of enhancers
appropriate for the particular cell system used, such as those
described in the literature. (Scharf, D. et al. (1994) Results
Probl. Cell Differ. 20:125-162.)
[0099] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the 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, Manassas, Va.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0100] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing UBCLE can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences. Resistant
clones of stably transformed cells may be proliferated using tissue
culture techniques appropriate to the cell type.
[0101] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thyridine kinase genes (Wigler, M. et al.
(1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes
(Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in
tk.sup.- or apr.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate
(Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als
or pat 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.
85:8047-51.) 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.
These markers can be used not only to identify transformants, but
also to quantify the amount of transient or stable protein
expression attributable to a specific vector system. (Rhodes, C. A.
et al. (1995) Methods Mol. Biol. 55:121-131.)
[0102] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding UBCLE is inserted within a marker gene
sequence, transformed cells containing sequences encoding UBCLE can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding UBCLE under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0103] Alternatively, host cells which contain the nucleic acid
sequence encoding UBCLE and express UBCLE may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0104] The presence of polynucleotide sequences encoding UBCLE can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
UBCLE. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding UBCLE
to detect transformants containing DNA or RNA encoding UBCLE.
[0105] A variety of protocols for detecting and measuring the
expression of UBCLE, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
UBCLE is preferred, but a competitive binding assay may be
employed. These and other assays are well described in the art, for
example, in Hampton, R. et al. (1990; Serological Methods, a
Laboratory Manual, APS Press, St. Paul, Minn.) and in Maddox, D. E.
et al. (1983; J. Exp. Med. 158:1211-1216).
[0106] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding UBCLE include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding UBCLE, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by 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.
[0107] Host cells transformed with nucleotide sequences encoding
UBCLE 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 UBCLE may be designed to
contain signal sequences which direct secretion of UBCLE through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding UBCLE to nucleotide sequences
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 the UBCLE encoding
sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing UBCLE and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMIAC; described in Porath, J. et al.
(1992) Prot. Exp. Purif. 3:263-281)), while the enterokinase
cleavage site provides a means for purifying UBCLE 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).
[0108] Fragments of UBCLE may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (Merrifield J. (1963) J. Am Chem. Soc. 85:2149-2154.)
Protein synthesis may be performed by manual techniques or by
automation. Automated synthesis may be achieved, for example, using
the Applied Biosystems 431A peptide synthesizer (Perkin Elmer).
Various fragments of UBCLE may be synthesized separately and then
combined to produce the full length molecule.
[0109] Therapeutics
[0110] Chemical and structural homology exists between UBCLE and
human UBCH5C (GI 1145690). In addition, UBCLE is expressed in
cancer, and developmental, immune, and neuronal disorders where
UBCLE plays a role in the cell cycle and in cell signaling.
Therefore, UBCLE appears to play a role in cancer, and
developmental, immune, and neuronal disorders.
[0111] Degradation of tumor suppressor proteins such as p53 by E2
enzymes may contribute to the development of cancer. Therefore, in
one embodiment, an antagonist of UBCLE may be administered to a
subject to treat or prevent a cancer. Such cancers can include, but
are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
and uterus.
[0112] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding UBCLE may be administered
to a subject to treat or prevent a cancer including, but not
limited to, those described above.
[0113] Abnormalities in processing of neural proteins (AP) by
enzymes of the UCS may contribute to neuronal disorders. Therefore,
in another embodiment, an antagonist which modulates the activity
of UBCLE may be administered to a subject to treat or prevent a
neuronal disorder. Such neuronal disorders can include, but are not
limited to, akathesia, Alzheimer's disease, amnesia, amyotrophic
lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms,
dementia, depression, Down's syndrome, tardive dyskinesia,
dystonias, epilepsy, Huntington's disease, multiple sclerosis,
Parkinson's disease, paranoid psychoses, schizophrenia, and
Tourette's disorder.
