U.S. patent application number 10/317460 was filed with the patent office on 2003-06-26 for human fatty acid beta-oxidation enzymes.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Bandman, Olga, Corley, Neil C., Guegler, Karl J., Hillman, Jennifer L., Shah, Purvi, Tang, Y. Tom.
Application Number | 20030119046 10/317460 |
Document ID | / |
Family ID | 21700131 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119046 |
Kind Code |
A1 |
Bandman, Olga ; et
al. |
June 26, 2003 |
Human fatty acid beta-oxidation enzymes
Abstract
The invention provides human fatty acid beta-oxidation enzymes
(HUFA) and polynucleotides which identify and encode HUFA. The
invention also provides expression vectors, host cells, antibodies,
agonists, and antagonists. The invention also provides methods for
treating or preventing disorders associated with expression of
HUFA.
Inventors: |
Bandman, Olga; (Mountain
View, CA) ; Hillman, Jennifer L.; (Santa Cruz,
CA) ; Guegler, Karl J.; (Menlo Park, CA) ;
Corley, Neil C.; (Castro Valley, CA) ; Tang, Y.
Tom; (San Jose, CA) ; Shah, Purvi; (San Jose,
CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
21700131 |
Appl. No.: |
10/317460 |
Filed: |
December 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10317460 |
Dec 10, 2002 |
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09481277 |
Jan 11, 2000 |
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6511833 |
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09481277 |
Jan 11, 2000 |
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09002298 |
Dec 31, 1997 |
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6046001 |
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Current U.S.
Class: |
435/6.14 ;
424/146.1; 424/93.6; 435/198; 435/320.1; 435/325; 435/69.1;
435/7.2; 530/388.26; 536/23.2 |
Current CPC
Class: |
C12N 9/001 20130101 |
Class at
Publication: |
435/6 ; 435/7.2;
435/69.1; 435/320.1; 435/325; 435/198; 530/388.26; 536/23.2;
424/146.1; 424/93.6 |
International
Class: |
C12Q 001/68; G01N
033/53; G01N 033/567; C07H 021/04; C12P 021/02; C12N 005/06; C07K
016/40; A61K 039/395; C12N 009/20 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:1 and SEQ ID NO:3, b) a
polypeptide comprising a naturally occurring amino acid sequence at
least 90% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO: 1 and SEQ ID NO:3, c) a biologically
active fragment of a polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:3,
and d) an immunogenic fragment of a polypeptide having an amino
acid sequence selected from the group consisting of SEQ ID NO: 1
and SEQ ID NO:3.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO:3.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:2 and SEQ ID NO:4.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID NO: 1
and SEQ ID NO:3.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4,
b) a polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4,
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 selected from the group consisting of SEQ ID
NO: 1 and SEQ ID NO:3.
19. A method for treating a disease or condition associated with
decreased expression of functional HUFA, 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 HUFA, 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 HUFA, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A method for a diagnostic test for a condition or disease
associated with the expression of HUFA 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 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 HUFA in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, further comprising a label.
35. A method of diagnosing a condition or disease associated with
the expression of HUFA in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO:3, or an immunogenic fragment thereof, under conditions to
elicit an antibody response, b) isolating antibodies from the
animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which
specifically binds to a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO:3.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3,
or an immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibody producing cells from the
animal, c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells, d)
culturing the hybridoma cells, and e) isolating from the culture
monoclonal antibody which specifically binds to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1 and SEQ ID NO:3.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO:3 in a sample, the method comprising: a) incubating the
antibody of claim 11 with the sample under conditions to allow
specific binding of the antibody and the polypeptide, and b)
detecting specific binding, wherein specific binding indicates the
presence of a polypeptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:3
in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1 and SEQ
ID NO:3 from a sample, the method comprising: a) incubating the
antibody of claim 11 with the sample under conditions to allow
specific binding of the antibody and the polypeptide, and b)
separating the antibody from the sample and obtaining the purified
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1 and SEQ ID NO:3.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO: 1.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
58. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:2.
59. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/481,277, filed Jan. 11, 2000, which is a
divisional application of U.S. application Ser. No. 09/002,298,
filed Dec. 31, 1997, now U.S. Pat. No. 6,046,001, issued Apr. 4,
2000, all of which applications and patents are hereby incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human fatty acid beta-oxidation enzymes and to the use
of these sequences in the diagnosis, treatment, and prevention of
genetic disorders, neuronal disorders, cancer, infectious diseases,
liver disorders, and cardiac and skeletal muscle disorders.
BACKGROUND OF THE INVENTION
[0003] Mitochondrial and peroxisomal beta-oxidation enzymes degrade
saturated and unsaturated fatty acids by sequential removal of
two-carbon units from Coenzyme A (CoA)-activated fatty acids. The
main beta-oxidation pathway degrades both saturated and unsaturated
fatty acids while the auxiliary pathway performs additional steps
required for the degradation of unsaturated fatty acids.
[0004] The pathways of mitchondrial and peroxisomal beta-oxidation
use similar enzymes, but have different substrate specificities and
functions. Mitochondria oxidize short-, medium-, and long-chain
fatty acids to produce energy for cells. Mitochondrial
beta-oxidation is a major energy source for cardiac and skeletal
muscle. In liver, it provides ketone bodies to the peripheral
circulation when glucose levels are low as in starvation, endurance
exercise, and diabetes. (Eaton, S. et al. (1996) Biochem. J.
320:345-357.) Peroxisomes oxidize medium-, long-, and
very-long-chain fatty acids, dicarboxylic fatty acids, branched
fatty acids, prostaglandins, xenobiotics, and bile acid
intermediates. The chief roles of peroxisomal beta-oxidation are to
shorten toxic lipophilic carboxylic acids to facilitate their
excretion and to shorten very-long-chain fatty acids prior to
mitochondrial beta-oxidation. (Mannaerts, G. P. and Van Veldhoven,
P. P. (1993) Biochimie 75:147-158.)
[0005] The auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA
reductase catalyzes the following reaction:
[0006] trans-2,
cis/trans-4-dienoyl-CoA+NADPH+H.sup.+--->trans-3-enoyl--
CoA+NADP.sup.+
[0007] This reaction removes even-numbered double bonds from
unsaturated fatty acids prior to their entry into the main
beta-oxidation pathway. (Koivuranta, K. T. et al. (1994) Biochem.
J. 304:787-792.) The enzyme may also remove odd-numbered double
bonds from unsaturated fatty acids. (Smeland, T. E. et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6673-6677.)
[0008] Rat 2,4-dienoyl-CoA reductase is located in both
mitochondria and peroxisomes. (Dommes, V. et al. (1981) J. Biol.
Chem. 256:8259-8262.) Two immunologically different forms of rat
mitochondrial enzyme exist with molecular masses of 60 kDa and 120
kDa. (Hakkola, E. H. and Hiltunen, J. K. (1993) Eur. J. Biochem.
215:199-204.) The 120 kDa mitochondrial rat enzyme is synthesized
as a 335 amino acid precursor with a 29 amino acid N-terminal
leader peptide which is cleaved to form the mature enzyme. (Hirose,
A. et al. (1990) Biochim. Biophys. Acta 1049:346-349.) A human
mitochondrial enzyme 83% similar to rat enzyme is synthesized as a
335 amino acid residue precursor with a 19 amino acid N-terminal
leader peptide. (Koivuranta, supra.) These cloned human and rat
mitochondrial enzymes function as homotetramers. (Koivuranta,
supra.) A Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA
reductase is 295 amino acids long, contains a C-terminal
peroxisomal targeting signal, and functions as a homodimer. (Coe,
J. G. S. et al. (1994) Mol. Gen. Genet. 244:661-672; and Gurvitz,
A. et al. (1997) J. Biol. Chem. 272:22140-22147.) All
2,4-dienoyl-CoA reductases have a fairly well conserved NADPH
binding site motif of sequence
-h-X-h-X-Gly-X-Gly-X-X-Gly-X-X-X-h-X-X-h- . . . Asp/Glu-, where
h=hydrophobic amino acid residue and X=any amino acid residue.
(Koivuranta, supra.)
[0009] The main pathway beta-oxidation enzyme enoyl-CoA hydratase
catalyzes the following reaction:
[0010] 2-trans-enoyl-CoA+H.sub.2O<--->3-hydroxyacyl-CoA This
reaction hydrates the double bond between C-2 and C-3 of
2-trans-enoyl-CoA, which is generated from saturated and
unsaturated fatty acids. (Engel, C. K. et al. (1996) EMBO J.
15:5135-5145.) This step is downstream from the step catalyzed by
2,4-dienoyl-reductase. Different enoyl-CoA hydratases act on
short-, medium-, and long-chain fatty acids. (Eaton, supra.)
Mitochondrial and peroxisomal enoyl-CoA hydratases occur as both
mono-functional enzymes and as part of multi-functional enzyme
complexes. Human liver mitochondrial short-chain enoyl-CoA
hydratase is synthesized as a 290 amino acid precursor with a 29
amino acid N-terminal leader peptide. (Kanazawa, M. et al. (1993)
Enzyme Protein 47:9-13; and Janssen, U. et al. (1997) Genomics
40:470-475.) Rat short-chain enoyl-CoA hydratase is 87% identical
to the human sequence in the mature region of the protein and
functions as a homohexamer. (Kanazawa, supra; and Engel, supra) A
mitochondrial trifunctional protein exists that has long-chain
enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and
long-chain 3-oxothiolase activities. (Eaton, supra.) In human
peroxisomes, enoyl-CoA hydratase activity is found in both a 327
amino acid residue mono-functional enzyme and as part of a
multi-functional enzyme, also known as bifunctional enzyme, which
possesses enoyl-CoA hydratase, enoyl-CoA isomerase, and
3-hydroxyacyl-CoA hydrogenase activities. (FitzPatrick, D. R. et
al. (1995) Genomics 27:457-466; and Hoefler, G. et al. (1994)
Genomics 19:60-67.) A 339 amino acid residue human protein with
short-chain enoyl-CoA hydratase activity also acts as an
AU-specific RNA binding protein. (Nakagawa, J. et al. (1995) Proc.
Natl. Acad. Sci. USA 92:2051-2055.) All enoyl-CoA hydratases share
homology near two active site glutamic acid residues, with 17 amino
acid residues highly conserved. (Wu, W.-J. et al. (1997)
Biochemistry 36:2211-2220.) Inherited deficiencies in mitochondrial
and peroxisomal beta-oxidation enzymes are associated with severe
diseases, some of which manifest themselves soon after birth and
lead to death within a few years. Mitochondrial beta-oxidation
associated deficiencies include, e.g., carnitine palmitoyl
transferase and carnitine deficiency, very-long-chain acyl-CoA
dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase
deficiency, short-chain acyl-CoA dehydrogenase deficiency, electron
transport flavoprotein and electron transport
flavoprotein:ubiquinone oxidoreductase deficiency, trifunctional
protein deficiency, and short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency. (Eaton, supra.) Mitochondrial trifunctional protein
(including enoyl-CoA hydratase) deficient patients have reduced
long-chain enoyl-CoA hydratase activities and suffer from
non-ketotic hypoglycemia, sudden infant death syndrome,
cardiomyopathy, hepatic dysfunction, and muscle weakness, and may
die at an early age. (Eaton, supra.) A patient with a deficiency in
mitochondrial 2,4-dienoyl-CoA reductase was hypotonic soon after
birth, had feeding difficulties, and died at four months from
respiratory acidosis. (Roe, C. R. et al. (1990) J. Clin. Invest.
85:1703-1707.)
