U.S. patent application number 10/443200 was filed with the patent office on 2004-03-25 for lipid metabolism transcription factor.
This patent application is currently assigned to Incyte Corporation. Invention is credited to Baughn, Mariah R., Kaser, Matthew R., Yue, Henry.
Application Number | 20040058358 10/443200 |
Document ID | / |
Family ID | 23240379 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040058358 |
Kind Code |
A1 |
Yue, Henry ; et al. |
March 25, 2004 |
Lipid metabolism transcription factor
Abstract
The invention provides a mammalian nucleic acid and fragments
thereof. It also provides for the use of these nucleic acids in a
model system for the characterization, diagnosis, evaluation,
treatment of conditions, diseases and disorders associated with
expression of the mammalian nucleic acid. The invention
additionally provides expression vectors and host cells for the
production of the protein encoded by the mammalian nucleic
acid.
Inventors: |
Yue, Henry; (Sunnyvale,
CA) ; Kaser, Matthew R.; (Castro Valley, CA) ;
Baughn, Mariah R.; (San Leandro, CA) |
Correspondence
Address: |
INCYTE CORPORATION
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Corporation
Palo Alto
CA
|
Family ID: |
23240379 |
Appl. No.: |
10/443200 |
Filed: |
May 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10443200 |
May 21, 2003 |
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09709976 |
Nov 10, 2000 |
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09709976 |
Nov 10, 2000 |
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09318978 |
May 26, 1999 |
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6245526 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 3/10 20180101; A61P
13/02 20180101; A61P 17/06 20180101; A61P 1/16 20180101; A61P 7/00
20180101; A61P 35/00 20180101; A61P 13/12 20180101; A61P 5/38
20180101; A61P 3/04 20180101; A61P 3/06 20180101; C07K 14/4702
20130101; A61P 35/02 20180101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 021/02; C12N 005/06; C07K 014/705; C07K 014/47 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO:2,
b) a polypeptide comprising a naturally occurring an amino acid
sequence at least 90% identical to an amino acid sequence of SEQ ID
NO:2, c) a biologically active fragment of a polypeptide having an
amino acid sequence of SEQ ID NO:2, and d) an immunogenic fragment
of a polypeptide having an amino acid sequence of SEQ ID NO:2.
2. An isolated polypeptide of claim 1, comprising an amino acid
sequence of SEQ ID NO:2.
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:1.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises the
amino acid sequence of SEQ ID NO:2.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence of SEQ
ID NO:1, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to a polynucleotide
sequence of SEQ ID NO:1, c) a polynucleotide complementary to a
polynucleotide of a), d) a polynucleotide complementary to a
polynucleotide of b) and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence of SEQ ID NO:2.
19. A method for treating a disease or condition associated with
decreased expression of functional LMTF, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional LMTF, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional LMTF, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of LMTF 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 LMTF 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 LMTF in a subject, comprising administering to
said subject all effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence of SEQ ID NO:2, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibodies
from said animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide comprising an amino acid sequence of
SEQ ID NO:2.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
of SEQ ID NO:2, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibody
producing cells from the animal, c) fusing the antibody producing
cells with immortalized cells to form monoclonal antibody-producing
hybridoma cells, d) culturing the hybridoma cells, and e) isolating
from the culture monoclonal antibody which binds specifically to a
polypeptide comprising an amino acid sequence of SEQ ID NO:2.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the monoclonal antibody is
produced by screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence of SEQ ID NO:2 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide comprising an amino acid sequence of
SEQ ID NO:2 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO:2 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide comprising an amino acid sequence of SEQ ID
NO:2.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 12.
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.
Description
[0001] This application is a contiunation of U.S. Ser. No.
09/709,976, filed Nov. 10, 2000, which is a divisional of U.S. Ser.
No. 09/318,978 filed May 26, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a new mammalian protein and to the use of these
sequences in the characterization, diagnosis, and treatment of cell
proliferative and lipid disorders.
BACKGROUND OF THE INVENTION
[0003] Phylogenetic relationships among organisms have been
demonstrated many times, and studies from a diversity of
prokaryotic and eukaryotic organisms suggest a more or less gradual
evolution of biochemical and physiological mechanisms and metabolic
pathways. Despite different evolutionary pressures, proteins that
regulate the cell cycle in yeast, nematode, fly, rat, and man have
common chemical or structural features and modulate the same
general cellular activity. Comparisons of human gene sequences with
those from other organisms where the structure and/or function may
be known allow researchers to draw analogies and to develop model
systems for testing hypotheses. These model systems are of great
importance in developing and testing diagnostic and therapeutic
agents for human conditions, diseases and disorders.
[0004] Fatty acids are required for phospholipid, glycolipid,
hormone, and intracellular messenger formation; to anchor proteins
to membranes; and as fuel molecules. Most cells can synthesize
fatty acids from acetate substrates, though many mammalian cells
also obtain fatty acids by hydrolysis of triglycerides. Synthesis
of phospholipids primarily occurs on the surface of the smooth
endoplasmic reticulum. Although most cells constitutively form
fatty acids, the level of synthesis varies with the needs of the
cell. During phases of rapid cell division, membrane formation
requires enhanced production of phospholipids. Animals that have
fasted and are then fed high-carbohydrate, low-fat diets show
marked increases in the amount and activity of enzymes responsible
for fatty acid synthesis. Increased synthesis of long chain fatty
acids also occurs in multiple common neoplasms, including those
arising in the breast, prostate, ovary, colon, and endometrium.
Overexpression of fatty acid synthase (FAS), a major enzyme of
fatty acid biosynthesis, is a marker for poor prognosis in breast
tumors and has been shown to be important for tumor growth (Moncur
et al. (1997) Proc Natl Acad Sci 95:6989-6994).
[0005] The transcriptional regulation of enzymes involved in fatty
acid synthesis is associated with Spot 14 (S14) protein. S14 is a
small, acidic nuclear protein with a carboxyterminal "zipper"
domain involved in homodimer formation. It is expressed in tissues
that produce lipids for use as metabolic fuels, such as lactating
mammary tissue, white and brown adipose tissue, and liver. The
expression of S14 is increased in response to insulin, dietary
carbohydrates, glucose, and thyroid hormone and reduced in response
to glucagon, fasting, and in diabetes mellitus. Expression of
antisense oligonucleotides has shown S14 induces tissue-specific
expression of several lipogenic enzymes including FAS and ATP
citrate lyase. The S14 gene is located on chromosome 11 at position
q13.5, a chromosomal region amplified in approximately 20% of
breast cancers, and is expressed in several breast cancer-derived
cell lines and in a majority of primary breast tumors (Cunningham
et al. (1998) Thyroid 8:815-825; Liaw and Towle (1984) J Biol Chem
259:7253-7260; Brown et al. (1997) J Biol Chem 272:2163-2166; and
Moncur, supra).
[0006] A zebrafish gastrulation protein, G12, shares features with
S14 including acidic pI (.about.4.9) and nearly identical size
(.about.17 kDa). The sequence similarity between the two proteins
is strongest at the carboxyterminus, including the zipper domain.
G12 is expressed in an outer, enveloping layer of cells (EVL),
analogous to the mammalian trophectoderm, during a period in
gastrulation in which the EVL layer expands to cover the
developing, embryonic deep cell layer. During this stage, apical
membrane turnover in the EVL increases and raises the requirement
for phospholipids used in plasma membranes (Conway (1995) Mech Dev
52:383-391; Fink and Cooper (1996) Dev Biol 174:180-189).
[0007] The discovery of a polynucleotide encoding a new mammalian
protein satisfies a need in the art by providing new compositions
which are useful in the characterization, diagnosis, and treatment
of cell proliferative and lipid disorders.
SUMMARY OF THE INVENTION
[0008] The invention is based on the discovery of a polynucleotide
encoding a mammalian protein, lipid metabolism transcription factor
(LMTF), which satisfies a need in the art by providing new
compositions useful in the characterization, diagnosis, and
treatment of cell proliferative and lipid disorders.
[0009] The invention provides an isolated and purified mammalian
polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1
or a fragment thereof. The invention also provides fragments
homologous to the mammalian polynucleotide from rat, mouse, and
monkey.
[0010] The invention further provides an isolated and purified
polynucleotide or a fragment thereof which hybridizes under high
stringency conditions to the polynucleotide of SEQ ID NO:1. The
invention also provides an isolated and purified polynucleotide
which is complementary to the polynucleotide of SEQ ID NO:1. In one
aspect, a single stranded complementary RNA or DNA molecule is used
as a probe which hybridizes under high stringency conditions to the
mammalian polynucleotide or a fragment thereof.
[0011] The invention further provides a method for detecting a
polynucleotide in a sample containing nucleic acids, the method
comprising the steps of: (a) hybridizing a probe to at least one of
the nucleic acids of the sample, thereby forming a hybridization
complex; and (b) detecting the hybridization complex, wherein the
presence of the hybridization complex correlates with the presence
of a polynucleotide in the sample. In one aspect, the method
further comprises amplifying the polynucleotide prior to
hybridization. The polynucleotide or fragment thereof may comprise
an element or target on a microarray. The invention also provides a
method for screening a library of molecules for specific binding to
a polynucleotide or a fragment thereof, the method comprising
providing a library of molecules, combining the polynucleotide of
claim 1 with a plurality of molecules under conditions which allow
specific binding, and detecting binding of the polynucleotide to
each of a plurality of molecules, thereby identifying at least one
molecule which specifically binds the polynucleotide. Such
molecules are potential regulators of polynucleotide function.