[0114] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding UBCLE may be administered
to a subject to treat or prevent a neuronal disorder including, but
not limited to, those disorders described above.
[0115] In a further embodiment, an antagonist of UBCLE may be
administered to a subject to treat or prevent a developmental
disorder. Such developmental disorders may include, but are not
limited to, renal tubular acidosis, Cushing's syndrome,
achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,
gonadal dysgenesis, myelodysplastic syndrome, hereditary
mucoepithelial dysplasia, hereditary keratodermas, hereditary
neuropathies such as Charcot-Marie-Tooth disease and
neurofibromatosis, hypothyroidism, hydrocephalus, seizure disorders
such as Syndenham's chorea and cerebral palsy, spinal bifida, and
congenital glaucoma, cataract, or sensorineural hearing loss.
[0116] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding UBCLE may be administered
to a subject to treat or prevent a developmental disorder
including, but not limited to, those disorders described above.
[0117] In a further embodiment, an antagonist of UBCLE may be
administered to a subject to treat or prevent an immune disorder.
Such immune disorder s may include, but are not limited to,
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis bronchitis, cholecystitis, contact dermayitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner
syndrome, and complications of cancer, hemodialysis, and
extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections; and trauma.
[0118] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding UBCLE may be administered
to a subject to treat or prevent an immune disorder including, but
not limited to, those disorders described above.
[0119] In one aspect, an antibody which specifically binds UBCLE
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 UBCLE.
[0120] 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.
[0121] An antagonist of UBCLE may be produced using methods which
are generally known in the art. In particular, purified UBCLE may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
UBCLE. Antibodies to UBCLE may also be generated using methods that
are well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, and single chain
antibodies, Fab fragments, and fragments produced by a Fab
expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are especially preferred for therapeutic
use.
[0122] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with UBCLE or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0123] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to UBCLE have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of UBCLE amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0124] Monoclonal antibodies to UBCLE 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.
80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120.)
[0125] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used.
(Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855;
Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S.
et al. (1985) Nature 314:452-454.) Alternatively, techniques
described for the production of single chain antibodies may be
adapted, using methods known in the art, to produce UBCLE-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. 88:11120-11123.)
[0126] 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. 86:3833-3837, and Winter, G. et al. (1991)
Nature 349:293-299.)
[0127] Antibody fragments which contain specific binding sites for
UBCLE may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (Huse, W. D. et al. (1989) Science
254:1275-1281.)
[0128] 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 UBCLE and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering UBCLE
epitopes is preferred, but a competitive binding assay may also be
employed. (Maddox, supra.)
[0129] In another embodiment of the invention, the polynucleotides
encoding UBCLE, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding UBCLE 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 UBCLE. Thus, complementary molecules or
fragments may be used to modulate UBCLE 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 UBCLE.
[0130] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequence complementary to the
polynucleotides of the gene encoding UBCLE. These techniques are
described, for example, in Sambrook (supra) and in Ausubel
(supra).
[0131] Genes encoding UBCLE can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide or fragment thereof encoding UBCLE. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector, and may last even longer if appropriate replication
elements are part of the vector system.
[0132] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding UBCLE. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (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.) A complementary sequence or
antisense molecule may also be designed to block translation of
mRNA by preventing the transcript from binding to ribosomes.
[0133] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding UBCLE.
[0134] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences:GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0135] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphorarnidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding UBCLE. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA constitutively or inducibly can
be introduced into cell lines, cells, or tissues.
[0136] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional 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.
[0137] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art, such as
those described in Goldman, C. K. et al. (1997; Nature
Biotechnology 15:462-466).
[0138] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0139] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of UBCLE, antibodies to UBCLE, and mimetics, agonists,
antagonists, or inhibitors of UBCLE. The compositions may be
administered alone or in combination with at least one other agent,
such as a stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0140] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0141] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0142] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0143] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylnethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0144] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0145] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0146] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0147] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0148] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0149] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0150] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of UBCLE, such
labeling would include amount, frequency, and method of
administration.