[0011] Defects in mitochondrial beta-oxidation are associated with
Reye's syndrome, a disease characterized by hepatic dysfunction and
encephalopathy that sometimes follows viral infection in children.
Reye's syndrome patients may have elevated serum levels of free
fatty acids. (Cotran, R. S. et al. (1994) Robbins Pathologic Basis
of Disease, W. B. Saunders Co., Philadelphia, Pa., p.866.) Patients
with mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency and medium-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency also exhibit Reye-like illnesses. (Eaton, supra; and
Egidio, R. J. et al. (1989) Am. Fam. Physician 39:221-226.)
[0012] Inherited conditions associated with peroxisomal
beta-oxidation include Zellweger-syndrome, neonatal
adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase
deficiency, peroxisomal thiolase deficiency, and bifunctional
protein deficiency. (Suzuki, Y. et al. (1994) Am. J. Hum. Genet.
54:36-43; Hoefler, supra) Patients with peroxisomal bifunctional
enzyme, including enoyl-CoA hydratase, deficiency suffer from
hypotonia, seizures, psychomotor defects, and defective neuronal
migration; accumulate very-long-chain fatty acids; and typically
die within a few years of birth. (Watkins, P. A. et al. (1989) J.
Clin. Invest. 83:771-777.)
[0013] Peroxisomal beta-oxidation is impaired in cancerous tissue.
Although neoplastic human breast epithelial cells have the same
number of peroxisomes as do normal cells, fatty acyl-CoA oxidase
activity is lower than in control tissue. (el Bouhtoury, F., et al.
(1992) J. Pathol. 166:27-35.) Human colon carcinomas have fewer
peroxisomes than normal colon tissue and have lower fatty-acyl-CoA
oxidase and bifunctional enzyme (including enoyl-CoA hydratase)
activities than normal tissue. (Cable, S., et al. (1992) Virchows
Arch. B Cell Pathol. Incl. Mol. Pathol. 62:221-226.)
[0014] The discovery of new human fatty acid beta-oxidation enzymes
and the polynucleotides encoding them satisfies a need in the art
by providing new compositions which are useful in the diagnosis,
treatment, and prevention of genetic disorders, neuronal disorders,
cancer, infectious diseases, liver disorders, and cardiac and
skeletal muscle disorders.
SUMMARY OF THE INVENTION
[0015] The invention features substantially purified polypeptides,
human fatty acid beta-oxidation enzymes, referred to collectively
as "HUFA" and individually as "HUFA-1" and "HUFA-2." In one aspect,
the invention provides a substantially purified polypeptide, HUFA,
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1,
and a fragment of SEQ ID NO:3.
[0016] The invention further provides a substantially purified
variant of HUFA having at least 90% amino acid identity to the
amino acid sequences of SEQ ID NO: 1 or SEQ ID NO:3, or to a
fragment of either of these sequences. The invention also provides
an isolated and purified polynucleotide sequence encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID
NO:1, and a fragment of SEQ ID NO:3. The invention also includes an
isolated and purified polynucleotide variant having at least 90%
polynucleotide identity to the polynucleotide sequence encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID
NO: 1, and a fragment of SEQ ID NO:3.
[0017] Additionally, the invention provides a composition
comprising a polynucleotide sequence encoding the polypeptide
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1,
and a fragment of SEQ ID NO:3. The invention further provides an
isolated and purified polynucleotide sequence which hybridizes
under stringent conditions to the polynucleotide sequence encoding
the polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID
NO: 1, and a fragment of SEQ ID NO:3, as well as an isolated and
purified polynucleotide sequence which is complementary to the
polynucleotide sequence encoding the polypeptide comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, and a fragment of SEQ
ID NO:3.
[0018] The invention also provides an isolated and purified
polynucleotide sequence comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a
fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4. The
invention further provides an isolated and purified polynucleotide
variant having at least 90% polynucleotide identity to the
polynucleotide sequence comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, a
fragment of SEQ ID NO:2, and a fragment of SEQ ID NO:4, as well as
an isolated and purified polynucleotide sequence which is
complementary to the polynucleotide sequence comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, and a fragment of
SEQ ID NO:4.
[0019] The invention further provides an expression vector
containing at least a fragment of the polynucleotide sequence
encoding the polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:3, a fragment
of SEQ ID NO:1, and a fragment of SEQ ID NO:3. In another aspect,
the expression vector is contained within a host cell.
[0020] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO:1, SEQ
ID NO:3, a fragment of SEQ ID NO:1, or a fragment of SEQ ID NO:3,
the method comprising the steps of: (a) culturing the host cell
containing an expression vector containing at least a fragment of a
polynucleotide sequence encoding HUFA under conditions suitable for
the expression of the polypeptide; and (b) recovering the
polypeptide from the host cell culture.
[0021] The invention also provides a pharmaceutical composition
comprising a substantially purified HUFA having the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ ID NO: 1,
or a fragment of SEQ ID NO:3 in conjunction with a suitable
pharmaceutical carrier.
[0022] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence of SEQ ID
NO:1, SEQ ID NO:3, a fragment of SEQ ID NO:1, or a fragment of SEQ
ID NO:3, as well as a purified agonist and a purified antagonist to
the polypeptide.
[0023] The invention also provides a method for treating or
preventing a genetic disorder, the method comprising administering
to a subject in need of such treatment an effective amount of a
pharmaceutical composition comprising substantially purified
HUFA.
[0024] The invention also provides a method for treating or
preventing a neuronal disorder, the method comprising administering
to a subject in need of such treatment an effective amount of a
pharmaceutical composition comprising substantially purified
HUFA-1.
[0025] The invention also provides a method for treating or
preventing a cancer, the method comprising administering to a
subject in need of such treatment an effective amount of a
pharmaceutical composition comprising substantially purified
HUFA-1.
[0026] The invention also provides a method for treating or
preventing an infectious disease, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising substantially
purified HUFA-2.
[0027] The invention also provides a method for treating or
preventing a liver disorder, the method comprising administering to
a subject in need of such treatment an effective amount of a
pharmaceutical composition comprising substantially purified
HUFA-2.
[0028] he invention also provides a method for treating or
preventing a cardiac or skeletal muscle disorder, the method
comprising administering to a subject in need of such treatment an
effective amount of a pharmaceutical composition comprising
substantially purified HUFA-2.
[0029] The invention also provides a method for detecting a
polynucleotide encoding HUFA in a biological sample containing
nucleic acids, the method comprising the steps of: (a) hybridizing
the complement of the polynucleotide sequence encoding the
polypeptide comprising SEQ ID NO: 1, SEQ ID NO:3, a fragment of SEQ
ID NO:1, or a fragment of SEQ ID NO:3 to at least one of the
nucleic acids of the biological sample, thereby forming a
hybridization complex; and (b) detecting the hybridization complex,
wherein the presence of the hybridization complex correlates with
the presence of a polynucleotide encoding HUFA in the biological
sample. In one aspect, the nucleic acids of the biological sample
are amplified by the polymerase chain reaction prior to the
hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ
ID NO: 1) and nucleic acid sequence (SEQ ID NO:2) of HUFA-1. The
alignment was produced using MACDNASIS PRO software (Hitachi
Software Engineering Co. Ltd., San Bruno, Calif.).
[0031] FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H show the amino acid
sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of
HUFA-2. The alignment was produced using MACDNASIS PRO
software.
[0032] FIGS. 3A, 3B, and 3C show the amino acid sequence alignments
among HUFA-1 (Incyte Clone 1995961; SEQ ID NO:1), Saccharomyces
cerevisiae peroxisomal 2,4-dienoyl-CoA reductase (GI 730864; SEQ ID
NO:5), human mitochondrial 2,4-dienoyl-CoA reductase (GI 602703;
SEQ ID NO:6), and rat 2,4-dienoyl-CoA reductase (GI 111287; SEQ ID
NO:7), produced using the multisequence alignment program of
LASERGENE software (DNASTAR Inc., Madison, Wis.).
[0033] FIGS. 4A, 4B, and 4C show the amino acid sequence alignments
among HUFA-2 (Incyte Clone 2595635; SEQ ID NO:3), human AU-binding
protein/enoyl-CoA hydratase (GI 780241; SEQ ID NO:8), human
enoyl-CoA hydratase (GI 1922287; SEQ ID NO:9), and human
peroxisomal enoyl-CoA hydratase-like protein (GI 564065; SEQ ID NO:
10), produced using the multisequence alignment program of
LASERGENE software.
DESCRIPTION OF THE INVENTION
[0034] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0035] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and which
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.
[0037] Definitions
[0038] "HUFA," as used herein, refers to the amino acid sequences
of substantially purified HUFA obtained from any species,
particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and preferably the human species, from any source,
whether natural, synthetic, semi-synthetic, or recombinant.
[0039] The term "agonist," as used herein, refers to a molecule
which, when bound to HUFA, increases or prolongs the duration of
the effect of HUFA. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of HUFA.
[0040] An "allele" or an "allelic sequence," as these terms are
used herein, is an alternative form of the gene encoding HUFA.
Alleles may result from at least one mutation in the nucleic acid
sequence and may result in altered mRNAs or in polypeptides whose
structure or function may or may not be altered. Any given natural
or recombinant gene may have none, one, or many allelic forms.
Common mutational changes which give rise to alleles are generally
ascribed to natural deletions, additions, or substitutions of
nucleotides. Each of these types of changes may occur alone, or in
combination with the others, one or more times in a given
sequence.
[0041] "Altered" nucleic acid sequences encoding HUFA, as described
herein, include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide the same HUFA or a polypeptide with at least one
functional characteristic of HUFA. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
HUFA, and improper or unexpected hybridization to alleles, with a
locus other than the normal chromosomal locus for the
polynucleotide sequence encoding HUFA. The encoded protein may also
be "altered," and may contain deletions, insertions, or
substitutions of amino acid residues which produce a silent change
and result in a functionally equivalent HUFA. Deliberate amino acid
substitutions may be made on the basis of similarity in polarity,
charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the residues, as long as the biological or
immunological activity of HUFA is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid,
positively charged amino acids may include lysine and arginine, and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; and phenylalanine and tyrosine.
[0042] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments", "immunogenic
fragments", or "antigenic fragments" refer to fragments of HUFA
which are preferably about 5 to about 15 amino acids in length and
which retain some biological activity or immunological activity of
HUFA. Where "amino acid sequence" is recited herein to refer to an
amino acid sequence of a naturally occurring protein molecule,
"amino acid sequence" and like terms are not meant to limit the
amino acid sequence to the complete native amino acid sequence
associated with the recited protein molecule.
[0043] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, pp. 1-5,
Cold Spring Harbor Press, Plainview, N.Y.)
[0044] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to HUFA, decreases the amount or the
duration of the effect of the biological or immunological activity
of HUFA. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of HUFA.
[0045] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind HUFA polypeptides can be
prepared using intact polypeptides or using fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0046] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0047] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to a specific nucleic acid sequence. The term
"antisense strand" is used in reference to a nucleic acid strand
that is complementary to the "sense" strand. Antisense molecules
may be produced by any method including synthesis or transcription.
Once introduced into a cell, the complementary nucleotides combine
with natural sequences produced by the cell to form duplexes and to
block either transcription or translation. The designation
"negative" can refer to the antisense strand, and the designation
"positive" can refer to the sense strand.