[0012] The invention also provides an expression vector containing
at least a fragment of the polynucleotide of SEQ ID NO:1. In
another aspect, the expression vector is contained within a host
cell. The invention further provides a method for producing a
protein, the method comprising the steps of culturing the host cell
for expression of the protein and recovering the protein from the
host cell culture. The invention also provides an isolated and
purified protein comprising the amino acid sequence of SEQ ID NO:2
or a portion thereof. Additionally, the invention provides a
composition comprising a purified protein having the sequence of
SEQ ID NO:2 or a portion thereof in conjunction with a
pharmaceutical carrier.
[0013] The invention further provides a method for using a portion
of the protein to produce antibodies. The invention also provides a
method for using a protein or a portion thereof to screen for
molecules which specifically bind the protein, the method
comprising the steps of combining the protein or a portion thereof
with a library of molecules under conditions which allow complex
formation and detecting complex formation, wherein the presence of
the complex identifies a molecule which specifically binds the
protein. In one aspect, a molecule identified using the method
increases the activity of the protein. In another aspect, a
molecule identified using the method decreases the activity of the
protein.
BRIEF DESCRIPTION OF THE FIGURES AND TABLE
[0014] FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show the nucleic acid
sequence (SEQ ID NO:1) encoding the amino acid sequence (SEQ ID
NO:2) of the mammalian protein. The alignment was produced using
MACDNASIS PRO software (Hitachi Software Engineering, South San
Francisco Calif.).
[0015] FIGS. 2A and 2B show the chemical and structural similarity
between SEQ ID NO:2, G12 (GI 861207; SEQ ID NO:26) and Spot14 (GI
1171574; SEQ ID NO:27), produced using the multisequence alignment
program of LASERGENE software (DNASTAR, Madison Wis.). The amino
acids of SEQ ID NO:2, from residue 45 to residue 59, may be used
for antibody production.
[0016] Table 1 shows the ESTs from human, rat, mouse, and monkey
which have homology with SEQ ID NO:1 and includes their nucleotide
length, biological source, region of overlap with SEQ ID NO:1, and
percent identity with SEQ ID NO:1.
DESCRIPTION OF THE INVENTION
[0017] It is understood that this invention is not limited to the
particular machines, materials and methods described. 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. As used herein, the singular forms "a",
"an", and "the" include plural reference unless the context clearly
dictates otherwise. For example, a reference to "a host cell"
includes a plurality of such host cells known to those skilled in
the art.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commnonly understood by one
of ordinary skill in the art to which this invention belongs. All
publications mentioned herein are cited for the purpose of
describing and disclosing the cell lines, protocols, reagents and
vectors which are reported in the publications and which might be
used in connection with the invention. Nothing herein is to be
construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
[0019] Definitions
[0020] "LMTF" refers to a purified protein, lipid metabolism
transcription factor, obtained from any mammalian species,
including murine, bovine, ovine, porcine, simian, and preferably
the human species, and from any source, whether natural, synthetic,
semi-synthetic, or recombinant.
[0021] "Agents, molecules, or compounds" are used interchangably
and refer to that which interacts with, specifically binds to, or
modifies the expression of the polynucleotides and proteins of the
invention; and may be composed of at least one of the following:
nucleic acids, proteins, carbohydrates, fats, lipids, organic and
inorganic substances.
[0022] "Biologically active" refers to a protein having structural,
immunological, regulatory, or chemical functions of a naturally
occurring, recombinant or synthetic molecule.
[0023] "Complementary" refer to the natural base pairing by
hydrogen bonding between purines and pyrimidines. For example, the
sequence A-C-G-T forms hydrogen bonds with its complement T-G-C-A
or U-G-C-A. Two single-stranded molecules may be considered
partially complementary, if only some of the nucleotides bond, or
completely complementary, if nearly all of the nucleotides bond.
The degree of complementarity between nucleic acid strands affects
the efficiency and strength of the hybridization and amplification
reactions.
[0024] "Derivative" refers to the chemical modification of a
polynucleotide or protein sequence. Chemical modifications of a
sequence can include replacement of hydrogen by an alkyl, acyl, or
amino group or glycosylation, pegylation, or any similar process
which retains or enhances biological activity or lifespan of the
molecule.
[0025] "Fragment" refers to an Incyte clone or any part of a
polynucleotide which retains a usable, functional characteristic.
Useful fragments include oligonucleotides which may be used in
hybridization or amplification technologies or in regulation of
replication, transcription or translation.
[0026] "Hybridization complex" refers to a complex between two
nucleic acid sequences by virtue of the formation of hydrogen bonds
between purines and pyrimidines.
[0027] "Polynucleotide" refers to a nucleic acid molecule,
oligonucleotide, or any fragment thereof. It may be DNA or RNA of
genomic or synthetic origin, double-stranded or single-stranded,
and combined with carbohydrate, lipids, protein or other materials
to perform a particular activity such as transformation or form a
useful composition such as a peptide nucleic acid (PNA).
"Oligonucleotide" is equivalent to the terms amplimer, primer,
oligomer, element, target, and probe and is preferably single
stranded.
[0028] "Protein" refers to an oligopeptide, peptide, or polypeptide
or portions thereof whether naturally occurring or synthetic.
[0029] "Portion", as used herein, refers to any part of a protein
used for any purpose, but especially for the screening of molecules
or compounds which specifically bind to that part or for the
production of antibodies.
[0030] "Sample" is used in its broadest sense. A sample containing
nucleic acids may comprise a bodily fluid; an extract from a cell,
chromosome, organelle, or membrane isolated from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; and the like.
[0031] Molecules or compounds which "specifically bind" the
mammalian polynucleotide or protein may include, nucleic acids,
carbohydrates, lipids, proteins, or any other organic or inorganic
molecules or their combinations which stabilize, increase, or
decrease the activity of the mammalian polynucleotide or protein.
"Purified" refers to nucleic acid or amino acid sequences that are
removed from their natural environment and are isolated or
separated, and are at least about 60% free, preferably about 75%
free, and most preferably about 90% free, from other components
with which they are naturally associated.
[0032] "Substrate" refers to any rigid or semi-rigid support to
which polynucleotides or proteins are bound and includes membranes,
filters, chips, sides, wafers, fibers, magnetic or nonmagnetic
beads, gels, capillaries or other tubing, plates, polymers, and
microparticles with a variety of surface forms including wells,
trenches, pins, channels and pores.
THE INVENTION
[0033] The invention is based on the discovery of a new mammalian
polynucleotide which encodes a mammalian protein, lipid metabolism
transcription factor, and the use of the nucleic acid sequence, or
fragments thereof, and amino acid sequences, or portions thereof,
as compositions in the characterization, diagnosis, or treatment,
of cell proliferative and lipid disorders.
[0034] Nucleic acids encoding the mammalian protein of the present
invention were identified by BLAST using Incyte clone 700145292H1
which was differentially expressed in male rat reproductive tissue.
A consensus sequence, SEQ ID NO:1, was assembled from the following
overlapping and/or extended nucleic acid fragments found in Incyte
Clones 1479946F6, 3241390F6, 1432520R1, 4534217H1, 2191992H1,
1320132T1, 1516707T1, 5595953H1, and 1988906R6; SEQ ID NOs:3-11,
respectively. FIGS. 1A-1F show the concensus sequence and
translation of SEQ ID NO:1.
[0035] In one embodiment, the protein comprising the amino acid
sequence of SEQ ID NO:2, LMTF, is 183 amino acids in length and has
one potential N-glycosylation site at residue N77; three potential
protein kinase C phosphorylation sites at residues S96, T162, and
T169; and a potential leucine zipper motif from residue L154
through L168. As shown in FIGS. 2A and 2B, the protein has chemical
and structural similarity with zebrafish G12 (GI 861207; SEQ ID
NO:26) and mouse S14 (GI 1171574; SEQ ID NO:27). In particular,
LMTF shares 48% identity with G12 protein and 32% identity with
S14. LMTF, G12, and S14 are similar in size (20 kDa, 17.5 kDa, and
17 kDa, respectively) and isoelectric point (5.3, 5.0, and 4.8,
respectively), as calculated using LASERGENE software (DNASTAR).
Furthermore, LMTF, G12, and S14 share conserved leucine residues
comprising a zipper motif at residues L154, L161, and L168 in
LMTF.
[0036] Table 1 shows the nucleic acid fragments from human, rat,
mouse, and monkey and their sequence coverage and identity with SEQ
ID NO:1. Columns 1 and 2 list the SEQ ID NO and Incyte clone
number, respectively, for each nucleic acid fragment. The fragments
of SEQ ID NO:1, SEQ ID NOs:3-11, are useful in hybridization or
amplification technologies to identify and distinguish between the
mammalian molecules disclosed herein and similar sequences
including SEQ ID NOs:12-25. Column 3 lists the nucleotide length
for each fragment. Columns 4 and 5 identify the source organism and
Incyte cDNA library from which the fragments were isolated,
respectively. Column 6 identifies the range of nucleotide residues
in SEQ ID NO:1 over which each fragment shows identity. Column 7
shows the percent sequence identity between each fragment and SEQ
ID NO:1 over the nucleotides set forth in column 6.