[0151] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0152] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays of neoplastic
cells, for example, or in animal models, usually mice, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0153] A therapeutically effective dose refers to that amount of
active ingredient, for example UBCLE or fragments thereof,
antibodies of UBCLE, and agonists, antagonists or inhibitors of
UBCLE, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED50 (the dose therapeutically effective in 50%
of the population) or LD50 (the dose lethal to 50% of the
population) statistics. The dose ratio between therapeutic and
toxic effects is the therapeutic index, and it can be expressed as
the LD50/ED50 ratio. Pharmaceutical compositions which exhibit
large therapeutic indices are preferred. The data obtained from
cell culture assays and animal studies are used 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, the sensitivity of the patient, and the route of
administration.
[0154] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, 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 the half-life and clearance rate of the particular
formulation.
[0155] Normal dosage amounts may vary from 0.1 .mu.g to 100,000
.mu.g, up to a total dose of about 1 gram, depending upon the route
of administration. Guidance as to particular dosages and methods of
delivery is provided in the literature and generally available to
practitioners in the art. Those skilled in the art will employ
different formulations for nucleotides than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides
will be specific to particular cells, conditions, locations,
etc.
[0156] Diagnostics
[0157] In another embodiment, antibodies which specifically bind
UBCLE may be used for the diagnosis of disorders characterized by
expression of UBCLE, or in assays to monitor patients being treated
with UBCLE or agonists, antagonists, and inhibitors of UBCLE.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as those described above for therapeutics. Diagnostic
assays for UBCLE include methods which utilize the antibody and a
label to detect UBCLE in human body fluids or in extracts of cells
or tissues. The antibodies may be used with or without
modification, and may be labeled by covalent or non-covalent
joining with a reporter molecule. A wide variety of reporter
molecules, several of which are described above, are known in the
art and may be used.
[0158] A variety of protocols for measuring UBCLE, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of UBCLE expression.
Normal or standard values for UBCLE expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to UBCLE under conditions
suitable for complex formation. The amount of standard complex
formation may be quantified by various methods, preferably by
photometric means. Quantities of UBCLE expressed in subject,
control, and disease samples from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0159] In another embodiment of the invention, the polynucleotides
encoding UBCLE 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 UBCLE may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
UBCLE, and to monitor regulation of UBCLE levels during therapeutic
intervention.
[0160] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genonic
sequences, encoding UBCLE or closely related molecules may be used
to identify nucleic acid sequences which encode UBCLE. The
specificity of the probe, whether it is made from a highly specific
region (e.g., the 5' regulatory region) or from a less specific
region (e.g., 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 UBCLE, alleles, or related
sequences.
[0161] 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 UBCLE encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
may be derived from the sequence of SEQ ID NO:2 or from genomic
sequences including promoter and enhancer elements and introns of
the naturally occurring UBCLE.
[0162] Means for producing specific hybridization probes for DNAs
encoding UBCLE include the cloning of polynucleotide sequences
encoding UBCLE or UBCLE derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0163] Polynucleotide sequences encoding UBCLE may be used for the
diagnosis of a disorder associated with expression of UBCLE.