[0048] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
HUFA, or of any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0049] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A." Complementarity between two single-stranded molecules may
be "partial," such that only some of the nucleic acids bind, or it
may be "complete," such that total complementarity exists between
the single stranded molecules. The degree of complementarity
between nucleic acid strands has significant effects on the
efficiency and strength of the hybridization between the nucleic
acid strands. This is of particular importance in amplification
reactions, which depend upon binding between nucleic acids strands,
and in the design and use of peptide nucleic acid (PNA)
molecules.
[0050] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation, an aqueous solution, or a sterile
composition. Compositions comprising polynucleotide sequences
encoding HUFA or fragments of HUFA may be employed as hybridization
probes. The probes may be stored in freeze-dried form and may be
associated with a stabilizing agent such as a carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution
containing salts (e.g., NaCl), detergents (e.g., SDS), and other
components (e.g., Denhardt's solution, dry milk, salmon sperm DNA,
etc.).
[0051] The phrase "consensus sequence," as used herein, refers to a
nucleic acid sequence which has been resequenced to resolve
uncalled bases, extended using XL-PCR (Perkin Elmer, Norwalk,
Conn.) in the 5' and/or the 3' direction, and resequenced, or which
has been assembled from the overlapping sequences of more than one
Incyte Clone using a computer program for fragment assembly, such
as the GELVIEW fragment assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0052] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding HUFA, by northern analysis is indicative of the presence
of nucleic acids encoding HUFA in a sample, and thereby correlates
with expression of the transcript from the polynucleotide encoding
HUFA.
[0053] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0054] The term "derivative," as used herein, refers to the
chemical modification of HUFA, of a polynucleotide sequence
encoding HUFA, or of a polynucleotide sequence complementary to a
polynucleotide sequence encoding HUFA. Chemical modifications of a
polynucleotide sequence can include, for example, replacement of
hydrogen by an alkyl, acyl, or amino group. A derivative
polynucleotide encodes a polypeptide which retains at least one
biological or immunological function of the natural molecule. A
derivative polypeptide is one modified by glycosylation,
pegylation, or any similar process that retains a at least one
biological or immunological function of the polypeptide from which
it was derived.
[0055] The term "homology," as used herein, refers to a degree of
complementarity. There may be partial homology or complete
homology. The word "identity" may substitute for the word
"homology." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially homologous."
The inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% homology or
identity). In the absence of non-specific binding, the
substantially homologous sequence or probe will not hybridize to
the second non-complementary target sequence.
[0056] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(LASERGENE software package, DNASTAR, Inc., Madison Wis.). The
MEGALIGN program can create alignments between two or more
sequences according to different methods, e.g., the Clustal Method.
(Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) The
Clustal algorithm groups sequences into clusters by examining the
distances between all pairs. The clusters are aligned pairwise and
then in groups. The percentage similarity between two amino acid
sequences, e.g., sequence A and sequence B, is calculated by
dividing the length of sequence A, minus the number of gap residues
in sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no homology between the two
amino acid sequences are not included in determining percentage
similarity. Percent identity between nucleic acid sequences can
also be calculated by the Clustal Method, or by other methods known
in the art, such as the Jotun Hein Method. (See, e.g., Hein, J.
(1990) Methods in Enzymology 183:626-645.) Identity between
sequences can also be determined by other methods known in the art,
e.g., by varying hybridization conditions.
[0057] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 6 kb to 10 Mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(Harrington, J. J. et al. (1997) Nat Genet. 15:345-355.) The term
"humanized antibody," as used herein, refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
[0058] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0059] As used herein, the term "hybridization complex" as used
herein, refers to a complex formed between two nucleic acid
sequences by virtue of the formation of hydrogen bonds between
complementary bases. A hybridization complex may be formed in
solution (e.g., Cot or Rot analysis) or formed between one nucleic
acid sequence present in solution and another nucleic acid sequence
immobilized on a solid support (e.g., paper, membranes, filters,
chips, pins or glass slides, or any other appropriate substrate to
which cells or their nucleic acids have been fixed).
[0060] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0061] The term "microarray," as used herein, refers to an array of
distinct polynucleotides or oligonucleotides arrayed on a
substrate, such as paper, nylon or any other type of membrane,
filter, chip, glass slide, or any other suitable solid support.
[0062] The term "modulate," as it appears herein, refers to a
change in the activity of HUFA. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of HUFA.
[0063] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to an oligonucleotide, nucleotide,
polynucleotide, or any fragment thereof, to DNA or RNA of genomic
or synthetic origin which may be single-stranded or double-stranded
and may represent the sense or the antisense strand, to peptide
nucleic acid (PNA), or to any DNA-like or RNA-like material. In
this context, "fragments" refers to those nucleic acid sequences
which are greater than about 60 nucleotides in length, and most
preferably are at least about 100 nucleotides, at least about 1000
nucleotides, or at least about 10,000 nucleotides in length.
[0064] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimers," "primers," "oligomers," and "probes," as
these terms are commonly defined in the art.
[0065] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (Nielsen, P. E. et al. (1993) Anticancer Drug
Des. 8:53-63.)
[0066] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding HUFA, or fragments thereof, or HUFA itself may comprise a
bodily fluid; an extract from a cell, chromosome, organelle, or
membrane isolated from a cell; a cell; genomic DNA, RNA, or cDNA
(in solution or bound to a solid support); a tissue; a tissue
print; and the like.
[0067] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein recognized by the binding molecule (i.e.,
the antigenic determinant or epitope). For example, if an antibody
is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0068] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotide
sequences and the claimed polynucleotide sequences. Suitably
stringent conditions can be defined by, for example, the
concentrations of salt or formamide in the prehybridization and
hybridization solutions, or by the hybridization temperature, and
are well known in the art. In particular, stringency can be
increased by reducing the concentration of salt, increasing the
concentration of formamide, or raising the hybridization
temperature.
[0069] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS, and 200 .mu.g/ml sheared and denatured
salmon sperm DNA. Hybridization could occur under reduced
stringency conditions as described above, but in 35% formamide at a
reduced temperature of 35.degree. C. The temperature range
corresponding to a particular level of stringency can be further
narrowed by calculating the purine to pyrimidine ratio of the
nucleic acid of interest and adjusting the temperature accordingly.
Variations on the above ranges and conditions are well known in the
art.
[0070] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0071] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0072] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, and refers to cells which transiently
express the inserted DNA or RNA for limited periods of time.
[0073] A "variant" of HUFA, as used herein, refers to an amino acid
sequence that is altered by one or more amino acids. The variant
may have "conservative" changes, wherein a substituted amino acid
has similar structural or chemical properties (e.g., replacement of
leucine with isoleucine). More rarely, a variant may have
"nonconservative" changes (e.g., replacement of glycine with
tryptophan). Analogous minor variations may also include amino acid
deletions or insertions, or both. Guidance in determining which
amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
LASERGENE software.
[0074] The Invention
[0075] The invention is based on the discovery of new human fatty
acid beta-oxidation enzymes (HUFA), the polynucleotides encoding
HUFA, and the use of these compositions for the diagnosis,
treatment, or prevention of genetic disorders, neuronal disorders,
cancer, infectious diseases, liver disorders, and cardiac and
skeletal muscle disorders.
[0076] Nucleic acids encoding the HUFA-1 of the present invention
were first identified in Incyte Clone 1995961 from the human breast
tumor cDNA library (BRSTTUT03) using a computer search for amino
acid sequence alignments. A consensus sequence, SEQ ID NO:2, was
derived from the following overlapping and/or extended nucleic acid
sequences: Incyte Clones 270674 (HNT2NOT01), 1995961 (BRSTTUT03),
and 2851728 (BRSTTUT13).
[0077] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 as shown in
FIGS. 1A-1D. HUFA-1 is 303 amino acids in length and has a
potential peroxisomal C-terminal targeting signal at A301KL. HUFA-1
has potential casein kinase II phosphorylation sites at residues
S57, S93, S 114, T208, S219, and T296 and potential protein kinase
C phosphorylation sites at residues S49, S120, and T296. HUFA-1 has
a potential NADPH-binding site motif at A21-E53, containing five of
the eight consensus amino acid residues: A21, V23, G25, 134, and
E53. As shown in FIGS. 3A-3C, HUFA-1 has chemical and structural
homology with Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA
reductase (GI 730864; SEQ ID NO:5), human mitochondrial
2,4-dienoyl-CoA reductase (GI 602703; SEQ ID NO:6), and rat
2,4-dienoyl-CoA reductase (GI 111287; SEQ ID NO:7). In particular,
HUFA-1 and Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA
reductase share 31% identity, HUFA-1 and human mitochondrial
2,4-dienoyl-CoA reductase share 31% identity, and HUFA-I and rat
2,4-dienoyl-CoA reductase share 30% identity. In the region of the
potential NADPH-binding site motif, A21-E53, HUFA-1 is 39%
identical to Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA
reductase, 52% identical to human mitochondrial 2,4-dienoyl-CoA
reductase, and 52% identical to rat 2,4-dienoyl-CoA reductase.
Northern analysis shows the expression of this sequence in various
libraries, at least 81% of which are immortalized or cancerous and
at least 9% of which involve immune response. Of particular note is
the expression of HUFA-1 in libraries from breast and brain
tissue.
[0078] Nucleic acids encoding the HUFA-2 of the present invention
were first identified in Incyte Clone 2595635 from the human
ovarian tumor cDNA library (OVARTUT02) using a computer search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO:4,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 077607 (SYNORAB01), 416055
(BRSTNOT01), 782840 (MYOMNOT01), 1421780 (KIDNNOT09), 1449639
(PLACNOT02), 1474617 (LUNGTUT03), 1485974 (CORPNOT02), 1617081
(BRAITUT12), 1987379 (LUNGAST01), and 2595635 (OVARTUT02).
[0079] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:3 as shown in FIGS.
2A-2H. HUFA-2 is 301 amino acids in length and has a potential
mitochondrial N-terminal leader peptide from residues M1 through
Y33. HUFA-2 has potential casein kinase II phosphorylation sites at
residues S107, T119, T161, S229, S235, and S263, and potential
protein kinase C phosphorylation sites at residues S12, T190, and
S263. As shown in FIGS. 4A-4C, HUFA-2 has chemical and structural
homology with human AU-binding protein/enoyl-CoA hydratase (GI
780241; SEQ ID NO:8), human enoyl-CoA hydratase (GI 1922287; SEQ ID
NO:9), and human peroxisomal enoyl-CoA hydratase-like protein (GI
564065; SEQ ID NO: 10). In particular, HUFA-2 and human AU-binding
protein/enoyl-CoA hydratase share 25% identity, HUFA-2 and human
enoyl-CoA hydratase share 20% identity, and HUFA-2 and human
peroxisomal enoyl-CoA hydratase-like protein share 21% identity. In
particular, 10 of the 17 highly conserved residues around the
enoyl-CoA hydratase active site are found in HUFA-2: G150, G154,
G155, G156, E158, D164, G182, P185, G189, and G198. In particular,
HUFA-2 contains the enoyl-CoA hydratase active site residue E158.
Northern analysis shows the expression of this sequence in various
libraries, at least 46% of which are immortalized or cancerous and
at least 26% of which involve immune response. Of particular note
is the expression of HUFA-2 in libraries prepared from heart and
liver tissue.
[0080] The invention also encompasses HUFA variants. A preferred
HUFA variant is one which has at least about 80%, more preferably
at least about 90%, and most preferably at least about 95% amino
acid sequence identity to the HUFA amino acid sequence, and which
contains at least one functional or structural characteristic of
HUFA.