[0037] Northern analysis shows the expression of LMTF in various
libraries, particularly in nervous tissues of human, rat, and
monkey. Of particular note is the expression of LMTF in conditions
associated with cell proliferation, such as cancer and
inflammation.
[0038] The mammalian fragments comprising SEQ ID NO:12-13 from
monkey, SEQ ID NO:14-15 from mouse, and SEQ ID NO:16-25 from rat
were identified using either SEQ ID NO:1 or SEQ ID NOs:3-11. These
fragments may be used to obtain the full length sequence for a
particular species which in turn can be used to produce transgenic
animals which mimic human diseases. The fragments are useful in
hybridization and amplication technologies to monitor animal
toxicological studies, clinical trials, and subject/patient
treatment profiles through time.
[0039] Characterization and Use of the Invention
[0040] In a particular embodiment disclosed herein, mRNA was
isolated from mammalian cells and tissues using methods which are
well known to those skilled in the art and used to prepare the cDNA
libraries. The Incyte clones listed above were isolated from
mammalian cDNA libraries. At least one library preparation
representative of the invention is described in the EXAMPLES below.
The consensus mammalian sequence was chemically and/or
electronically assembled from fragments including Incyte clones,
extension, and/or shotgun sequences using computer programs such as
the AUTOASSEMBLER application (Applied Biosystems, Foster City
Calif.).
[0041] Methods for sequencing nucleic acids are well known in the
art and may be used to practice any of the embodiments of the
invention. The methods may employ such enzymes as the Klenow
fragment of DNA polymerase I, T7 SEQUENASE DNA polymerase, Taq DNA
polymerase, and THERMOSEQUENASE DNA polymerase (Amersham Pharmacia
Biotech (APB), Picataway N.J.) or combinations of polymerases and
proofreading exonucleases such as those found in the ELONGASE
amplification system (Life Technologies, Rockville Md.).
Preferably, sequence preparation is automated with machines such as
the HYDRA microdispenser (Robbins Scientific, Sunnyvale Calif.),
MICROLAB 2200 system (Hamilton, Reno Nev.), and the DNA ENGINE
thermal cycler (MJ Research, Watertown Mass.). Machines used for
sequencing include the ABI 3700, 377 or 373 DNA sequencing systems
(Applied Biosystems), the MEGABACE 1000 DNA sequencing system
(APB), and the like. The sequences may be analyzed using a variety
of algorithms which are well known in the art and described in
Ausubel (1997; Short Protocols in Molecular Biology, John Wiley
& Sons, New York N.Y., unit 7.7) and in Meyers (1995; Molecular
Biology and Biotechnology, Wiley VCH, New York N.Y., pp.
856-853).
[0042] Shotgun sequencing is used to generate more sequence from
cloned inserts derived from multiple sources. Shotgun sequencing
methods are well known in the art and use thermostable DNA
polymerases, heat-labile DNA polymerases, and primers chosen from
representative of regions flanking the nucleic acid sequences of
interest. Prefinished sequences (incomplete assembled sequences)
are inspected for identity using various algorithms or programs
well known in the art (Gordon (1998) Genome Res 8:195-202).
Contaminating sequences including vector or chimeric sequences or
deleted sequences can be removed or restored, respectively,
organizing the prefinished sequences into finished sequences.
[0043] The sequences of the invention may be extended using various
PCR-based methods known in the art. For example, the XL-PCR kit
(Applied Biosystems), nested primers, and commercially available
EDNA or genomic DNA libraries (Life Technologies; Clontech, Palo
Alto Calif., respectively) may be used to extend the nucleotide
sequence. For all PCR-based methods, primers may be designed using
commercially available software, such as OLIGO software (Molecular
Biology Insights, Cascade Colo.) to be about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal
to a target sequence at temperatures of about 68.degree. C. to
72.degree. C. When extending a sequence to recover regulatory
elements, it is preferable to use genomic, rather than cDNA
libraries.
[0044] The polynucleotide sequence of SEQ ID NO:1 and fragments
thereof can be used in various hybridization technologies for
various purposes. Hybridization probes may be designed or derived
from SEQ ID NO:1. Such probes maybe made from a highly specific
region such as the 5' regulatory region or from a conserved motif,
and used in protocols to identify naturally occurring sequences
encoding the mammalian protein, allelic variants, or related
sequences, and should preferably have at least 50% sequence
identity to any of the protein sequences. The hybridization probes
of the subject invention may be DNA or RNA and maybe derived from
the sequence of SEQ ID NO:1 or from genomic sequences including
promoters, enhancers, and introns of the mammalian gene.
Hybridization or PCR probes may be produced using oligolabeling,
nick translation, end-labeling, or PCR amplification in the
presence of the labeled nucleotide. A vector containing the nucleic
acid sequence may be used to produce an mRNA probe in vitro by
addition of an RNA polymerase and labeled nucleotides. These
procedures may be conducted using commercially available kits such
as those provided by APB.
[0045] The stringency of hybridization is determined by G+C content
of the probe, salt concentration, and temperature. In particular,
stringency can be increased by reducing the concentration of salt
or raising the hybridization temperature. In solutions used for
some membrane based hybridizations, additions of an organic solvent
such as formamide allows the reaction to occur at a lower
temperature. Hybridization can be performed at low stringency with
buffers, such as 5.times.SSC with 1% sodium dodecyl sulfate (SDS)
at 60.degree. C., which permits the formation of a hybridization
complex between nucleotide sequences that contain some mismatches.
Subsequent washes are performed at higher stringency with buffers
such as 0.2.times.SSC with 0.1% SDS at either 45.degree. C. (medium
stringency) or 68.degree. C. (high stringency). At high stringency,
hybridization complexes will remain stable only where the nucleic
acid sequences are completely complementary. In some membrane-based
hybridizations, preferably 35% or most preferably 50%, formamide
can be added to the hybridization solution to reduce the
temperature at which hybridization is performed, and background
signals call be reduced by the use of other detergents such as
Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a
blocking agent such as salmon sperm DNA. Selection of components
and conditions for hybridization are well known to those skilled in
the art and are reviewed in Ausubel (supra) and Sambrook et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview N.Y.
[0046] Microarrays may be prepared and analyzed using methods known
in the art. Oligonucleotides may be used as either probes or
targets in a microarray. The microarray can be used to monitor the
expression level of large numbers of genes simultaneously and to
identify genetic variants, mutations, and single nucleotide
polymorphisms. Such information may be used to determine gene
function; to understand the genetic basis of a condition, disease,
or disorder; to diagnose a condition, disease, or disorder; and to
develop and monitor the activities of therapeutic agents. (See,
e.g., Brennan et al. (1995) U.S. Pat. No. 5,474,796; Schena et al.
(1996) Proc Natl Acad Sci 93:10614-10619; Baldeschweiler et al.
(1995) PCT application WO95/251116; Shalon et al. (1995) PCT
application WO95/35505; Heller et al. (1997) Proc Natl Acad Sci
94:2150-2155; and Heller et al. (1997) U.S. Pat. No.
5,605,662.)
[0047] Hybridization probes are also useful in mapping the
naturally occurring genomic sequence. The sequences may be mapped
to a particular chromosome, to a specific region of a chromosome,
or to artificial chromosome constructions, e.g., human artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs), bacterial P1 constructions, or
single chromosome DNA libraries.
[0048] A multitude of polynucleotide sequences capable of encoding
the mammalian protein may be cloned into a vector and used to
express the protein, or portions thereof, in host cells. The
nucleotide sequence can be engineered by such methods as DNA
shuffling (Stenmier and Crameri (1996) U.S. Pat. No. 5,830,721) and
site-directed mutagenesis to create new restriction sites, alter
glycosylation patterns, change codon preference to increase
expression in a particular host, produce splice variants, extend
half-life, and the like. The expression vector may contain
transcriptional and translational control elements (promoters,
enhancers, specific initiation signals, and 3'untranslated regions)
from various sources which have been selected for their efficiency
in a particular host. The vector, nucleic acid sequence, and
regulatory elements are combined using in vitro recombinant DNA
techniques, synthetic techniques, and/or in vivo genetic
recombination techniques well known in the art and described in
Sambrook (supra, ch. 4, 8, 16 and 17).
[0049] A variety of host systems may be transformed with an
expression vector. These include, but are not limited to, bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression
vectors; insect cell systems transformed with baculovirus
expression vectors; plant cell systems transformed with expression
vectors containing viral and/or bacterial elements, or animal cell
systems (Ausubel supra, unit 16). For example, an adenovirus
transcription/translation complex may be utilized in mammalian
cells, Sequences may be ligated into the non-essential E1 or E3
region of the viral genome, and the infective virus used to
transform and express the protein in host cells. The Rous sarcoma
virus enhancer or SV40 or EBV-based vectors may also be used for
high-level protein expression
[0050] Routine cloning, subcloning, and propagation of
polynucleotide sequences can be achieved using the multifunctional
PBLUESCRIPT vector (Stratagene, La Jolla Calif.) or PSPORT1 plasmid
(Life Technologies). Introduction of a polynucleotide sequence into
the multiple cloning site of these vectors disrupts the lacZ gene
and allows colorimetric screening for transformed bacteria. In
addition, these vectors may be useful for in vitro transcription,
dideoxy sequencing, single strand rescue with helper phage, and
creation of nested deletions in the cloned sequence.
[0051] For long term production of recombinant proteins, the vector
can be stably transformed into cell lines along with a selectable
or visible marker gene on the same or on a separate vector. After
transformation, cells are allowed to grow for about 1 to 2 days in
enriched media and then are transferred to selective media.