Disorders include, but are not limited to, cancers such as
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen, testis, thymus, thyroid, and uterus; neuronal
disorders such as akathesia, Alzheimer's disease, amnesia,
amyotrophic lateral sclerosis, bipolar disorder, catatonia,
cerebral neoplasms, dementia, depression, Down's syndrome, tardive
dyskinesia, dystonias, epilepsy, Huntington's disease, multiple
sclerosis, Parkinson's disease, paranoid psychoses, schizophrenia,
and Tourette's disorder; developmental disorders such as renal
tubular acidosis, Cushing's syndrome, achondroplastic dwarfism,
Duchenne and Becker muscular dystrophy, gonadal dysgenesis,
myelodysplastic syndrome, hereditary mucoepithelial dysplasia,
hereditary keratodermas, hereditary neuropathies such as
Charcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,
hydrocephalus, seizure disorders such as Syndenham's chorea and
cerebral palsy, spinal bifida, and congenital glaucoma, cataract,
or sensorineural hearing loss; and immune disorders such as
Addison's disease, adult respiratory distress syndrome, allergies,
ankylosing spondylitis, amyloidosis, anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis ,bronchitis, cholecystitis, contact dermayitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, lupus erythematosus, multiple sclerosis, myasthenia
gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, ulcerative colitis, Werner
syndrome, and complications of cancer, hemodialysis, and
extracorporeal circulation; viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections; and trauma. The
polynucleotide sequences encoding UBCLE may be used in Southern or
northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in dipstick, pin, and ELISA assays; and in
microarrays utilizing fluids or tissues from patient biopsies to
detect altered UBCLE expression. Such qualitative or quantitative
methods are well known in the art.
[0164] In a particular aspect, the nucleotide sequences encoding
UBCLE may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding UBCLE may be labeled by standard
methods and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantitated and compared with a standard value. If the
amount of signal in the patient sample is significantly altered
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 UBCLE
in the sample indicates the presence of the associated disorder.
Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies, in
clinical trials, or in monitoring the treatment of an individual
patient.
[0165] In order to provide a basis for the diagnosis of a disorder
associated with expression of UBCLE, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding UBCLE, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for a disorder. Deviation from
standard values is used to establish the presence of a
disorder.
[0166] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to evaluate whether the level of
expression in the patient begins to approximate that which is
observed in the normal subject. The results obtained from
successive assays may be used to show the efficacy of treatment
over a period ranging from several days to months.
[0167] With respect to cancer, the presence of 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.
[0168] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding UBCLE may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding UBCLE, or a fragment of a
polynucleotide complementary to the polynucleotide encoding UBCLE,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0169] Methods which may also be used to quantitate the expression
of UBCLE include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (Melby, P.C. et al. (1993) J.
Immunol. Methods 159:235-244, and Duplaa, C. et al. (1993) Anal.
Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid
quantitation.
[0170] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously (to produce a transcript image) and to identify
genetic variants, mutations, and polymorphisms. This information
may be used in determining gene function, in understanding the
genetic basis of a disorder, in diagnosing a disorder, and in
developing and monitoring the activities of therapeutic agents.
[0171] In one embodiment, the microarray is prepared and used
according to methods known in the art, such as those described in
published PCT application WO95/11995 (Chee et al.), Lockhart, D. J.
et al. (1996; Nat. Biotech. 14:1675-1680), and Schena, M. et al.
(1996; Proc. Natl. Acad. Sci. 93:10614-10619).
[0172] 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 to
60 nucleotides in length, more preferably about 15 to 30
nucleotides in length, and most preferably about 20 to 25
nucleotides in length. For a certain type of microarray, it may be
preferable to use oligonucleotides which are about 7 to 10
nucleotides in length. The microarray may contain oligonucleotides
which cover the known 5' or 3' sequence, or may contain 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 specific to a gene or genes of interest in which
at least a fragment of the sequence is known or oligonucleotides
specific to one or more unidentified cDNAs common to a particular
cell or tissue type or to a normal, developmental, or disease
state. In certain situations, it may be appropriate to use pairs of
oligonucleotides on a microarray. The pairs will be identical,
except for one nucleotide preferably 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 about 2 to 1,000,000.
[0173] 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' end, 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 one aspect, the oligomers are synthesized at
designated areas on a substrate using a light-directed chemical
process. The substrate may be paper, nylon, any other type of
membrane, filter, chip, glass slide, or any other suitable solid
support.