[0081] The invention also encompasses polynucleotides which encode
HUFA. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:2, as
shown in FIG. 1, which encodes a HUFA-1. In a further embodiment,
the invention encompasses the polynucleotide sequence comprising
the sequence of SEQ ID NO:4, as shown in FIG. 2, which encodes a
HUFA-2. The invention also encompasses a variant of a
polynucleotide sequence encoding HUFA. In particular, such a
variant polynucleotide sequence will have at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding HUFA. A particular aspect of the invention encompasses a
variant of SEQ ID NO:2 which has at least about 80%, more
preferably at least about 90%, and most preferably at least about
95% polynucleotide sequence identity to SEQ ID NO:2. The invention
further encompasses a polynucleotide variant of SEQ ID NO:4 having
at least about 80%, more preferably at least about 90%, and most
preferably at least about 95% polynucleotide sequence identity to
SEQ ID NO:4. Any one of the polynucleotide variants described above
can encode an amino acid sequence which contains at least one
functional or structural characteristic of HUFA.
[0082] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding HUFA, some bearing minimal
homology to the polynucleotide sequences of any known and naturally
occurring gene, may be produced. Thus, the invention contemplates
each and every possible variation of polynucleotide sequence that
could be made by selecting combinations based on possible codon
choices. These combinations are made in accordance with the
standard triplet genetic code as applied to the polynucleotide
sequence of naturally occurring HUFA, and all such variations are
to be considered as being specifically disclosed.
[0083] Although nucleotide sequences which encode HUFA and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HUFA under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HUFA or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding HUFA and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0084] The invention also encompasses production of DNA sequences
which encode HUFA and HUFA derivatives, or fragments thereof,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding HUFA or any fragment
thereof.
[0085] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:2, SEQ ID NO:4, a fragment of SEQ ID NO:2, or a fragment of
SEQ ID NO:4 under various conditions of stringency as taught in
Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407)
and Kimmel, A. R. (1987; Methods Enzymol. 152:507-511).
[0086] Methods for DNA sequencing are well known and generally
available in the art and may be used to practice any of the
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE (US
Biochemical Corp., Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE amplification system marketed by
GIBCO/BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the MICROLAB 2200 (Hamilton, Reno, Nev.),
Peltier thermal cycler (PTC200; MJ Research, Watertown, Mass.) and
the ABI CATALYST and 373 and 377 DNA sequencers (Perkin Elmer).
[0087] The nucleic acid sequences encoding HUFA may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences, such as
promoters and regulatory elements. For example, one method which
may be employed, restriction-site PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus. (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322.) In particular, genomic DNA
is first amplified in the presence of a primer to a linker sequence
and a primer specific to the known region. The amplified sequences
are then subjected to a second round of PCR with the same linker
primer and another specific primer internal to the first one.
Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced using reverse transcriptase.
[0088] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region. (Triglia, T. et
al. (1988) Nucleic Acids Res. 16:8186.) The primers may be designed
using commercially available software such as OLIGO 4.06 primer
analysis software (National Biosciences Inc., Plymouth, Minn.) or
another appropriate program to be about 22 to 30 nucleotides in
length, to have a GC content of about 50% or more, and to anneal to
the target sequence at temperatures of about 68.degree. C. to
72.degree. C. The method uses several restriction enzymes to
generate a suitable fragment in the known region of a gene. The
fragment is then circularized by intramolecular ligation and used
as a PCR template.
[0089] Another method which may be used is capture PCR, which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA. (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119.) In this method,
multiple restriction enzyme digestions and ligations may be used to
place an engineered double-stranded sequence into an unknown
fragment of the DNA molecule before performing PCR. Another method
which may be used to retrieve unknown sequences is that of Parker,
J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060). Additionally,
one may use PCR, nested primers, and PROMOTERFINDER libraries to
walk genomic DNA (Clontech, Palo Alto, Calif.). This process avoids
the need to screen libraries and is useful in finding intron/exon
junctions.
[0090] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable in that they will
include more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into 5' non-transcribed regulatory regions.
[0091] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and a charge coupled device
camera for detection of the emitted wavelengths. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin Elmer),
and the entire process from loading of samples to computer analysis
and electronic data display may be computer controlled. Capillary
electrophoresis is especially preferable for the sequencing of
small pieces of DNA which might be present in limited amounts in a
particular sample.
[0092] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HUFA may be used in
recombinant DNA molecules to direct expression of HUFA, or
fragments or functional equivalents thereof, in appropriate host
cells. Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence may be produced, and these sequences
may be used to clone and express HUFA.
[0093] As will be understood by those of skill in the art, it may
be advantageous to produce HUFA-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0094] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HUFA encoding sequences for a variety of reasons including,
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0095] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HUFA may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of HUFA activity, it may
be useful to encode a chimeric HUFA protein that can be recognized
by a commercially available antibody. A fusion protein may also be
engineered to contain a cleavage site located between the HUFA
encoding sequence and the heterologous protein sequence, so that
HUFA may be cleaved and purified away from the heterologous
moiety.
[0096] In another embodiment, sequences encoding HUFA may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, the protein itself may be
produced using chemical methods to synthesize the amino acid
sequence of HUFA, or a fragment thereof. For example, peptide
synthesis can be performed using various solid-phase techniques
(Roberge, J. Y. et al. (1995) Science 269:202-204) and automated
synthesis may be achieved using the ABI 431A peptide synthesizer
(Perkin Elmer).
[0097] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography. (See, e.g.,
Chiez, R. M. and Regnier, F. Z. (1990) Methods Enzymol.
182:392-421.) The composition of the synthetic peptides may be
confirmed by amino acid analysis or by sequencing. (See, e.g., the
Edman degradation procedure described in Creighton, T. (1983)
Proteins, Structures and Molecular Principles, W H Freeman and Co.,
New York, N.Y.). Additionally, the amino acid sequence of HUFA, or
any part thereof, may be altered during direct synthesis and/or
combined with sequences from other proteins, or any part thereof,
to produce a variant polypeptide.
[0098] In order to express a biologically active HUFA, the
nucleotide sequences encoding HUFA or derivatives thereof may be
inserted into appropriate expression vector, i.e., a vector which
contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0099] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HUFA and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989; Molecular Cloning, A Laboratory Manual, ch. 4, 8, and 16-17,
Cold Spring Harbor Press, Plainview, N.Y.) and Ausubel, F. M. et
al. (1995 and periodic supplements; Current Protocols in Molecular
Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,
N.Y.).
[0100] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HUFA. These include, but
are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0101] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector (i.e., enhancers, promoters,
and 5' and 3' untranslated regions) which interact with host
cellular proteins to carry out transcription and translation. Such
elements may vary in their strength and specificity. Depending on
the vector system and host utilized, any number of suitable
transcription and translation elements, including constitutive and
inducible promoters, may be used. For example, when cloning in
bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.)
or PSPORT1 plasmid (GIBCO/BRL), and the like, may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO, and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferable. If it is
necessary to generate a cell line that contains multiple copies of
the sequence encoding HUFA, vectors based on SV40 or EBV may be
used with an appropriate selectable marker.
[0102] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for HUFA. For example,
when large quantities of HUFA are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, multifunctional E. coli cloning
and expression vectors such as BLUESCRIPT (Stratagene), in which
the sequence encoding HUFA may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced, pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509), and the like. pGEX vectors (Promega,
Madison, Wis.) may also be used to express foreign polypeptides as
fusion proteins with glutathione S-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems may be designed to include heparin, thrombin, or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0103] In the yeast Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters, such as alpha
factor, alcohol oxidase, and PGH, may be used. For reviews, see
Ausubel (supra) and Grant et al. (1987; Methods Enzymol.
153:516-544).
[0104] In cases where plant expression vectors are used, the
expression of sequences encoding HUFA may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.
6:307-311.) Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used. (Coruzzi,
G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105.) These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews. (See, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196.)
[0105] An insect system may also be used to express HUFA. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding HUFA may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of HUFA will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which HUFA may be expressed. (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227.)
[0106] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HUFA may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing HUFA in
infected host cells. (Logan, J. and T. Shenk (1984) Proc. Natl.
Acad. Sci. 81:3655-3659.) In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0107] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of about 6 kb to 10 Mb are constructed and
delivered via conventional delivery methods (liposomes,
polycationic amino polymers, or vesicles) for therapeutic
purposes.
[0108] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding HUFA. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding HUFA and its initiation codon and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers appropriate for the particular cell
system used, such as those described in the literature. (Scharf, D.
et al. (1994) Results Probl. Cell Differ. 20:125-162.)
[0109] In addition, a host cell strain may be chosen for its
ability to modulate expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding, and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and W138), are available from the American
Type Culture Collection (ATCC, Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0110] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
capable of stably expressing HUFA can be transformed using
expression vectors which may contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene
on the same or on a separate vector. Following the introduction of
the vector, cells may be allowed to grow for about 1 to 2 days in
enriched media before being switched to selective media. The
purpose of the selectable marker is to confer resistance to
selection, and its presence allows growth and recovery of cells
which successfully express the introduced sequences.
[0111] Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0112] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase genes (Wigler, M. et al.
(1977) Cell 11:223-32) and adenine phosphoribosyltransferase genes
(Lowy, I. et al. (1980) Cell 22:817-23), which can be employed in
tk.sup.- or apr.sup.- cells, respectively. Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate
(Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and als
or pat confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase, respectively (Murry, supra). Additional
selectable genes have been described, for example, trpB, which
allows cells to utilize indole in place of tryptophan, or hisD,
which allows cells to utilize histinol in place of histidine.
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl.
[0113] Acad. Sci. 85:8047-51.) Recently, the use of visible markers
has gained popularity with such markers as anthocyanins, B
glucuronidase and its substrate GUS, and luciferase and its
substrate luciferin. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131.)
[0114] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding HUFA is inserted within a marker gene
sequence, transformed cells containing sequences encoding HUFA can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding HUFA under the control of a single promoter.
Expression of the marker gene in response to induction or selection
usually indicates expression of the tandem gene as well.
[0115] Alternatively, host cells which contain the nucleic acid
sequence encoding HUFA and express HUFA may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, or chip based technologies for the
detection and/or quantification of nucleic acid or protein
sequences.
[0116] The presence of polynucleotide sequences encoding HUFA can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
HUFA. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding HUFA
to detect transformants containing DNA or RNA encoding HUFA.
[0117] A variety of protocols for detecting and measuring the
expression of HUFA, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell
sorting (FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
HUFA is preferred, but a competitive binding assay may be employed.
These and other assays are well described in the art, for example,
in Hampton, R. et al. (1990; Serological Methods, a Laboratory
Manual, Section IV, APS Press, St Paul, Minn.) and in Maddox, D. E.
et al. (1983; J. Exp. Med. 158:1211-1216).
[0118] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HUFA include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HUFA, or any
fragments thereof, may be cloned into a vector for the production
of an mRNA probe. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Pharmacia & Upjohn (Kalamazoo, Mich.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0119] Host cells transformed with nucleotide sequences encoding
HUFA may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode HUFA may be designed to
contain signal sequences which direct secretion of HUFA through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding HUFA to nucleotide sequences
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences, such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.), between the purification domain and the HUFA encoding
sequence may be used to facilitate purification. One such
expression vector provides for expression of a fusion protein
containing HUFA and a nucleic acid encoding 6 histidine residues
preceding a thioredoxin or an enterokinase cleavage site. The
histidine residues facilitate purification on immobilized metal ion
affinity chromatography (IMAC; described in Porath, J. et al.
(1992) Prot. Exp. Purif. 3: 263-281)), while the enterokinase
cleavage site provides a means for purifying HUFA from the fusion
protein. A discussion of vectors which contain fusion proteins is
provided in Kroll, D. J. et al. (1993; DNA Cell Biol.