Selectable markers, antimetabolite, antibiotic, or herbicide
resistance genes, confer resistance to the relevant selective agent
and allow growth and recovery of cells which successfully express
the introduced sequences. Resistant clones identified either by
survival on selective media or by the expression of visible
markers, such as anthocyanins, green fluorescent protein (GFP),
.beta. glucuronidase, luciferase and the like, may be propagated
using tissue culture techniques. Visible markers are also used to
quantify the amount of protein expressed by the introduced genes.
Verification that the host cell contains the desired mammalian
polynucleotide is based on DNA-DNA or DNA-RNA hybridizations or PCR
amplification techniques.
[0052] The host cell may be chosen for its ability to modify a
recombinant protein in a desired fashion. Such modifications
include acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, acylation and the like. Post-translational processing
which cleaves a "prepro" form may also be used to specify protein
targeting, folding, and/or activity. Different host cells which
have specific cellular machinery and characteristic mechanisms for
post-translational activities (CHO, HEK293, and WVI38; American
Type Culture Collection, Manassas Va.) may be chosen to ensure the
correct modification and processing of the foreign protein.
[0053] Heterologous moieties engineered into a vector for ease of
purification include glutathione S-transferase (GST), calmodulin
binding peptide (CBP), 6-His, FLAG, c-myc, and the like. GST, CBP,
and 6-His are purified using commercially available affinity
matrices such as immobilized glutathione, calmodulin, and
metal-chelate resins, respectively. FLAG and c-myc are purified
using commercially available monoclonal and polyclonal antibodies.
A proteolytic cleavage site may be located between the desired
protein sequence and the heterologous moiety for ease of separation
following purification. Methods for recombinant protein expression
and purification are discussed in Ausubel (supra, unit 16) and are
commercially available.
[0054] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker group. A linker group such
as methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-.alpha.-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluorloacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or
N,N-dimethylformamide. The peptide is cleaved between the peptide
carboxyterminus and the linker group to yield a peptide acid or
amide (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook,
San Diego Calif. pp. S1-S20). Automated synthesis may also be
carried out on machines such as the ABI 431A peptide synthesizer
(Applied Biosystems). A protein or portion thereof may be purified
by preparative high performance liquid chromatography and its
composition confirmed by amino acid analysis or by sequencing
(Creighton (1984) Proteins, Structures and Molecular Properties, WH
Freeman, New York N.Y.).
[0055] Various hosts including goats, rabbits, rats, mice, humans,
and others may be immunized by injection with mammalian protein or
any portion thereof. Adjuvants such as Freund's, mineral gels, and
surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemacyanlin
(KLH), and dinitroplhenol may be used to increase immunological
response. The oligopeptide, peptide, or portion of protein used to
induce antibodies should consist of at least about five amino
acids, more preferably ten amino acids, which are identical to a
portion of the natural protein. Oligopeptides may be fused with
proteins such as KLH in order to produce antibodies to the chimeric
molecule.
[0056] Monoclonal antibodies may be prepared using any technique
which provides for the production of antibodies by continuous cell
lines in culture. These include, but are not limited to, the
hybridoma technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique. (See, e.g., Kohler et al. (1975) Nature
256:495-497; Kozbor et al. (1985) J Immunol Methods 81:31-42; Cote
et al. (1983) Proc Natl Acad Sci 80:2026-2030; and Cole et al.
(1984) Mol Cell Biol 62:109-120.)
[0057] Alternatively, techniques described for the production of
single chain antibodies may be adapted, using methods known in the
art, to produce epitope-specific single chain antibodies. Antibody
fragments which contain specific binding sites for epitopes of the
mammalian protein may also be generated. For example, such
fragments include, but are not limited to, F(ab')2 fragments
produced by pepsin digestion of the antibody molecule and Fab
fragments generated by reducing the disulfide bridges of the
F(ab')2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity. (See, e.g., Huse et al.
(1989) Science 246:1275-1281.)
[0058] The mammalian protein may be used in screening assays of
phagemid or B-lymphocyte immunoglobulin libraries to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoassays 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 the protein and its specific antibody. A
two-site, monoclonal-based immunoassay utilizing monoclonal
antibodies reactive to two non-interfering epitopes is preferred,
but a competitive binding assay may also be employed (Pound (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.).
[0059] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid, amino acid, and antibody assays. Synthesis of labeled
molecules may be achieved using Promega (Madison Wis.) or APB kits
for incorporation of a labeled nucleotide such as .sup.32P-dCTP,
Cy3-dCTP or Cy5-dCTP or amino acid such as .sup.35S-methionine
(APB). Nucleic acids and amino acids may be directly labeled with a
variety of substances including fluorescent, chemiluminescent, or
chromogenic agents, and the like, by chemical conjugation to
amines, thiols and other groups present in the molecules using
reagents such as BIODIPY or FITC (Molecular Probes, Eugene
Oreg.).
[0060] Diagnostics
[0061] The polynucleotides, fragments, oligonucleotides,
complementary RNA and DNA molecules, and PNAs may be used to detect
and quantify altered gene expression, absence/presence vs. excess,
expression of mRNAs or to monitor mRNA levels during therapeutic
intervention. Condition, diseases or disorders associated with
altered expression of LMTF include, but are not limited to, a cell
proliferative disorder such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective
tissue disease, myelofibrosis, paroxysmal nocturnal hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia, and cancers
including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, teratocarcinoma, and, in particular, cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
and a lipid disorder such as fatty liver, cholestasis, carnitine
deficiency, carnitine palmitoyltransferase deficiency, myoadenylate
deaminase deficiency, hypertriglyceridemia, lipid storage disorders
such Fabry's disease, Gaucher's disease, Niemann-Pick's disease,
metachromatic leukodystrophy, adrenoleukodystrophy, GM.sub.2
gangliosidosis, and ceroid lipofuscinosis, abetalipoproteinemia,
Tangier disease, hyperlipoproteinemia, diabetes mellitus,
lipodystrophy, lipomatoses, acute panniculitis, disseminated fat
necrosis, adiposis dolorosa, lipoid adrenal hyperplasia, minimal
change disease, lipomas, hypercholesterolemia, hypercholesterolemia
with hypertriglyceridemia, primary hypoalphalipoproteinemia,
hypothyroidism, renal disease, liver disease, lecithin:cholesterol
acyltransferase deficiency, cerebrotendinous xanthomatosis,
sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's
disease, hyperlipidemia, hyperlipemia, lipid myopathies, and
obesity. The diagnostic assay may use hybridization or
amplification technology to compare gene expression in a biological
sample from a patient to standard samples in order to detect
altered gene expression. Qualitative or quantitative methods for
this comparison are well known in the art.
[0062] For example, the nucleotide sequence may be labeled by
standard methods and added to a biological sample from a patient.
After an incubation period in which hybridization complexes form,
the sample is washed and the amount of label, or its signal, is
quantified and compared with a standard value. If the amount of
label in the patient sample is significantly altered in comparison
to the standard value, then the presence of the associated
condition, disease or disorder is indicated.
[0063] In order to provide a basis for the diagnosis of a
condition, disease or disorder associated with gene expression, a
normal or standard expression profile is established. This may be
accomplished by combining a biological sample taken from normal
subjects, either animal or human, with a sequence or a fragment
thereof under conditions 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 an isolated and purified polynucleotide is
used. Standard values obtained in this manner may be compared with
values obtained from samples from patients who are symptomatic for
a particular condition, disease, or disorder. Deviation from
standard values toward those associated with a particular condition
is used to diagnose that condition.
[0064] Such assays may also be used to evaluate the efficacy of a
particular therapeutic treatment regimen in animal studies and in
clinical trial or to monitor the treatment of an individual
patient. Once the presence of a condition is established and a
treatment protocol is initiated, diagnostic assays may be repeated
on a regular basis to determine if the level of expression in the
patient begins to approximate that which is observed in a 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.
[0065] Therapeutics
[0066] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of the mammalian LMTF,
zebrafish G12, and mouse Spot14. In addition, expression is closely
associated with nervous tissue and appears to play a role in cell
proliferative and inflammatory disorders. In the treatment of
conditions associated with increased expression or activity, it is
desirable to decrease expression or protein activity. In the
treatment of conditions associated with decreased expression or
activity, it is desirable to increase expression or protein
activity.
[0067] In one embodiment, the mammalian protein or a portion or
derivative thereof may be administered to a subject to treat or
prevent a condition associated with altered expression or activity
of the mammalian protein. Examples of such conditions include, but
are not limited to, a cell proliferative disorder such as actinic
keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis,
hepatitis, mixed connective tissue disease, myelofibrosis,
paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis,
primary thrombocythemia, and cancers including adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus; and a lipid disorder
such as fatty liver, cholestasis, carnitine deficiency, carnitine
palmitoyltransferase deficiency, myoadenylate deaminase deficiency,
hypertriglyceridemia, lipid storage disorders such Fabry's disease,
Gaucher's disease, Niemann-Pick's disease, metachromatic
leukodystrophy, adrenoleukodystrophy, GM.sub.2 gangliosidosis, and
ceroid lipofuscinosis, abetalipoproteinemia, Tangier disease,
hyperlipoproteinemia, diabetes mellitus, lipodystrophy,
lipomatoses, acute panniculitis, disseminated fat necrosis,
adiposis dolorosa, lipoid adrenal hyperplasia, minimal change
disease, lipomas, hypercholesterolemia, hypercholesterolemia with
hypertriglyceridemia, primary hypoalphalipoproteinemia,
hypothyroidism, renal disease, liver disease, lecithin:cholesterol
acyltransferase deficiency, cerebrotendinous xanthomatosis,
sitosterolemia, hypocholesterolemia, Tay-Sachs disease, Sandhoff's
disease, hyperlipidemia, hyperlipemia, lipid myopathies, and
obesity.