[0174] In one aspect, the oligonucleotides may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, such as that described in
published PCT application WO95/251116 (Baldeschweiler et al.). In
another aspect, a grid array analogous to a dot or slot blot
(HYBRIDOT apparatus, GIBCO/BRL) may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system or thermal, UV, mechanical or chemical
bonding procedures. In yet another aspect, an array may be produced
by hand or by using available devices, materials, and machines
(including BRINKMANN multichannel pipettors or robotic
instruments), and may contain 8, 24, 96, 384, 1536, or 6144
oligonucleotides, or any other multiple from 2 to 1,000,000 which
lends itself to the efficient use of commercially available
instrumentation.
[0175] In order to conduct sample analysis using the microarrays,
polynucleotides are extracted from a biological sample. The
biological samples may be obtained from any bodily fluid (blood,
urine, saliva, phlegm, gastric juices, etc.), cultured cells,
biopsies, or other tissue preparations. To produce probes, the
polynucleotides extracted from the sample are used to produce
nucleic acid sequences which are complementary to the nucleic acids
on the microarray. If the microarray consists of cDNAs, antisense
RNAs (aRNA) are appropriate probes. Therefore, in one aspect, nRNA
is used to produce cDNA which, in turn and in the presence of
fluorescent nucleotides, is used to produce fragment or
oligonucleotide aRNA probes. These fluorescently labeled probes are
incubated with the microarray so that the probe sequences hybridize
to the cDNA oligonucleotides of the microarray. In another aspect,
nucleic acid sequences used as probes can include polynucleotides,
fragments, and complementary or antisense sequences produced using
restriction enzymes, PCR technologies, and OLIGOLABELING or
TRANSPROBE kits (Pharmacia & Upjohn) well known in the area of
hybridization technology.
[0176] 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 the
degree of complementarity and the relative abundance of each
oligonucleotide sequence on the microarray. 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 or for
functional analysis of the sequences, mutations, variants, or
polymorphisms among samples. (Heller, R. A. et al. (1997) Proc.
Natl. Acad. Sci. 94:2150-2155.)
[0177] In another embodiment of the invention, nucleic acid
sequences encoding UBCLE may be used to generate hybridization
probes 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, such as those reviewed in Price, C. M. (1993; Blood Rev.
7:127-134) and Trask, B. J. (1991; Trends Genet. 7:149-154).
[0178] 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 the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the location of the gene encoding UBCLE on a
physical chromosomal map and a specific disorder, or predisposition
to a specific disorder, may help define the region of DNA
associated with that disorder. The nucleotide sequences of the
subject invention may be used to detect differences in gene
sequences between normal, carrier, and affected individuals.
[0179] 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.
[0180] In another embodiment of the invention, UBCLE, 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 UBCLE and the agent being tested may be
measured.
[0181] 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, 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 UBCLE, or fragments thereof, and washed.
Bound UBCLE is then detected by methods well known in the art.
Purified UBCLE 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.
[0182] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding UBCLE specifically compete with a test compound for binding
UBCLE. In this manner, antibodies can be used to detect the
presence of any peptide which shares one or more antigenic
determinants with UBCLE.
[0183] In additional embodiments, the nucleotide sequences which
encode UBCLE 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.
[0184] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0185] I. ADRETUTo5 cDNA Library Construction
[0186] The ADRETUT05 cDNA library was constructed from tumor tissue
obtained from the right adrenal gland of a 52 year-old female
during a unilateral adrenalectomy. Pathology indicated a
pheochromocytoma. Patient history included benign hypertension,
depressive disorder, chronic sinusitis, idiopathic proctocolitis,
urinary tract infection and irritable colon. Family history
included benign hypertension in a sibling, cerebrovascular disease,
secondary Parkinsonism and irritable colon in the mother;
atherosclerotic coronary artery disease, hyperlipidemia and
malignant brain neoplasm in siblings and secondary Parkinsonism in
the father.