12:441-453).
[0120] Fragments of HUFA may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.)
Protein synthesis may be performed by manual techniques or by
automation. Automated synthesis may be achieved, for example, using
the Applied Biosystems 431A peptide synthesizer (Perkin Elmer).
Various fragments of HUFA may be synthesized separately and then
combined to produce the full length molecule.
[0121] Therapeutics
[0122] Chemical and structural homology exists among HUFA-1 and
peroxisomal 2,4-dienoyl-CoA reductase from Saccharomyces cerevisiae
(GI 730864), mitochondrial 2,4-dienoyl-CoA reductase from human (GI
602703), and 2,4-dienoyl-CoA reductase from rat (GI 111287). In
addition, HUFA-1 is expressed in cancerous and brain tissue.
Therefore, HUFA-1 appears to play a role in genetic disorders,
neuronal disorders, and cancer.
[0123] Chemical and structural homology exists among HUFA-2 and
AU-binding protein/enoyl-CoA hydratase from human (GI 780241),
enoyl-CoA hydratase from human (GI 1922287), and peroxisomal
enoyl-CoA hydratase-like protein from human (GI 564065). In
addition, HUFA-2 is expressed in heart and liver tissue. Therefore,
HUFA-2 appears to play a role in genetic disorders, infectious
diseases, liver disorders, and cardiac and skeletal muscle
disorders.
[0124] Therefore, in one embodiment, HUFA or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a genetic disorder. Such disorders can include, but are not
limited to, adrenoleukodystrophy, Alport's syndrome, choroideremia,
Duchenne and Becker muscular dystrophy, Down's syndrome, cystic
fibrosis, chronic granulomatous disease, Gaucher's disease,
Huntington's chorea, Marfan's syndrome, muscular dystrophy,
myotonic dystrophy, pycnodysostosis, Refsum's syndrome,
retinoblastoma, sickle cell anemia, thalassemia, Werner syndrome,
von Willebrand's disease, Wilm's tumor, Zellweger syndrome,
peroxisomal acyl-CoA oxidase deficiency, peroxisomal thiolase
deficiency, peroxisomal bifunctional protein deficiency,
mitochondrial carnitine palmitoyl transferase and carnitine
deficiency, mtiochondrial very-long-chain acyl-CoA dehydrogenase
deficiency, mitochondrial medium-chain acyl-CoA dehydrogenase
deficiency, mitochondrial short-chain acyl-CoA dehydrogenase
deficiency, mitochondrial electron transport flavoprotein and
electron transport flavoprotein:ubiquinone oxidoreductase
deficiency, mitochondrial trifunctional protein deficiency, and
mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency.
[0125] In another embodiment, a vector capable of expressing HUFA
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a genetic disorder including, but not
limited to, those described above.
[0126] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a genetic disorder including, but not limited to,
those provided above.
[0127] In still another embodiment, an agonist which modulates the
activity of HUFA may be administered to a subject to treat or
prevent a genetic disorder including, but not limited to, those
listed above.
[0128] In another embodiment, HUFA-1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
neuronal disorder. Such disorders can include, but are not limited
to, akathesia, Alzheimer's disease, amnesia, amyotrophic lateral
sclerosis, bipolar disorder, catatonia, cerebral neoplasms,
dementia, depression, diabetic neuropathy, Down's syndrome, tardive
dyskinesia, dystonias, epilepsy, Huntington's disease, multiple
sclerosis, neurofibromatosis, Parkinson's disease, paranoid
psychoses, postherpetic neuralgia, schizophrenia, and Tourette's
disorder.
[0129] In another embodiment, a vector capable of expressing HUFA-1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a neuronal disorder including, but not
limited to, those described above.
[0130] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA-1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a neuronal disorder including, but not limited to,
those provided above.
[0131] In still another embodiment, an agonist which modulates the
activity of HUFA-1 may be administered to a subject to treat or
prevent a neuronal disorder including, but not limited to, those
listed above.
[0132] In another embodiment, HUFA-1 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
cancer. Cancers can include, but are not limited to,
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen, testis, thymus, thyroid, and uterus.
[0133] In another embodiment, a vector capable of expressing HUFA-1
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a cancer including, but not limited to,
those described above.
[0134] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA-1 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a cancer including, but not limited to, those
provided above.
[0135] In still another embodiment, an agonist which modulates the
activity of HUFA may be administered to a subject to treat or
prevent a cancer including, but not limited to, those listed
above.
[0136] In another embodiment, HUFA-2 or a fragment or derivative
thereof may be administered to a subject to treat or prevent an
infectious disease. Such diseases can include, but are not limited
to, viral infections: adenoviruses (ARD, pneumonia), arenaviruses
(lymphocytic choriomeningitis), bunyaviruses (Hantavirus),
coronaviruses (pneumonia, chronic bronchitis), hepadnaviruses
(hepatitis), herpesviruses (HSV, VZV, Epstein-Barr virus,
cytomegalovirus), flaviviruses (yellow fever), orthomyxoviruses
(influenza), papillomaviruses (cancer), paramyxoviruses (measles,
mumps), picornoviruses (rhinovirus, poliovirus, coxsackie-virus),
polyomaviruses (BK virus, JC virus), poxviruses (smallpox),
reovirus (Colorado tick fever), retroviruses (HIV, HTLV),
rhabdoviruses (rabies), rotaviruses (gastroenteritis), and
togaviruses (encephalitis, rubella); bacterial infections, fungal
infections, parasitic infections, protozoal infections, and
helminthic infections.
[0137] In another embodiment, a vector capable of expressing HUFA-2
or a fragment or derivative thereof may be administered to a
subject to treat or prevent an infectious disease including, but
not limited to, those described above.
[0138] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA-2 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent an infectious disease including, but not limited
to, those provided above.
[0139] In still another embodiment, an agonist which modulates the
activity of HUFA-2 may be administered to a subject to treat or
prevent an infectious disease including, but not limited to, those
listed above.
[0140] In another embodiment, HUFA-2 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
liver disorder. Such disorders can include, but are not limited to,
cirrhosis, jaundice, cholestasis, hereditary hyperbilirubinemia,
hepatic encephalopathy, hepatorenal syndrome, hepatitis, hepatic
steatosis, hemochromatosis, Wilson's disease, alpha-antitrypsin
deficiency, Reye's syndrome, primary sclerosing cholangitis, liver
infarction, portal vein obstruction and thrombosis, passive
congestion, centrilobular necrosis, peliosis hepatis, hepatic vein
thrombosis, veno-occlusive disease, preeclampsia, eclampsia, acute
fatty liver of pregnancy, intrahepatic cholestasis of pregnancy,
and hepatic tumors including nodular hyperplasias, adenomas, and
carcinomas.
[0141] In another embodiment, a vector capable of expressing HUFA-2
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a liver disorder including, but not
limited to, those described above.
[0142] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA-2 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a liver disorder including, but not limited to,
those provided above.
[0143] In still another embodiment, an agonist which modulates the
activity of HUFA-2 may be administered to a subject to treat or
prevent a liver disorder including, but not limited to, those
listed above.
[0144] In another embodiment, HUFA-2 or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
cardiac or skeletal muscle disorder. Such disorders can include,
but are not limited to, cardiomyopathy, myocarditis, Duchenne's
muscular dystrophy, Becker's muscular dystrophy, myotonic
dystrophy, central core disease, nemaline myopathy, centronuclear
myopathy, lipid myopathy, mitochondrial myopathy, infectious
myositis, polymyositis, dermatomyositis, inclusion body myositis,
thyrotoxic myopathy, and ethanol myopathy.
[0145] In another embodiment, a vector capable of expressing HUFA-2
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a cardiac or skeletal muscle disorder
including, but not limited to, those described above.
[0146] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUFA-2 in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a cardiac or skeletal muscle disorder including,
but not limited to, those provided above.
[0147] In still another embodiment, an agonist which modulates the
activity of HUFA-2 may be administered to a subject to treat or
prevent a cardiac or skeletal muscle disorder including, but not
limited to, those listed above.
[0148] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0149] An antagonist of HUFA may be produced using methods which
are generally known in the art. In particular, purified HUFA may be
used to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind HUFA. Antibodies
to HUFA may also be generated using methods that are well known in
the art. Such antibodies may include, but are not limited to,
polyclonal, monoclonal, chimeric, and single chain antibodies, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies (i.e., those which inhibit dimer formation)
are especially preferred for therapeutic use.
[0150] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others may be immunized by
injection with HUFA or with any fragment or oligopeptide thereof
which has immunogenic properties. Depending on the host species,
various adjuvants may be used to increase immunological response.
Such adjuvants include, but are not limited to, Freund's, mineral
gels such as aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable.
[0151] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to HUFA have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 10 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of HUFA amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0152] Monoclonal antibodies to HUFA may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120.) In addition, techniques developed for the production
of "chimeric antibodies," such as the splicing of mouse antibody
genes to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used.
(Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855;
Neuberger, M. S. et al. (1984) Nature 312:604-608; and Takeda, S.
et al. (1985) Nature 314:452-454.) Alternatively, techniques
described for the production of single chain antibodies may be
adapted, using methods known in the art, to produce HUFA-specific
single chain antibodies. Antibodies with related specificity, but
of distinct idiotypic composition, may be generated by chain
shuffling from random combinatorial immunoglobulin libraries.
(Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci.
88:11120-11123.)
[0153] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86: 3833-3837, and Winter, G. et al. (1991)
Nature 349:293-299.)
[0154] Antibody fragments which contain specific binding sites for
HUFA may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (Huse, W. D. et al. (1989) Science
254:1275-1281.)
[0155] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between HUFA and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering HUFA epitopes
is preferred, but a competitive binding assay may also be employed.
(Maddox, supra.)
[0156] In another embodiment of the invention, the polynucleotides
encoding HUFA, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding HUFA may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding HUFA. Thus, complementary molecules or
fragments may be used to modulate HUFA activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding HUFA.
[0157] Expression vectors derived from retroviruses, adenoviruses,
or herpes or vaccinia viruses, or from various bacterial plasmids,
may be used for delivery of nucleotide sequences to the targeted
organ, tissue, or cell population. Methods which are well known to
those skilled in the art can be used to construct vectors which
will express nucleic acid sequence complementary to the
polynucleotides of the gene encoding HUFA. These techniques are
described, for example, in Sambrook (supra) and in Ausubel
(supra).
[0158] Genes encoding HUFA can be turned off by transforming a cell
or tissue with expression vectors which express high levels of a
polynucleotide or fragment thereof encoding HUFA. Such constructs
may be used to introduce untranslatable sense or antisense
sequences into a cell. Even in the absence of integration into the
DNA, such vectors may continue to transcribe RNA molecules until
they are disabled by endogenous nucleases. Transient expression may
last for a month or more with a non-replicating vector, and may
last even longer if appropriate replication elements are part of
the vector system.
[0159] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding HUFA. Oligonucleotides derived from
the transcription initiation site, e.g., between about positions
-10 and +10 from the start site, are preferred. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (Gee, J. E. et al. (1994) in
Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,
pp. 163-177, Futura Publishing Co., Mt. Kisco, N.Y.) A
complementary sequence or antisense molecule may also be designed
to block translation of mRNA by preventing the transcript from
binding to ribosomes.
[0160] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding HUFA.
[0161] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0162] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding HUFA. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA constitutively or inducibly can
be introduced into cell lines, cells, or tissues.