[0068] In another embodiment, a composition comprising the purified
mammalian protein in conjunction with a pharmaceutical carrier may
be administered to a subject to treat or prevent a condition
associated with altered expression or activity of the mammalian
protein including, but not limited to, those provided above.
[0069] In a further embodiment, an agonist which modulates the
activity of the mammalian protein may be administered to a subject
to treat or prevent a condition associated with altered expression
or activity of the protein including, but not limited to, those
listed above.
[0070] In an additional embodiment, a vector capable of expressing
the mammalian protein or a portion or derivative thereof may be
administered to a subject to treat or prevent a condition
associated with altered expression or activity of protein
including, but not limited to, those described above.
[0071] In yet another embodiment, an antagonist or inhibitor of the
mammalian protein may be administered to a subject to treat or
prevent a condition associated with altered expression or activity
of the protein. In one aspect, an antibody which specifically binds
the mammalian protein 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 the mammalian
protein.
[0072] In a still further embodiment, a vector expressing the
complement of the polynucleotide encoding the mammalian protein may
be administered to a subject to treat or prevent a condition
associated with altered expression or activity of the protein
including, but not limited to, those described above.
[0073] Any of the nucleic acids, complementary sequences, vectors,
proteins, agonists, antagonists, or antibodies of the invention may
be administered in combination with other therapeutic agents.
Selection of the agents for use in combination therapy may be made
by one of ordinary skill in the art according to conventional
pharmaceutical principles. A combination of therapeutic agents may
act synergistically to effect treatment of a particular condition
at a lower dosage of each agent.
[0074] Gene expression may be modified by designing complementary
sequences or antisense molecules (DNA, RNA, or PNA) to the control,
5', or regulatory regions of the gene encoding the mammalian
protein. Oligonucleotides designed with reference to the
transcription initiation site are preferred. Similarly, inhibition
can be achieved using triple helix base-pairing which inhibits the
binding of polymerases, transcription factors, or regulatory
molecules (Gee et al. In: Huber and Carr (1994) Molecular and
Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.
163-177.) A complementary sequence may also be designed to block
translation by preventing binding between ribosomes and mRNA.
[0075] 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 at sites such as GUA, GUU, and GUC. Once such sites are
identified, an oligonucleotide with the same sequence may be
evaluated for secondary structural features which would render the
oligonucleotide inoperable. The suitability of candidate targets
may also be evaluated by testing their hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0076] Complementary nucleic acids and ribozymes of the invention
may be prepared via recombinant expression, in vitro or in vivo, or
using solid phase phosphoramidite chemical synthesis. In addition,
RNA molecules may be modified to increase intracellular stability
and half-life by addition of flanking sequences at the 5' and/or 3'
ends of the molecule or by the use of phosphorothioate or 2'
O-ethyl rather than phosphodiesterase linkages within the backbone
of the molecule. Modification is inherent in the production of PNAs
and can be extended to other nucleic acid molecules. Either the
inclusion of nontraditional bases such as inosine, queosine, and
wybutosine, and or the modification of adenine, cytidine, guanine,
thymine, and uridine with acetyl-, methyl-, thio- groups renders
the molecule less available to endogenous endonucleases.
[0077] The nucleic acid sequence encoding the mammalian protein may
be used to screen a library of molecules for specific binding
affinity. The assay can be used to screen a library of DNA
molecules, RNA molecules, PNAs, peptides, or proteins including
transcription factors, enhancers, repressors, and the like which
regulate the activity of the nucleic acid sequence in the
biological system. The assay involves providing a library of
molecules, combining the mammalian nucleic acid sequence or a
fragment thereof with the library of molecules under conditions to
allow specific binding, and detecting specific binding to identify
at least one molecule which specifically binds the nucleic acid
sequence.
[0078] Similarly the mammalian protein or a portion thereof may be
used to screen libraries of molecules in any of a variety of
screening assays. The portion of the protein employed in such
screening may be free in solution, affixed to an abiotic or biotic
substrate (e.g. borne on a cell surface), or located
intracellularly. Specific binding between the protein and molecule
may be measured. Depending oil the kind of library being screened,
the assay may be used to identify DNA, RNA, or PNA molecules,
agonists, antagonists, antibodies, immunoglobulins, inhibitors,
peptides, proteins, drugs and the like, which specifically bind the
protein. One method for high throughput screening using very small
assay volumes and very small amounts of test compound is described
in U.S. Pat. No. 5,876,946, which screens large numbers of
molecules for enzyme inhibition or receptor binding.
[0079] Pharmaceutical compositions are those substances wherein the
active ingredients are contained in an effective amount to achieve
a desired and intended purpose. The determination of an effective
dose is well within the capability of those skilled in the art. For
any compound, the therapeutically effective dose may be estimated
initially either in cell culture assays or in animal models. The
animal model is also used to achieve a desirable concentration
range and route of administration. Such information may then be
used to determine useful doses and routes for administration in
humans.
[0080] A therapeutically effective dose refers to that amount of
protein or inhibitor which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity of such agents may be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., ED50 (the dose therapeutically
effective in 50% of the population) and LD50 (the dose lethal to
50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, and it may be
expressed as the ratio, LD50/ED50. Pharmaceutical compositions
which exhibit large therapeutic indexes are preferred. The data
obtained from cell culture assays and animal studies are used in
formulating a range of dosage for human use.
[0081] Model Systems
[0082] Animal models may be used as bioassays where they exhibit a
toxic response similar to that of humans and where exposure
conditions are relevant to human exposures. Mammals are the most
common models and most toxicity studies are performed on rodents
such as rats or mice because of low cost, availability, and
abundant reference toxicologic. Inbred rodent strains provide a
convenient model for investigation of the physiological
consequences of under- or over-expression of genes of interest and
for the development of methods for diagnosis and treatment of
diseases. A rodent strain inbred to over-express a particular gene
may also serve as a convenient source of the protein expressed by
that gene.
[0083] Toxicology is the study of the effects of agents on living
systems. The majority of toxicity studies are performed on rats or
mice to help predict the effects of these agents on human health.
Observation of qualitative and quantitative changes in physiology,
behavior, homeostatic processes, and lethality are used to generate
a toxicity profile and to assess the consequences on human health
following exposure to the agent.
[0084] Toxicological tests measure the effects of a single,
repeated, or long-term exposure of a subject to an agent. Agents
may be tested for specific endpoints such as cytotoxicity,
mutagenicity, carcinogenicity and teratogenicity. Degree of
response varies according to the route of exposure (contact,
ingestion, injection, or inhalation), age, sex, genetic makeup, and
health status of the subject. Toxicokinetic studies trace the
absorption, distribution, metabolism, storage, and excretion of the
agent in subject tissues, and toxicodynamic studies chart
biological responses that are consequences of the presence of the
agent in subject tissues.
[0085] Genetic toxicology identifies and analyzes the ability of an
agent to produce genetic mutations Genotoxic agents usually have
common chemical or physical properties that facilitate interaction
with nucleic acids and are most harmful when chromosomal
aberrations are passed along to progeny. Toxicological studies may
identify agents that increase the frequency of structural or
functional abnormalities in progeny if administered to either
parent before conception, to the mother during pregnancy, or to the
developing organism. Mice and rats are most frequently used in
these tests because of their short reproductive cycle and their
capacity to be raised in numbers sufficient to satisfy statistical
requirements.
[0086] All toxicology studies on experimental animals involve the
preparation of a form of the agent for administration, the
selection of the route of administration, and the selection of the
species to resemble the species of pharmacological interest. Dose
concentrations are varied to investigate a range of dose-related
effects which are identified, measured, and related to
exposure.
[0087] Acute toxicity tests are based on a single administration of
the agent to the subject to determine the symptomology or lethality
of the agent. Three experiments are conducted: 1) an initial
dose-range-finding experiment, 2) an experiment to narrow the range
of effective doses, and 3) a final experiment for establishing the
dose-response curve.
[0088] Prolonged toxicity tests are based on the repeated
administration of the agent. Rat and dog are commonly used in these
studies to provide data from species in different families. With
the exception of carcinogenesis, there is considerable evidence
that daily administration of an agent at high-dose concentrations
for periods of three to four months will reveal most forms of
toxicity in adult animals.
[0089] Chronic toxicity tests, with a duration of a year or more,
are used to demonstrate either the absence of toxicity or the
carcinogenic potential of an agent. When studies are conducted on
rats, a minimum of three test groups plus one control group are
used, and animals are examined and monitored at the outset and at
intervals throughout the experiment.
[0090] Transgenic rodents which over-express or under-express a
gene of interest may be inbred and used to model human diseases or
to test therapeutic or toxic agents. (See, e.g., van Beusechem and
Valerio, In: Murray (1992) Transgenesis: Applications of Gene
Transfer, John Wiley & Sons Ltd. Chichester, England, pp.