[0187] The frozen tissues were homogenized and lysed in TRIZOL
reagent (1 gm tissue/10 ml TRIZOL; Cat. #10296-028; GIBCO/BRL), a
monophasic solution of phenol and guanidine isothiocyanate, using a
Brinkmann POLYTRON Homogenizer PT-3000 (Brinkmann Instruments,
Westbury, N.Y.). After a brief incubation on ice, chloroform was
added (1:5 v/v), and the lysate was centrifuged. The upper
chloroform layer was removed to a fresh tube and the RNA extracted
with isopropanol, resuspended in DEPC-treated water, and DNase
treated for 25 min at 37.degree. C. The RNA was re-extracted once
with acid phenol-chloroform pH 4.7 and precipitated using 0.3M
sodium acetate and 2.5 volumes ethanol. The mRNA was then isolated
with the OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used
to construct the cDNA library.
[0188] The mRNA was handled according to the recommended protocols
in the (Cat. #18248-013, GIBCO/BRL). The cDNAs were fractionated on
a SEPHAROSE CL4B column (Cat. #275105-01; Pharmacia), and those
cDNAs exceeding 400 bp were ligated into pINCY 1. The plasmid pINCY
1 was subsequently transformed into DH5 .alpha. competent cells
(Cat. #18258-012; GIBCO/BRL).
[0189] II Isolation and Sequencing of cDNA Clones
[0190] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 Plasmid Kit (Catalog #26173, QIAGEN). The
recommended protocol was employed except for the following changes:
1) the bacteria were cultured in 1 ml of sterile Terrific Broth
(Catalog #22711, GIBCO/BRL) with carbenicillin at 25 mg/L and
glycerol at 0.4%; 2) the cultures were incubated for 19 hours after
the wells were inoculation and then lysed with 0.3 ml of lysis
buffer; 3) following isopropanol precipitation, the DNA pellet was
resuspended in 0.1 ml of distilled water. After the last step in
the protocol, samples were transferred to a 96-well block for
storage at 4.degree. C.
[0191] The cDNAs were sequenced by the method of Sanger et al.
(1975, J. Mol. Biol. 94:441f), using a Hamilton MICROLAB 2200
(Hamilton, Reno, Nev.) in combination with Peltier thermal cyclers
(PTC200 from MJ Research, Watertown, Mass.) and Applied Biosystems
377 DNA sequencing systems.
[0192] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0193] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and
Altschul et al. (1990) J. Mol. Biol. 215:403-410.)
[0194] BLAST produced alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST was especially useful in
determining exact matches or in identifying homologs which may be
of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant)
origin. Other algorithms such as the one described in Smith, T. et
al. (1992; Protein Engineering 5:35-51), could have been used when
dealing with primary sequence patterns and secondary structure gap
penalties. The sequences disclosed in this application have lengths
of at least 49 nucleotides and have no more than 12% uncalled bases
(where N is recorded rather than A, C, G, or T).
[0195] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10.sup.-25 for nucleotides and
10.sup.-10 or peptides.
[0196] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mamalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0197] IV. Northern Analysis
[0198] 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, supra).
[0199] Analogous computer techniques applying BLAST 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.
[0200] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0201] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0202] The results of northern analysis are reported as a list of
libraries in which the transcript encoding UBCLE 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.
[0203] V. Extension of UBCLE Encoding Polynucleotides
[0204] The nucleic acid sequence of Incyte Clone 2501808 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 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the target sequence at temperatures of about
68.degree. C. to about 72.degree. C. Any stretch of nucleotides
which would result in hairpin structures and primer-primer
dimerizations was avoided.
[0205] 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.
[0206] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier thermal cycler (PTC200; M. J. Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with 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 steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0207] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK
(QIAGEN), and trimmed of overhangs using Klenow enzyme to
facilitate religation and cloning.
[0208] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (Sambrook, supra). After
incubation for one hour at 37.degree. C., the E. coli mixture was
plated on Luria Bertani (LB) agar (Sambrook, 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.of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0209] 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 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0210] 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.
[0211] 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.