[0163] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or
2'O-methyl rather than phosphodiesterase linkages within the
backbone of the molecule. This concept is inherent in the
production of PNAs and can be extended in all of these molecules by
the inclusion of nontraditional bases such as inosine, queosine,
and wybutosine, as well as acetyl-, methyl-, thio-, and similarly
modified forms of adenine, cytidine, guanine, thymine, and uridine
which are not as easily recognized by endogenous endonucleases.
[0164] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art, such as
those described in Goldman, C. K. et al. (1997; Nature
Biotechnology 15:462-466).
[0165] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0166] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of HUFA, antibodies to HUFA, and mimetics,
agonists, antagonists, or inhibitors of HUFA. The compositions may
be administered alone or in combination with at least one other
agent, such as a stabilizing compound, which may be administered in
any sterile, biocompatible pharmaceutical carrier including, but
not limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0167] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0168] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0169] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0170] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0171] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0172] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0173] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks's solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0174] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0175] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0176] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acids. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0177] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of HUFA, such
labeling would include amount, frequency, and method of
administration.
[0178] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art.
[0179] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays of neoplastic
cells, for example, or in animal models, usually mice, rabbits,
dogs, or pigs. An animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0180] A therapeutically effective dose refers to that amount of
active ingredient, for example HUFA or fragments thereof,
antibodies of HUFA, and agonists, antagonists or inhibitors of
HUFA, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED50 (the dose therapeutically effective in 50%
of the population) or LD50 (the dose lethal to 50% of the
population) statistics. The dose ratio of toxic to therapeutic
effects is the therapeutic index, which can be expressed as the
LD.sub.50/ED.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies is used in formulating
a range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage form
employed, the sensitivity of the patient, and the route of
administration.
[0181] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on the half-life and clearance rate of the particular
formulation.
[0182] Normal dosage amounts may vary from 0.1 .mu.g to 100,000
.mu.g, up to a total dose of about 1 gram, depending upon the route
of administration. Guidance as to particular dosages and methods of
delivery is provided in the literature and generally available to
practitioners in the art. Those skilled in the art will employ
different formulations for nucleotides than for proteins or their
inhibitors. Similarly, delivery of polynucleotides or polypeptides
will be specific to particular cells, conditions, locations,
etc.
[0183] Diagnostics
[0184] In another embodiment, antibodies which specifically bind
HUFA may be used for the diagnosis of disorders characterized by
expression of HUFA, or in assays to monitor patients being treated
with HUFA or agonists, antagonists, and inhibitors of HUFA.
Antibodies useful for diagnostic purposes may be prepared in the
same manner as those described above for therapeutics. Diagnostic
assays for HUFA include methods which utilize the antibody and a
label to detect HUFA in human body fluids or in extracts of cells
or tissues. The antibodies may be used with or without
modification, and may be labeled by covalent or non-covalent
joining with a reporter molecule. A wide variety of reporter
molecules, several of which are described above, are known in the
art and may be used.
[0185] A variety of protocols for measuring HUFA, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of HUFA expression. Normal or
standard values for HUFA expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to HUFA under conditions suitable
for complex formation. The amount of standard complex formation may
be quantified by various methods, preferably by photometric means.
Quantities of HUFA expressed in subject, control, and disease
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0186] In another embodiment of the invention, the polynucleotides
encoding HUFA may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HUFA may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
HUFA, and to monitor regulation of HUFA levels during therapeutic
intervention.
[0187] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HUFA or closely related molecules may be used
to identify nucleic acid sequences which encode HUFA. The
specificity of the probe, whether it is made from a highly specific
region (e.g., the 5' regulatory region) or from a less specific
region (e.g., the 3' coding region), and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding HUFA, alleles, or related
sequences.
[0188] Probes may also be used for the detection of related
sequences, and should preferably contain at least 50% of the
nucleotides from any of the HUFA encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
may be derived from the sequences of SEQ ID NO:2 and SEQ ID NO:4 or
from genomic sequences including promoter and enhancer elements and
introns of the naturally occurring HUFA.
[0189] Means for producing specific hybridization probes for DNAs
encoding HUFA include the cloning of polynucleotide sequences
encoding HUFA or HUFA derivatives into vectors for the production
of mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32p or 35S, or by enzymatic labels, such
as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0190] Polynucleotide sequences encoding HUFA may be used for the
diagnosis of a disorder associated with expression of HUFA.
Examples of such disorders include, but are not limited to, genetic
disorders, such as adrenoleukodystrophy, Alport's syndrome,
choroideremia, Duchenne and Becker muscular dystrophy, Down's
syndrome, cystic fibrosis, chronic granulomatous disease, Gaucher's
disease, Huntington's chorea, Marfan's syndrome, muscular
dystrophy, myotonic dystrophy, pycnodysostosis, Refsum's syndrome,
retinoblastoma, sickle cell anemia, thalassemia, Werner syndrome,
von Willebrand's disease, Wilm's tumor, Zellweger syndrome,
peroxisomal acyl-CoA oxidase deficiency, peroxisomal thiolase
deficiency, peroxisomal bifunctional protein deficiency,
mitochondrial carnitine palmitoyl transferase and carnitine
deficiency, mtiochondrial very-long-chain acyl-CoA dehydrogenase
deficiency, mitochondrial medium-chain acyl-CoA dehydrogenase
deficiency, mitochondrial short-chain acyl-CoA dehydrogenase
deficiency, mitochondrial electron transport flavoprotein and
electron transport flavoprotein:ubiquinone oxidoreductase
deficiency, mitochondrial trifunctional protein deficiency, and
mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase
deficiency.
[0191] Polynucleotide sequences encoding HUFA-1 may be used for the
diagnosis of a disorder associated with expression of HUFA-1.
Examples of such disorders include, but are not limited to,
neuronal disorders, such as akathesia, Alzheimer's disease,
amnesia, amyotrophic lateral sclerosis, bipolar disorder,
catatonia, cerebral neoplasms, dementia, depression, diabetic
neuropathy, Down's syndrome, tardive dyskinesia, dystonias,
epilepsy, Huntington's disease, multiple sclerosis,
neurofibromatosis, Parkinson's disease, paranoid psychoses,
postherpetic neuralgia, schizophrenia, and Tourette's disorder; 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.
[0192] Polynucleotide sequences encoding HUFA-2 may be used for the
diagnosis of a disorder associated with expression of HUFA-2.
Examples of such disorders include, but are not limited to,
infectious diseases, including viral infections: adenoviruses (ARD,
pneumonia), arenaviruses (lymphocytic choriomeningitis),
bunyaviruses (Hantavirus), coronaviruses (pneumonia, chronic
bronchitis), hepadnaviruses (hepatitis), herpesviruses (HSV, VZV,
Epstein-Barr virus, cytomegalovirus), flaviviruses (yellow fever),
orthomyxoviruses (influenza), papillomaviruses (cancer),
paramyxoviruses (measles, mumps), picornoviruses (rhinovirus,
poliovirus, coxsackie-virus), polyomaviruses (BK virus, JC virus),
poxviruses (smallpox), reovirus (Colorado tick fever), retroviruses
(HIV, HTLV), rhabdoviruses (rabies), rotaviruses (gastroenteritis),
and togaviruses (encephalitis, rubella); bacterial infections,
fungal infections, parasitic infections, protozoal infections, and
helminthic infections; liver disorders, including cirrhosis,
jaundice, cholestasis, hereditary hyperbilirubinemia, hepatic
encephalopathy, hepatorenal syndrome, hepatitis, hepatic steatosis,
hemochromatosis, Wilson's disease, alpha,-antitrypsin deficiency,
Reye's syndrome, primary sclerosing cholangitis, liver infarction,
portal vein obstruction and thrombosis, passive congestion,
centrilobular necrosis, peliosis hepatis, hepatic vein thrombosis,
veno-occlusive disease, preeclampsia, eclampsia, acute fatty liver
of pregnancy, intrahepatic cholestasis of pregnancy, and hepatic
tumors including nodular hyperplasias, adenomas, and carcinomas;
and cardiac and skeletal muscle disorders, including
cardiomyopathy, myocarditis, Duchenne's muscular dystrophy,
Becker's muscular dystrophy, myotonic dystrophy, central core
disease, nemaline myopathy, centronuclear myopathy, lipid myopathy,
mitochondrial myopathy, infectious myositis, polymyositis,
dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and
ethanol myopathy.
[0193] The polynucleotide sequences encoding HUFA may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; in dipstick, pin, and ELISA
assays; and in microarrays utilizing fluids or tissues from patient
biopsies to detect altered HUFA expression. Such qualitative or
quantitative methods are well known in the art.
[0194] In a particular aspect, the nucleotide sequences encoding
HUFA may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding HUFA may be labeled by standard methods and
added to a fluid or tissue sample from a patient under conditions
suitable for the formation of hybridization complexes. After a
suitable incubation period, the sample is washed and the signal is
quantitated and compared with a standard value. If the amount of
signal in the patient sample is significantly altered from that of
a comparable control sample, the nucleotide sequences have
hybridized with nucleotide sequences in the sample, and the
presence of altered levels of nucleotide sequences encoding HUFA in
the sample indicates the presence of the associated disorder. Such
assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies, in clinical
trials, or in monitoring the treatment of an individual
patient.
[0195] In order to provide a basis for the diagnosis of a disorder
associated with expression of HUFA, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
encoding HUFA, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for a disorder. Deviation from
standard values is used to establish the presence of a
disorder.
[0196] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to evaluate whether the level of
expression in the patient begins to approximate that which is
observed in the normal subject. The results obtained from
successive assays may be used to show the efficacy of treatment
over a period ranging from several days to months.
[0197] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0198] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HUFA may involve the use of PCR. These
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably contain a fragment
of a polynucleotide encoding HUFA, or a fragment of a
polynucleotide complementary to the polynucleotide encoding HUFA,
and will be employed under optimized conditions for identification
of a specific gene or condition. Oligomers may also be employed
under less stringent conditions for detection or quantitation of
closely related DNA or RNA sequences.
[0199] Methods which may also be used to quantitate the expression
of HUFA include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (Melby, P. C. et al. (1993) J.
Immunol. Methods 159:235-244, and Duplaa, C. et al. (1993) Anal.
Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in an ELISA format
where the oligomer of interest is presented in various dilutions
and a spectrophotometric or colorimetric response gives rapid
quantitation.
[0200] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously (to produce a transcript image) and to identify
genetic variants, mutations, and polymorphisms. This information
may be used in determining gene function, in understanding the
genetic basis of a disorder, in diagnosing a disorder, and in
developing and monitoring the activities of therapeutic agents.
[0201] In one embodiment, the microarray is prepared and used
according to methods known in the art, such as those described in
published PCT application WO95/11995 (Chee et al.), Lockhart, D. J.
et al. (1996; Nat. Biotech. 14:1675-1680), and Schena, M. et al.
(1996; Proc. Natl. Acad. Sci. 93:10614-10619).
[0202] The microarray is preferably composed of a large number of
unique single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6 to
60 nucleotides in length, more preferably about 15 to 30
nucleotides in length, and most preferably about 20 to 25
nucleotides in length. For a certain type of microarray, it may be
preferable to use oligonucleotides which are about 7 to 10
nucleotides in length. The microarray may contain oligonucleotides
which cover the known 5' or 3' sequence, or may contain sequential
oligonucleotides which cover the full length sequence or unique
oligonucleotides selected from particular areas along the length of
the sequence. Polynucleotides used in the microarray may be
oligonucleotides specific to a gene or genes of interest in which
at least a fragment of the sequence is known or oligonucleotides
specific to one or more unidentified cDNAs common to a particular
cell or tissue type or to a normal, developmental, or disease
state. In certain situations, it may be appropriate to use pairs of
oligonucleotides on a microarray. The pairs will be identical,
except for one nucleotide preferably located in the center of the
sequence. The second oligonucleotide in the pair (mismatched by
one) serves as a control. The number of oligonucleotide pairs may
range from about 2 to 1,000,000.