283-289.) To produce the rat or mouse model, a gene candidate which
mimics a human disease is coupled to a strong promoter and injected
into a fertilized egg, and the egg transferred into a
pseudopregnant dam. The promoter may be activated at a specific
time in a specific tissue type during fetal development or
postnatally. Expression of the transgene is monitored by analysis
of phenotype, tissue-specific mRNA expression, and challenged with
experimental drug therapies. Examples of transgenes used as models
of human disease include the investigation of the mutant amyloid
precursor protein and apolipoprotein E genes in familial
Alzheimer's Disease (Price and Sisodia (1998) Ann Rev Neurosci
21:479-505).
[0091] Embryonic stem cells (ES) isolated from rodent embryos
retain the potential to form an embryo. When ES cells are placed
inside a carrier embryo, they resume normal development and
contribute to all tissues of the live-born animal. ES cells are the
preferred cells used in the creation of experimental knockout and
knockin rodent strains. Mouse ES cells, such as the mouse 129/SvJ
cell line, are derived from the early mouse embryo and are grown
under culture conditions well known in the art. Vectors for
knockout strains contain a disease gene candidate modified to
include a marker gene sequence which disrupts transcription and/or
translation in vivo. The vector is introduced into ES cells by
transformation methods such as electroporation, liposome delivery,
microinjection, and the like which are well known in the art. The
endogenous rodent gene is replaced by the disrupted disease gene
through homologous recombination and integration during cell
division. Then transformed ES cells are selected, identified, and
preferably microinjected into mouse cell blastocysts such as those
from the C57BL/6 mouse strain. The blastocysts are surgically
transferred to pseudopregnant dams and the resulting chimeric
progeny are genotyped and bred to produce heterozygous or
homozygous strains.
[0092] ES cells are also used to study the differentiation of
various cell types and tissues in vitro, such as neural cells,
hematopoietic lineages, and cardiomyocytes (Bain et al. (1995) Dev
Biol 168:342-357; Wiles and Keller (1991) Development 111:259-267;
and Klug et al. (1996) J Clin Invest 98:216-224). Recent
developments demonstrate that ES cells derived from human
blastocysts may also be manipulated in vitro to differentiate into
eight separate cell lineages, including endoderm, mesoderm, and
ectodermal cell types (Thomson (1998) Science 282:1145-1147).
[0093] As described herein, the uses of the nucleotide sequences,
provided in the Sequence Listing of this application, are exemplary
of known techniques and are not intended to reflect any limitation
on their use in any technique that would be known to the person of
average skill in the art. Furthermore, the nucleotide sequences
provided in this application may be used in molecular biology
techniques that have not yet been developed, provided the new
techniques rely on properties of nucleotide sequences that are
currently known to the person of ordinary skill in the art.
[0094] In gene knockout analysis, a region of a human disease gene
candidate is enzymatically modified to include a non-mammalian gene
such as the neomycin phosphotransferase gene (neo; Capecchi (1989)
Science 244:1288-1292). The inserted coding sequence disrupts
transcription and translation of the targeted gene and prevents
biochemical synthesis of the disease candidate protein. The
modified gene is transformed into cultured embryonic stem cells
(described above), the transformed cells are injected into rodent
blastulae, and the blastulae are implanted into pseudopregnant
dams. Transgenic progeny are crossbred to obtain homozygous inbred
lines.
[0095] Totipotent ES cells, present in the early stages of
embryonic development, can be used to create knockin humanized
animals (pigs) or transgenic animal models (mice or rats) of human
diseases. With knockin technology, a region of a human gene is
injected into animal ES cells, and the human sequence integrates
into the animal cell genome by recombination. Totipotent ES cells
which contain the integrated human gene are handled as described
above. Inbred animals are studied and treated to obtain information
on the analogous human condition. These methods have been used to
model several human diseases. (See, e.g., Lee et al. (1998) Proc
Natl Acad Sci 95:11371-11376; Baudoin et al. (1998) Genes Dev
12:1202-1216; and Zhuang et al. (1998) Mol Cell Biol
18:3340-3349).
[0096] The field of animal testing deals with data and methodology
from basic sciences such as physiology, genetics, chemistry,
pharmacology and statistics. These data are paramount in evaluating
the effects of therapeutic agents on non-human primates as they can
be related to human health. Monkeys are used as human surrogates in
vaccine and drug evaluations, and their responses are relevant to
human exposures under similar conditions. Cynomolgus monkeys
(Macaca fascicularis, M. mulatta) and common marmosets (Callithrix
jacchlus) are the most common non-human primates (NHPs) used in
these investigations. Since great cost is associated with
developing and maintaining a colony of NHPs, early research and
toxicological studies are usually carried out in rodent models. In
studies using behavioral measures such as drug addiction, NHPs are
the first choice test animal. In addition, NHPs and individual
humans exhibit differential sensitivities to many drugs and toxins
and can be classified as "extensive metabolizers" and "poor
metabolizers" of these agents. For this reason, NHPs are the
favored models for studying metabolism and toxicology of agents
acted upon by the cytochrome P.sub.450 family of enzymes.
[0097] In additional embodiments, the nucleotide sequences which
encode the mammalian protein 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. All
patents and publications cited are incorporated herein by
reference.
EXAMPLES
[0098] It is to be understood that this invention is not limited to
the particular machines, materials and methods described. Although
particular embodiments are described, equivalent embodiments may be
used to practice the invention. The described embodiments are not
intended to limit the scope of the invention which is limited only
by the appended claims. The examples below are provided to
illustrate the subject invention and are not included for the
purpose of limiting the invention. For purposes of example, the
preparation of the human corpus callosum cDNA library, CORPNOT02,
is described.
[0099] I Representative cDNA Sequence Preparation
[0100] The human corpus callosum cDNA library CORPNOT02 was
constructed from tissue obtained from a 74-year-old Caucasian male
(specimen #RA95-09-0670; International Institute for the
Advancement of Medicine, Exton Pa.) who died from Alzheimer's
disease. The frozen tissue was homogenized and lysed in guanidinium
isothiocyanate solution using a POLYTRON homogenizer (PT-3000;
Brinkmann Instruments, Westbury N.J.). The lysate was centrifuged
over a 5.7 M CsCl cushion using an SW28 rotor in an L8-70M
ultracentrifuge (Beckman Coulter, Fullerton Calif.) for 18 hours at
25,000 rpm at ambient temperature. The RNA was extracted with acid
phenol, pH 4.7, precipitated using 0.3 M sodium acetate and 2.5
volumes of ethanol, resuspended in RNAse-free water, and treated
with DNAse (Life Technologies) at 37.degree. C. The RNA extraction
and precipitation were repeated as before.
[0101] Messenger RNA (mRNA) was isolated using the OLIGOTEX kit
(Qiagen, Valencia Calif.) and used to construct the cDNA library.
The mRNA was handled according to the recommended protocols in the
SUPERSCRIPT plasmid system (Life Technologies) which contains a
NotI primer-adaptor designed to prime the first strand cDNA
synthesis at the poly(A) tail of mRNAs. Double stranded cDNA was
blunted, ligated to EcoRI adaptors and digested with NotI (New
England Biolabs, Beverly Mass.). The cDNAs were fractionated on a
SEPHAROSE CL-4B column (APB), and those cDNAs exceeding 400 bp were
ligated into the NotI and EcoRI sites of the pINCY plasmid (Incyte
Genomics, Palo Alto Calif.). The plasmid was transformed into
DH5.alpha. or ELECTROMAX DH10B competent cells (Life
Technologies).
[0102] Plasmid DNA was released from the cells and purified using
the REAL PREP 96 plasmid kit (Qiagen). The recommended protocol was
employed except for the following changes: 1) the bacteria were
cultured in 1 ml of sterile TERRIFIC BROTH (BD Biosciences, Sparks
Md.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) after
inoculation, the cultures were incubated for 19 hours and then
lysed with 0.3 ml of lysis buffer; and 3) following isopropanol
precipitation, the plasmid 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 at 4.degree. C.
[0103] The cDNAs were prepared using either a MICROLAB 2200 system
(Hamilton) or a HYDRA microdispenser (Robbins Scientific) in
combination with the DNA ENGINE thermal cyclers (MJ Research) and
sequenced by the method of Sanger and Coulson (1975; J Mol Biol
94:441-448) using either ABI PRISM 377 (Applied Biosystems) or
MEGABACE 1000 (APB) sequencing, systems. Most of the isolates were
sequenced according to standard ABI protocols and kits (Applied
Biosytems). The solution volumes were used at 0.25x-1.0x
concentrations. In the alternative, cDNAs were sequenced using
solutions and dyes from APB.
[0104] II Identification, Extension, Assembly, and Analyses of the
Sequences
[0105] Incyte clone 700145292 from ZOOSEQ database (Incyte
Genomics) was used to identify Incyte Clone 5595953 from the
LIFESEQ database (Incyte Genomics). The first pass and extended
cDNAs, SEQ ID NOs:3-11, which cluster with Incyte Clone 5595953
were assembled using Phred/Phrap or CONSED (Green, University of
Washington, Seattle Wash.) or the GCG Fragment assembly system
(Genetics Computer Group (GCG), Madison Wis.). The assembled
sequence was searched for open reading frames, and the coding
region was translated using MACDNASIS PRO software (Hitachi
Software Engineering). The full length nucleotide and amino acid
sequences were analyzed by BLAST queries against databases such as
the GenBank databases, SwissProt, BLOCKS, PRINTS, Prosite, and PFAM
and by LASERGENE software (DNASTAR). Functional analyses of the
amino acid sequences were performed using MOTIFS (GCG) and HMM
algorithms. Antigenic index (Jameson-Wolf analysis) of the amino
acid sequences were determined using LASERGENE software (DNASTAR).