[0212] VI. Labeling and Use of Individual Hybridization Probes
[0213] 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 (National Biosciences)
and 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 using a SEPHADEX G-25
superfine resin column (Pharmacia & Upjohn). An aliquot
containing 10.sup.7 counts per minute of the labeled probe is used
in a typical membrane-based hybridization analysis of human genomic
DNA digested with one of the following endonucleases:Ase I, Bgl II,
Eco RI, Pst I, Xba 1, or Pvu II (DuPont NEN).
[0214] 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
autoradiography film (Kodak, Rochester, N.Y.) is exposed to the
blots in a PHOSPHOIMAGER cassette (Molecular Dynamnics, Sunnyvale,
Calif.) for several hours, hybridization patterns are compared
visually.
[0215] VII. Microarrays
[0216] To produce oligonucleotides for a microarray, one of the
nucleotide sequences of the present invention 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
approximately 20 sequence-specific oligonucleotides of 20
nucleotides in length (20-mers). A matched set of oligonucleotides
are 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, such as that described in Chee (supra).
[0217] In the alternative, a chemical coupling procedure and an ink
jet device are used to synthesize oligomers on the surface of a
substrate. (See Baldeschweiler, supra.) In another alternative, a
grid 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 or thermal, UV, mechanical, or
chemical bonding procedures. A typical 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 image is examined to determine the
degree of complementarity and the relative abundance/expression
level of each oligonucleotide sequence in the microarray.
[0218] VIII. Complementary Polynucleotides
[0219] Sequences complementary to the UBCLE-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring UBCLE. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 primer analysis software and the coding
sequence of UBCLE. To inhibit transcription, a conplementary
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 UBCLE-encoding transcript.
[0220] IX. Expression of UBCLE
[0221] Expression of UBCLE 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
UBCLE in E. coli. This vector contains a promoter for
.beta.-galactosidase upstream of the cloning site, 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.
[0222] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of UBCLE into bacterial growth media which can be used
directly in the following assay for activity.
[0223] X. Demonstration of UBCLE Activity
[0224] UBCLE activity is demonstrated by the formation of
di-ubiquitin conjugates from free ubiquitin (van Nocker et al.
supra). UBCLE is incubated together with 75 pmol .sup.125 I-labeled
ubiquitin, 20 nM wheat E1, 2 mM Mg ATP, 0.1 mM dithiothreitol, and
50 mM Tris-HCl, pH 8.0. The reaction is incubated for 2 minutes at
4.degree. C. and the di-ubiquitin product separated from free
ubiquitin by polyacrylamide gel electrophoresis. Di-ubiquitin is
visualized by autoradiography, removed from the gel, and counted in
a gamma radioisotope counter. The amount of di-ubiquitin formed in
the reaction is proportional to the activity of UBCLE in the
assay.
[0225] XI. Production of UBCLE Specific Antibodies
[0226] UBCLE substantially purified using PAGE electrophoresis
(Sambrook, supra), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols. The UBCLE amino acid sequence 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 (upra) and by
others.
[0227] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an Applied Biosystems 431A peptide
synthesizer using fmoc-chemistry and coupled to KLH (Sigma, St.
Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccimide
ester (MBS), following the procedure described in Ausubel et al.,
supra. Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. Resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
[0228] XII. Purification of Naturally Occurring UBCLE Using
Specific Antibodies
[0229] Naturally occurring or recombinant UBCLE is substantially
purified by immunoaffinity chromatography using antibodies specific
for UBCLE. An immunoaffinity column is constructed by covalently
coupling UBCLE 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.
[0230] Media containing UBCLE are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of UBCLE (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/UBCLE binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and UBCLE is collected.
[0231] XIII. Identification of Molecules Which Interact with
UBCLE
[0232] UBCLE or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent. (Bolton et al. (1973)
Biochem. J. 133:529-538.) Candidate molecules previously arrayed in
the wells of a multi-well plate are incubated with the labeled
UBCLE, washed, and any wells with labeled UBCLE complex are
assayed. Data obtained using different concentrations of UBCLE are
used to calculate values for the number, affinity, and association
of UBCLE with the candidate molecules.
[0233] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
* * * * *