[0203] In order to produce oligonucleotides to a known sequence for
a microarray, the gene of interest is examined using a computer
algorithm which starts at the 5' end, or, more preferably, at the
3' end of the nucleotide sequence. The algorithm identifies
oligomers of defined length that are unique to the gene, have a GC
content within a range suitable for hybridization, and lack
predicted secondary structure that may interfere with
hybridization. In one aspect, the oligomers are synthesized at
designated areas on a substrate using a light-directed chemical
process. The substrate may be paper, nylon, any other type of
membrane, filter, chip, glass slide, or any other suitable solid
support.
[0204] In one aspect, the oligonucleotides may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, such as that described in
published PCT application WO95/251116 (Baldeschweiler et al.). In
another aspect, a grid array analogous to a dot or slot blot
(HYBRIDOT apparatus, GIBCO/BRL) may be used to arrange and link
cDNA fragments or oligonucleotides to the surface of a substrate
using a vacuum system or thermal, UV, mechanical or chemical
bonding procedures. In yet another aspect, an array may be produced
by hand or by using available devices, materials, and machines
(including Brinkmann multichannel pipettors or robotic
instruments), and may contain 8, 24, 96, 384, 1536, or 6144
oligonucleotides, or any other multiple from 2 to 1,000,000 which
lends itself to the efficient use of commercially available
instrumentation.
[0205] In order to conduct sample analysis using the microarrays,
polynucleotides are extracted from a biological sample. The
biological samples may be obtained from any bodily fluid (blood,
urine, saliva, phlegm, gastric juices, etc.), cultured cells,
biopsies, or other tissue preparations. To produce probes, the
polynucleotides extracted from the sample are used to produce
nucleic acid sequences which are complementary to the nucleic acids
on the microarray. If the microarray consists of cDNAs, antisense
RNAs (aRNA) are appropriate probes. Therefore, in one aspect, mRNA
is used to produce cDNA which, in turn and in the presence of
fluorescent nucleotides, is used to produce fragment or
oligonucleotide aRNA probes. These fluorescently labeled probes are
incubated with the microarray so that the probe sequences hybridize
to the cDNA oligonucleotides of the microarray. In another aspect,
nucleic acid sequences used as probes can include polynucleotides,
fragments, and complementary or antisense sequences produced using
restriction enzymes, PCR technologies, and Oligolabeling or
TransProbe kits (Pharmacia & Upjohn) well known in the area of
hybridization technology.
[0206] Incubation conditions are adjusted so that hybridization
occurs with precise complementary matches or with various degrees
of less complementarity. After removal of nonhybridized probes, a
scanner is used to determine the levels and patterns of
fluorescence. The scanned images are examined to determine the
degree of complementarity and the relative abundance of each
oligonucleotide sequence on the microarray. A detection system may
be used to measure the absence, presence, and amount of
hybridization for all of the distinct sequences simultaneously.
This data may be used for large scale correlation studies or for
functional analysis of the sequences, mutations, variants, or
polymorphisms among samples. (Heller, R. A. et al. (1997) Proc.
Natl. Acad. Sci. 94:2150-2155.)
[0207] In another embodiment of the invention, nucleic acid
sequences encoding HUFA may be used to generate hybridization
probes useful for mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, such as human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries, such as those reviewed in Price, C. M. (1993; Blood Rev.
7:127-134) and Trask, B. J. (1991; Trends Genet. 7:149-154).
[0208] Fluorescent in situ hybridization (FISH, as described, e.g.,
in Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular
Biology and Biotechnology, pp. 965-968, VCH Publishers New York,
N.Y.) may be correlated with other physical chromosome mapping
techniques and genetic map data. Examples of genetic map data can
be found in various scientific journals or at the Online Mendelian
Inheritance in Man (OMIM) site. Correlation between the location of
the gene encoding HUFA on a physical chromosomal map and a specific
disorder, or predisposition to a specific disorder, may help define
the region of DNA associated with that disorder. The nucleotide
sequences of the subject invention may be used to detect
differences in gene sequences between normal, carrier, and affected
individuals.
[0209] In situ hybridization of chromosomal preparations and
physical mapping techniques, such as linkage analysis using
established chromosomal markers, may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, ataxia-telangiectasia (AT) to 11q22-23
(Gatti, R. A. et al. (1988) Nature 336:577-580), any sequences
mapping to that area may represent associated or regulatory genes
for further investigation. The nucleotide sequence of the subject
invention may also be used to detect differences in the chromosomal
location due to translocation, inversion, etc., among normal,
carrier, or affected individuals.
[0210] In another embodiment of the invention, HUFA, its catalytic
or immunogenic fragments, or oligopeptides thereof can be used for
screening libraries of compounds in any of a variety of drug
screening techniques. The fragment employed in such screening may
be free in solution, affixed to a solid support, borne on a cell
surface, or located intracellularly. The formation of binding
complexes between HUFA and the agent being tested may be
measured.
[0211] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564 (Geysen, et al.). In this
method, large numbers of different small test compounds are
synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with HUFA, or
fragments thereof, and washed. Bound HUFA is then detected by
methods well known in the art. Purified HUFA can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0212] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HUFA specifically compete with a test compound for binding
HUFA. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
HUFA.
[0213] In additional embodiments, the nucleotide sequences which
encode HUFA 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.
[0214] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0215] I. cDNA Library Construction
[0216] BRSTTUT03
[0217] The BRSTTUT03 cDNA library was constructed from cancerous
breast tissue removed from a 58-year-old Caucasian female who had
undergone unilateral extended simple mastectomy following diagnosis
of multicentric invasive grade 4 mammary lobular carcinoma.
[0218] The frozen tissue was homogenized and lysed using a Polytron
PT-3000 homogenizer (Brinkmann Instruments, Westbury, N.J.) in
guanidinium isothiocyanate solution. The lysate was centrifuged
over a 5.7 M CsCl cushion using an SW28 rotor in an L8-70M
ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at
ambient temperature. The RNA was extracted with acid phenol pH 4.0,
precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,
resuspended in RNAse-free water and DNase treated at 37.degree. C.
The RNA extraction and precipitation were repeated as before. The
mRNA was then isolated using the OLIGOTEX kit (QIAGEN Inc.,
Chatsworth, Calif.) and used to construct the cDNA library.
[0219] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system for cDNA synthesis and plasmid
cloning (Catalog #18248-013; Gibco/BRL), cDNAs were fractionated on
a SEPHAROSE CL4B column (Catalog #275105-01; Pharmacia), and those
cDNAs exceeding 400 bp were ligated into PSPORT1. The plasmid
PSPORT1 was subsequently transformed into DH5.alpha. competent
cells (Catalog #18258-012; Gibco/BRL).
[0220] OVARTUT02
[0221] The OVARTUT02 library was constructed from tumorous ovary
tissue obtained from a 51-year-old Caucasian female during a total
abdominal hysterectomy.
[0222] The frozen tissue was homogenized and lysed in TRIZOL
reagent (1 g tissue/10 ml TRIZOL; Catalog #10296-028; Gibco/BRL), a
monoplastic solution of phenol and guanidine isothiocyanate, using
a PT-3000 homogenizer (Brinkmann Instruments, Westbury, N.Y.).
After a brief incubation on ice, chloroform was added (1:5 v/v) and
the lysate was centrifuged. The upper chloroform layer was removed
to a fresh tube and the RNA extracted with isopropanol, resuspended
in DEPC-treated water, and DNase treated for 25 min at 37.degree.
C. The RNA was extracted once with acid phenol-chloroform pH 4.7
and precipitated using 0.3M sodium acetate and 2.5 volumes ethanol.
The mRNA was then isolated using the OLIGOTEX kit (QIAGEN) and used
to construct the cDNA library.
[0223] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system for cDNA Synthesis and plasmid
cloning (Catalog #18248-013, Gibco/BRL). The cDNAs were
fractionated on a SEPHAROSE CL4B column (Catalog #275105-01;
Pharmacia), and those cDNAs exceeding 400 bp were ligated into
pINCY 1 (Incyte). The plasmid pINCY 1 was subsequently transformed
into DH5a competent cells (Catalog #18258-012; Gibco/BRL).
[0224] II. Isolation and Sequencing of cDNA Clones
[0225] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 plasmid kit (Catalog #26173; QIAGEN). This kit
enables the simultaneous purification of 96 samples in a 96-well
block using multi-channel reagent dispensers. The recommended
protocol was employed except for the following changes: 1) the
bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog
#22711, Gibco/BRL) with carbenicillin at 25 mg/L and glycerol at
0.4%; 2) after inoculation, the cultures were incubated for 19
hours and at the end of incubation, the cells were 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 storage at 4.degree. C.
[0226] The cDNAs were sequenced by the method of Sanger et al.
(1975; J. Mol. Biol. 94:441f), using a MICROLAB 2200 (Hamilton,
Reno, Nev.) in combination with Peltier PTC200 thermal cyclers (MJ
Research, Watertown, Mass.) and Applied Biosystems 377 DNA
sequencing systems; and the reading frame was determined.
[0227] III. Homology Searching of cDNA Clones and Their Deduced
Proteins
[0228] The nucleotide sequences and/or amino acid sequences of the
Sequence Listing were used to query sequences in the GenBank,
SwissProt, BLOCKS, and Pima II databases. These databases, which
contain previously identified and annotated sequences, were
searched for regions of homology using BLAST (Basic Local Alignment
Search Tool). (Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and
Altschul et al. (1990) J. Mol. Biol. 215:403-410.) BLAST produced
alignments of both nucleotide and amino acid sequences to determine
sequence similarity. Because of the local nature of the alignments,
BLAST was especially useful in determining exact matches or in
identifying homologs which may be of prokaryotic (bacterial) or
eukaryotic (animal, fungal, or plant) origin. Other algorithms such
as the one described in Smith, T. et al. (1992; Protein Engineering
5:35-51), could have been used when dealing with primary sequence
patterns and secondary structure gap penalties. The sequences
disclosed in this application have lengths of at least 49
nucleotides and have no more than 12% uncalled bases (where N is
recorded rather than A, C, G, or T).
[0229] The BLAST approach searched for matches between a query
sequence and a database sequence. BLAST evaluated the statistical
significance of any matches found, and reported only those matches
that satisfy the user-selected threshold of significance. In this
application, threshold was set at 10-25 for nucleotides and
10.sup.-10 for peptides.
[0230] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and other
mammalian sequences (mam), and deduced amino acid sequences from
the same clones were then searched against GenBank functional
protein databases, mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp), for homology.
[0231] IV. Northern Analysis
[0232] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and 16.)
[0233] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as the GenBank or the LIFESEQ database (Incyte
Pharmaceuticals). This analysis is much faster than multiple
membrane-based hybridizations. In addition, the sensitivity of the
computer search can be modified to determine whether any particular
match is categorized as exact or homologous.