Then, the clones and assembled sequence were compared using BLAST
across all mammalian libraries to identify homologous nucleic acid
sequences, SEQ ID NOs:12-25.
[0106] III Sequence Similarity
[0107] Sequence similarity was calculated as percent identity based
on comparisons between at least two nucleic acid or amino acid
sequences using the clustal method of the MEGALIGN program
(DNASTAR). The clustal method uses an algorithm which groups
sequences into clusters by examining the distances between all
pairs. After the clusters are aligned pairwise, they are realigned
in groups. Percent similarity between two sequences, sequence A and
sequence B, is calculated by dividing the length of sequence A,
minus the number of gap residues in sequence A, minus the number of
gap residues in sequence B, into the sum of the residue matches
between sequence A and sequence B, times one hundred. Gaps of very
low or zero similarity between the two sequences are not
included.
[0108] IV Northern Analysis
[0109] 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.
[0110] Analogous computer techniques applying BLAST were used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ (Incyte Genomics). Sequence-based
analysis is much faster than membrane-based hybridization, and the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or similar.
The basis of the search is the product score which is defined as:
(percent sequence identity.times.percent mammalian BLAST score)
divided by 100. 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
at least 70, the match will be exact. Similar or related molecules
are usually identified by selecting those which show product scores
between 8 and 40.
[0111] The results of northern analyses are reported is a
percentage distribution of libraries in which the transcript
encoding the mammalian protein occurred. Analysis involved the
categorization of cDNA libraries by organ/tissue and disease. The
organ/tissue categories included cardiovascular, dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous, reproductive, and urologic. The disease
categories included cancer, inflammation/trauma, cell
proliferation, and neurological. For each category, the number of
libraries expressing the sequence was counted and divided by the
total number of libraries across all categories.
[0112] V Extension of Polynucleotides
[0113] The nucleic acid sequence of SEQ ID NO:1 was produced by
extension of Incyte cDNA clones using oligonucleotide primers. One
primer was synthesized to initiate 5' extension of the known
fragment, and the other, to initiate 3' extension of the known
fragment. The initial primers were designed using OLIGO software
(Molecular Biology Insights) to be about 22 to 30 nucleotides in
length, to have a GC content of about 50%, and to anneal to the
target sequence at temperatures of about 68.degree. C. to about
72.degree. C. Any fragment which would result in hairpin structures
and primer-primer dimerizations was avoided. Selected human cDNA
libraries were used to extend the sequence. If more than one
extension is needed, additional or nested sets of primers are
designed.
[0114] High fidelity amplification was obtained by performing PCR
in 96-well plates using the DNA ENGINE thermal cycler (MJ
Research). The reaction mix contained DNA template, 200 nmol of
each primer, reaction buffer containing Mg.sup.2+,
(NH.sub.4).sub.2SO.sub.4, and .beta.-mercaptoethanol, TAQ DNA
polymerase (APB), ELONGASE enzyme (Life Technologies), and Pfu DNA
polymerase (Stratagene), with the following parameters for primer
pair selected from the plasmid: Step 1: 94.degree. C., 3 min; Step
2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times;
Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree. C. In
the alternative, parameters for the primer pair, T7 and
SK+(Stratagene), were as follows: Step 1: 94.degree. C., 3 min;
Step 2: 94.degree. C., 15 sec; Step 3: 57.degree. C., 1 min; Step
4: 68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated 20
times; Step 6: 68.degree. C., 5 min; and Step 7: storage at
4.degree. C.
[0115] The concentration of DNA in each well was determined by
dispensing 100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v);
Molecular Probes) dissolved in 1.times. TE and 0.5 .mu.l of
undiluted PCR product into each well of an opaque fluorimeter plate
(Corning Life Sciences, Acton Mass.) and allowing the DNA to bind
to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose mini-gel to determine which
reactions were successful in producing longer sequence.
[0116] The extended sequences were desalted, concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector (APB).
For shotgun sequencing, the digested fragments were separated on
about 0.6-0.8% agarose gels, fragments were excised as visualized
under UV light, and agar removed/digested with AGARACE (Promega).
Extended fragments were religated using T4 ligase (New England
Biolabs, Beverly Mass.) into pUC 18 vector (APB), treated with Pfu
DNA polymerase (Stratagene) to fill-in restriction site overhangs,
and transformed into competent E. coli cells. Transformed cells
were selected on antibiotic-containing media, and individual
colonies were picked and cultured overnight at 37.degree. C. in
384-well plates in LB/2.times. carbenicillin liquid media.
[0117] The cells were lysed, and DNA was amplified using Taq DNA
polymerase (APB) and Pfu DNA polymerase (Stratagene) with the
following parameters: Step 1: 94.degree. C., 3 min; Step 2:
94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29 times;
Step 6: 72.degree. C., 5 min; and Step 7: storage at 4.degree. C.
DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the conditions described above. Samples were diluted with 20%
dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing primers and the DYENAMIC DIRECT kit (APB) or
the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit
(Applied Biosystems).
[0118] In like manner, the nucleotide sequence of SEQ ID NO:1 is
used to obtain regulatory sequences using the procedure above,
oligonucleotides designed for outward extension, and a genomic
library.
[0119] VI Labeling of Probes anti Hybridization Analyses
[0120] Polynucleotide sequences are isolated from a biological
source and applied to a substrate for standard nucleic acid
hybridization protocols by one of the following methods. A mixture
of target nucleic acids, a restriction digest of genomic DNA, is
fractionated by electrophoresis through all 0.7% agarose gel in
1.times. TAE [Tris-acetate-ethylenediamin- e tetraacetic acid
(EDTA)] running buffer and transferred to a nylon membrane by
capillary transfer using 20.times. saline sodium citrate (SSC).
Alternatively, the target nucleic acids are individually ligated to
a vector and inserted into bacterial host cells to form a library.
Target nucleic acids are arranged on a substrate by one of the
following methods. In the first method, bacterial cells containing
individual clones are robotically picked and arranged on a nylon
membrane. The membrane is placed on bacterial growth medium, LB
agar containing carbenicillin, and incubated at 37.degree. C. for
16 hours. Bacterial colonies are denatured, neutralized, and
digested with proteinase K. Nylon membranes are exposed to UV
irradiation in a STRATALINKER UV-crosslinker (Stratagene) to
cross-link DNA to the membrane.
[0121] In the second method, target nucleic acids are amplified
from bacterial vectors by thirty cycles of PCR using primers
complementary to vector sequences flanking the insert. Amplified
target nucleic acids are purified using SEPHACRYL-400 beads (APB).
Purified target nucleic acids are robotically arrayed onto a glass
microscope slide (Corning Life Sciences). The slide was previously
coated with 0.05% aminopropyl silane (Sigma-Aldrich) and cured at
110.degree. C. The arrayed glass slide (microarray) is exposed to
UV irradiation in a STRATALINKER UV-crosslinker (Stratagene).
[0122] cDNA probe sequences are made from mRNA templates. Five
micrograms of mRNA is mixed with 1 .mu.g random primer (Life
Technologies), incubated at 70.degree. C. for 10 minutes, and
lyophilized. The lyophilized sample is resuspended in 50 .mu.l of
1.times. first strand buffer (cDNA Synthesis systems; Life
Technologies) containing a dNTP mix, [.alpha.-.sup.32P]dCTP,
dithiothreitol, and MMLV reverse transcriptase (Stratagene), and
incubated at 42.degree. C. for 1-2 hours. After incubation, the
probe is diluted with 42 .mu.l dH.sub.2O, heated to 95.degree. C.
for 3 minutes, and cooled on ice. mRNA in the probe is removed by
alkaline degradation. The probe is neutralized, and degraded mRNA
and unincorporated nucleotides are removed using a PROBEQUANT G-50
MicroColumn (APB). Probes can be labeled with fluorescent
nucleotides, Cy3-dCTP or Cy5-dCTP (APB), in place of the
radiolabeled nucleotide, [.sup.32P]dCTP.
[0123] Hybridization is carried out at 65.degree. C. in a
hybridization buffer containing 0.5 M sodium phosphate (pH 7.2), 7%
SDS, and 1 mM EDTA. After the substrate is incubated in
hybridization buffer at 65.degree. C. for at least 2 hours, the
buffer is replaced with 10 ml of fresh buffer containing the probe
sequences. After incubation at 65.degree. C. for 18 hours, the
hybridization buffer is removed, and the substrate is washed
sequentially under increasingly stringent conditions, up to 40 mM
sodium phosphate, 1% SDS, 1 mM EDTA at 65.degree. C. To detect
signal produced by a radiolabeled probe hybridized on a membrane,
the substrate is exposed to a PHOSPHORIMAGER cassette (APB), and
the image is analyzed using IMAGEQUANT data analysis software
(APB). To detect signals produced by a fluorescent probe hybridized
on a microarray, the substrate is examined by confocal laser
microscopy, and images are collected and analyzed using GEMTOOLS
gene expression analysis software (Incyte Genomics).
[0124] VII Complementary Polynucleotides
[0125] Sequences complementary to the polynucleotide, or a fragment
thereof, are used to detect, decrease, or inhibit gene expression.
Although use of oligonucleotides comprising from about 15 to about
30 base pairs is described, essentially the same procedure is used
with larger or smaller fragments or their derivatives (PNAs).
Oligonucleotides are designed using OLIGO software (Molecular
Biology Insights) and SEQ ID NO:1 or its fragments, SEQ ID NO:3-9.
To inhibit transcription by preventing promoter binding, a
complementary oligonucleotide is designed to bind to the most
unique 5' sequence, most preferably about 10 nucleotides before the
initiation codon of the open reading frame. To inhibit translation,
a complementarily oligonucleotide is designed to prevent ribosomal
binding to the mRNA encoding the mammalian protein.
[0126] VIII Expression of the Mammalian Protein
[0127] Expression and purification of the mammalian protein are
achieved using bacterial or virus-based expression systems. For
expression in bacteria, cDNA is subcloned into a vector containing
an antibiotic resistance gene and an inducible promoter that
directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into bacterial hosts, e.g., BL21(DE3). Antibiotic
resistant bacteria express the mammalian protein upon induction
with isopropyl beta-D-thiogalactopyranoside. Expression in
eukaryotic cells is achieved by infecting Spodoptera frugiperda
(Sf9) insect cells with recombinant baculovirus, Autographica
californica nuclear polyhedrosis virus. The nonessential polyhedrin
gene of baculovirus is replaced with the mammalian cDNA by either
homologous recombination or bacterial-mediated transposition
involving transfer plasmid intermediates. Viral infectivity is
maintained and the strong polyhedrin promoter drives high levels of
cDNA transcription.
[0128] In most expression systems, the mammalian protein is
synthesized as a fusion protein with GST or FLAG, which permits
rapid, single-step, affinity-based purification of recombinant
fusion protein from crude cell lysates. GST enables the
purification of fusion proteins on immobilized glutathione under
conditions that maintain protein activity and antigenicity (APB).
Following purification, the GST moiety can be proteolytically
cleaved from the mammalian protein at specifically engineered
sites. FLAG, an 8-amino acid peptide, enables immunoaffinity
purification using commercially available monoclonal and polyclonal
anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.). 6-His, a
stretch of six consecutive histidine residues, enables purification
on metal-chelate resins (Qiagen). Methods for protein expression
and purification are discussed in Ausubel (supra, unit 16).
Purified mammalian protein obtained by these methods can be used
directly in the following activity assay.
[0129] IX Functional Assays
[0130] Protein function is assessed by expressing the sequences
encoding LMTF at physiologically elevated levels in mammalian cell
culture. The polynucleotide is subcloned into pCMV SPORT vector
(Life Technologies), which contains the strong cytomegalovirus
promoter, and 5-10 .mu.g of the vector is transformed into a
endothelial or hematopoietic human cell line using electroporation.
An additional 1-2 .mu.g of a plasmid containing sequence encoding
CD64-GFP (Clontech) is co-transformed to provide an fluorescent
marker to identify transformed cells using flow cytometry.
[0131] The influence of the introduced genes on expression can be
assessed using purified populations of these transformed cells.
Since CD64-GFP, which is expressed on the surface of transformed
cells, binds to conserved regions of human immunoglobulin G (IgG),
the transformed cells is separated using magnetic beads coated with
either human IgG or antibody against CD64 (DYNAL, Lake Success
N.Y.). mRNA is purified from the cells and analyzed by
hybridization techniques.
[0132] X Production of LMTF Specific Antibodies
[0133] LMTF is purified using polyacrylamide gel electrophoresis is
used to immunize rabbits and to produce antibodies using standard
protocols.
[0134] Alternatively, the amino acid sequence of LMTF is analyzed
using LASERGENE software (DNASTAR) to determine regions of high
immunogenicity. An immunogenic epitope such as those near the
C-terminus or in hydrophilic regions is selected, synthesized, and
used to raise antibodies by means known to those of skill in the
art.
[0135] Typically, epitopes of about 15 residues in length are
produced using an ABI 431A peptide synthesizer (Applied Biosystems)
using Fmoc-chemistry and coupled to KLH (Sigma-Aldrich) by reaction
with N-maleimidobenzoyl-N-hydroxysuccinimide ester to increase
immunogenicity. Rabbits are immunized with the epitope-KLH complex
in complete Freund's adjuvant. Immunizations are repeated at
intervals thereafter in incomplete Freund's adjuvant. After a
sufficient period of time, antisera are drawn and tested for
antipeptide activity. Testing involves binding the peptide to
plastic, blocking with 1% bovine serum albumin, reacting with
rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG. Methods well known in the art are used to
determine antibody titer and the amount of complex formation.
[0136] XI Purification of Naturally Occurring Protein Using
Specific Antibodies
[0137] Naturally occurring or recombinant mammalian protein is
purified by immunoaffinty chromatography using antibodies specific
for the protein. An immunoaffinity column is constructed by
covalently coupling the antibody to CNBr-activated SEPHAROSE resin
(APB). Media containing the protein is passed over the
immunoaffinity column, and the column is washed using high ionic
strength buffers in the presence of detergent to allow preferential
absorbance of the protein. After coupling, the column is eluted
using a buffer of pH 2-3 or a high concentration of urea or
thiocyanate ion to disrupt antibody/protein binding, and the
protein is collected.
[0138] XII Screening Molecules for Specific Binding with the
Polynucleotide or Protein
[0139] The nucleic acid sequence, or fragments thereof, or the
protein, or portions thereof, are labeled with .sup.32P-dCTP,
Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes),
respectively. Libraries of candidate molecules previously arranged
on a substrate are incubated in the presence of labeled nucleic
acid sequence or protein. After incubation, the substrate is
washed, and any position on the substrate retailing label, which
indicates specific binding or complex formation, is assayed, and
the binding molecule is identified. Data obtained using different
concentrations of the nucleic acid or protein are used to calculate
affinity between the labeled nucleic acid or protein and the bound
molecule.
[0140] XIII Demonstration of Protein Activity
[0141] LMTF activity is measured by its ability to modulate
transcription of a reporter gene. The assay entails the use of a
reporter gene construct that consists of a transcription factor
response element fused upstream to sequences encoding the E. coli
.beta.-galactosidase enzyme (LacZ). Sequences encoding LMTF are
subcloned into a mammalian expression vector containing a strong
promoter that drives high levels of cDNA expression. Vectors of
choice include PCMV SPORT (Life Technologies) and PCR 3.1
(Invitrogen, Carlsbad Calif.), both of which contain the
cytomegalovirus promoter. The recombinant vector and reporter gene
construct are co-transformed into a human cell line, preferably of
neuronal origin, using either liposome formulations or
electroporation. The amount of .beta.-galactosidase enzyme activity
associated with LMTF transfectcd cells, relative to control cells
transformed with the reporter construct alone, is proportional to
the amount of transcription modulated by the LMTF gene product.
[0142] All patents and publications mentioned in the specification
are incorporated by reference herein. Various modifications and
variations of the described method and system of the invention will
be apparent to those skilled in the art without departing from the
scope and spirit of the invention. Although the invention has been
described in connection with specific preferred embodiments, it
should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
that are obvious to those skilled in the field of molecular biology
or related fields are intended to be within the scope of the
following claims.
1TABLE 1 Nucleic Acid Incyte Nucleotide Percent SEQ ID NO: Clone
Number Length Source Library Coverage Identity 3 1479946F6 502 Homo
sapiens CORPNOT02 1-502 n/a 4 3241390F6 529 Homo sapiens COLAUCT01
281-801 n/a 5 1432520R1 562 Homo sapiens BEPINON01 599-1178 n/a 6
4534217H1 254 Homo sapiens OVARNOT12 1152-1414 n/a 7 2191992H1 238
Homo sapiens THYRTUT03 1200-1437 n/a 8 1320132T1 661 Homo sapiens
BLADNOT04 1299-1957 n/a 9 1516707T1 624 Homo sapiens PANCTUT01
1445-2070 n/a 10 5595953H1 252 Homo sapiens COLCDTT03 1592-1845 n/a
11 1988906R6 302 Homo sapiens LUNGAST01 1851-2092 n/a 12
700712962H1 144 Macaca fascicularis MNBFNOTO2 12-156 92.4 13
700715135H1 274 Macaca fascicularis NBCNOT01 292-564 93.1 14
701253541H1 273 Mus musculus MOLUDIT07 560-1032 71.2 15 701252210H1
250 Mus musculus MOLUDIT07 1564-1831 81.0 16 700545683H1 272 Rattus
norvegicus RASPNOT01 1-271 52.2 17 700145292H1 257 Rattus
norvegicus RAPRNOT01 191-488 44.7 18 700861443H1 239 Rattus
norvegicus RABGNOT02 338-591 63.2 19 700225363H1 302 Rattus
norvegicus RAKINOT01 479-780 86.4 20 700643425H1 286 Rattus
norvegicus RABUNOT01 593-879 81.1 21 700525920H1 285 Rattus
norvegicus RABMNOT01 783-1066 77.2 22 700773927H1 270 Rattus
norvegicus RABONOT01 1001-1197 54.8 23 700513679H1 283 Rattus
norvegicus RASNNOT01 1318-1594 72.1 24 700767486H1 266 Rattus
norvegicus RAHYNOT01 1594-1876 73.7 25 700327166H1 199 Rattus
norvegicus RASNNOT01 1813-2008 74.4
[0143]
Sequence CWU 0
0
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