[0234] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0235] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Homologous molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0236] The results of northern analysis are reported as a list of
libraries in which the transcript encoding HUFA occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0237] V. Extension of HUFA Encoding Polynucleotides
[0238] The nucleic acid sequences of Incyte Clones 1995961 and
2595635 were used to design oligonucleotide primers for extending
partial nucleotide sequences to full length. For each nucleic acid
sequence, one primer was synthesized to initiate extension of an
antisense polynucleotide, and the other was synthesized to initiate
extension of a sense polynucleotide. Primers were used to
facilitate the extension of the known sequence "outward" generating
amplicons containing new unknown nucleotide sequence for the region
of interest. The initial primers were designed from the cDNA using
OLIGO 4.06 software (National Biosciences), or another appropriate
program, to be about 22 to 30 nucleotides in length, to have a GC
content of about 50% or more, and to anneal to the target sequence
at temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0239] Selected human cDNA libraries (GIBCO/BRL) were used to
extend the sequence. If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0240] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
Peltier PTC200 thermal cycler (MJ Research, Watertown, Mass.),
beginning with 40 pmol of each primer and the recommended
concentrations of all other components of the kit, with the
following parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0241] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK
(QIAGEN), and trimmed of overhangs using Klenow enzyme to
facilitate religation and cloning.
[0242] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (Sambrook, supra, Appendix A,
p. 2.) After incubation for one hour at 37.degree. C., the E. coli
mixture was plated on Luria Bertani (LB) agar (Sambrook, supra,
Appendix A, p. 1) containing 2.times.Carb. The following day,
several colonies were randomly picked from each plate and cultured
in 150 .mu.l of liquid LB/2.times.Carb medium placed in an
individual well of an appropriate commercially-available sterile
96-well plate. The following day, 5 .mu.l of each overnight culture
was transferred into a non-sterile 96-well plate and, after
dilution 1:10 with water, 5 .mu.l from each sample was transferred
into a PCR array.
[0243] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0244] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0245] In like manner, the nucleotide sequences of SEQ ID NO:2 and
SEQ ID NO:4 are used to obtain 5' regulatory sequences using the
procedure above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0246] VI. Labeling and Use of Individual Hybridization Probes
[0247] Hybridization probes derived from SEQ ID NO:2 and SEQ ID
NO:4 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although
the labeling of oligonucleotides, consisting of about 20 base
pairs, is specifically described, essentially the same procedure is
used with larger nucleotide fragments. Oligonucleotides are
designed using state-of-the-art software such as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of
each oligomer and 250 .mu.Ci of [.gamma.-.sup.32p] adenosine
triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN,
Boston, Mass.). The labeled oligonucleotides are substantially
purified using a SEPHADEX G-25 superfine resin column (Pharmacia
& Upjohn). An aliquot containing 10.sup.7 counts per minute of
the labeled probe is used in a typical membrane-based hybridization
analysis of human genomic DNA digested with one of the following
endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II
(DuPont NEN).
[0248] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (NYTRAN Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1 .times.saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film
(Kodak, Rochester, N.Y.) is exposed to the blots or the blots are
exposed to a Phosphorlmager cassette (Molecular Dynamics,
Sunnyvale, Calif.), hybridization patterns are compared
visually.
[0249] VII. Microarrays
[0250] To produce oligonucleotides for a microarray, one of the
nucleotide sequences of the present invention is examined using a
computer algorithm which starts at the 3' end of the nucleotide
sequence. The algorithm identifies oligomers of defined length that
are unique to the gene, have a GC content within a range suitable
for hybridization, and lack predicted secondary structure that
would interfere with hybridization. The algorithm identifies
approximately 20 sequence-specific oligonucleotides of 20
nucleotides in length (20-mers). A matched set of oligonucleotides
are created in which one nucleotide in the center of each sequence
is altered. This process is repeated for each gene in the
microarray, and double sets of twenty 20-mers are synthesized and
arranged on the surface of the silicon chip using a light-directed
chemical process, such as that described in Chee (supra).
[0251] In the alternative, a chemical coupling procedure and an ink
jet device are used to synthesize oligomers on the surface of a
substrate. (See Baldeschweiler, supra.) In another alternative, a
grid array analogous to a dot or slot blot is used to arrange and
link cDNA fragments or oligonucleotides to the surface of a
substrate using a vacuum system or thermal, UV, mechanical, or
chemical bonding procedures. A typical array may be produced by
hand or using available materials and machines and contain grids of
8 dots, 24 dots, 96 dots, 384 dots, 1536 dots, or 6144 dots. After
hybridization, the microarray is washed to remove nonhybridized
probes, and a scanner is used to determine the levels and patterns
of fluorescence. The scanned image is examined to determine the
degree of complementarity and the relative abundance/expression
level of each oligonucleotide sequence in the microarray.
[0252] VIII. Complementary Polynucleotides
[0253] Sequences complementary to the HUFA-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring HUFA. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 software and the coding sequence of HUFA.
To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the HUFA-encoding transcript.
[0254] IX. Expression of HUFA
[0255] Expression of HUFA is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector is also used to express
HUFA in E. coli. This vector contains a promoter for
.beta.-galactosidase upstream of the cloning site, followed by
sequence containing the amino-terminal Met and the subsequent seven
residues of .beta.-galactosidase. Immediately following these eight
residues is a bacteriophage promoter useful for transcription and a
linker containing a number of unique restriction sites.
[0256] Induction of an isolated, transformed bacterial strain with
isopropyl beta-D-thiogalactopyranoside (IPTG) using standard
methods produces a fusion protein which consists of the first 8
residues of .beta.-galactosidase, about 5 to 15 residues of linker,
and the full length protein. The signal residues direct the
secretion of HUFA into bacterial growth media which can be used
directly in the following assay for activity.
[0257] X. Demonstration of HUFA Activity
[0258] HUFA-1
[0259] HUFA-1 is assayed by measuring the disappearance of
5-phenyl-2,4-pentadienoyl-CoA and NADPH, which absorb at 340 nm.
(Nada, M. A. et al. (1994) Lipids 29:517-521.) A solution of 0.2 M
potassium phosphate (pH 8), 0.1 mM NADPH, and 25 .mu.M
5-phenyl-2,4,-pentadienoyl-C- oA is mixed in an optical cuvette.
The assay is started by the addition of sample to the cuvette. The
change in absorbance at 340 nm is measured using an ultraviolet
spectrophotometer. The amount of HUFA-1 in the sample is
proportional to the decrease in absorbance at 340 nm at 25.degree.
C.
[0260] HUFA-2
[0261] HUFA-2 is assayed in a coupled assay measuring the hydration
and subsequent dehydrogenation of either a short-chain enoyl-CoA
(crotonyl-CoA) or a long-chain enoyl-CoA (trans-2-dodecenoyl-CoA).
(Wanders, R. J. A. et al. (1992) Biochem. Biophys. Res. Commun.
188:1139-1145.) The dehydrogenation reaction converts the NAD
analog acetylpyridine adenine dinucleotide (APAD) to the reduced
form APADH, which absorbs at 365 nm. A solution of 100 mM Tris-HCl
(pH 8), 1 mM APAD, 0.1% (w/v) Triton X-100, 7.1 units/ml
3-hydroxyacyl-CoA dehydrogenase from pig heart, and sample is mixed
in an optical cuvette and incubated for two minutes at 37.degree.
C. Crotonyl-CoA or trans-2-dodecenoyl-CoA at a final concentration
of 100 .mu.M is added to start the reaction. The change in
absorbance at 365 nm is measured using an ultraviolet
spectrophotometer. The amount of HUFA-2 in the sample is
proportional to the increase in absorbance at 365 nm at 37.degree.
C.
[0262] XI. Production of HUFA Specific Antibodies
[0263] HUFA substantially purified using PAGE electrophoresis
(Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or other
purification techniques, is used to immunize rabbits and to produce
antibodies using standard protocols. The HUFA amino acid sequence
is analyzed using LASERGENE software (DNASTAR Inc.) to determine
regions of high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions, is described by
Ausubel F. M. et al. (1995 and periodic supplements) Current
Protocols in Molecular Biology, ch. 11, John Wiley & Sons, New
York, N.Y. and by others.
[0264] Typically, the oligopeptides are 15 residues in length, and
are synthesized using an Applied Biosystems 431A peptide
synthesizer using fmoc-chemistry and coupled to KLH (Sigma, St.
Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), following the
procedure described in Ausubel et al., supra. Rabbits are immunized
with the oligopeptide-KLH complex in complete Freund's adjuvant.
Resulting antisera are tested for antipeptide activity, for
example, by binding the peptide to plastic, blocking with 1% BSA,
reacting with rabbit antisera, washing, and reacting with
radio-iodinated goat anti-rabbit IgG.
[0265] XII. Purification of Naturally Occurring HUFA Using Specific
Antibodies
[0266] Naturally occurring or recombinant HUFA is substantially
purified by immunoaffinity chromatography using antibodies specific
for HUFA. An immunoaffinity column is constructed by covalently
coupling HUFA antibody to an activated chromatographic resin, such
as CNBr-activated SEPHAROSE (Pharmacia & Upjohn). After the
coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0267] Media containing HUFA are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of HUFA (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/HUFA binding (e.g., a buffer of pH
2 to pH 3, or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and HUFA is collected.
[0268] XIII. Identification of Molecules Which Interact with
HUFA
[0269] HUFA or biologically active fragments thereof are labeled
with 1251 Bolton-Hunter reagent. (Bolton et al. (1973) Biochem. J.
133:529.) Candidate molecules previously arrayed in the wells of a
multi-well plate are incubated with the labeled HUFA, washed, and
any wells with labeled HUFA complex are assayed. Data obtained
using different concentrations of HUFA are used to calculate values
for the number, affinity, and association of HUFA with the
candidate molecules.
[0270] Various modifications and variations of the described
methods and systems of the invention will be apparent to those
skilled in the art without departing from the scope and spirit of
the invention. Although the invention has been described in
connection with specific preferred embodiments, it should be
understood that the invention as claimed should not be unduly
limited to such specific embodiments. Indeed, various modifications
of the described modes for carrying out the invention which are
obvious to those skilled in molecular biology or related fields are
intended to be within the scope of the following claims.
Sequence CWU 1
1
10 1 303 PRT Homo sapiens misc_feature Incyte ID No 1995961 1 Met
Ala Ser Trp Ala Lys Gly Arg Ser Tyr Leu Ala Pro Gly Leu 1 5 10 15
Leu Gln Gly Gln Val Ala Ile Val Thr Gly Gly Ala Thr Gly Ile 20 25
30 Gly Lys Ala Ile Val Lys Glu Leu Leu Glu Leu Gly Ser Asn Val 35
40 45 Val Ile Ala Ser Arg Lys Leu Glu Arg Leu Lys Ser Ala Ala Asp
50 55 60 Glu Leu Gln Ala Asn Leu Pro Pro Thr Lys Gln Ala Arg Val
Ile 65 70 75 Pro Ile Gln Cys Asn Ile Arg Asn Glu Glu Glu Val Asn
Asn Leu 80 85 90 Val Lys Ser Thr Leu Asp Thr Phe Gly Lys Ile Asn
Phe Leu Val 95 100 105 Asn Asn Gly Gly Gly Gln Phe Leu Ser Pro Ala
Glu His Ile Ser 110 115 120 Ser Lys Gly Trp His Ala Val Leu Glu Thr
Asn Leu Thr Gly Thr 125 130 135 Phe Tyr Met Cys Lys Ala Val Tyr Ser
Ser Trp Met Lys Glu His 140 145 150 Gly Gly Ser Ile Val Asn Ile Ile
Val Pro Thr Lys Ala Gly Phe 155 160 165 Pro Leu Ala Val His Ser Gly
Ala Ala Arg Ala Gly Val Tyr Asn 170 175 180 Leu Thr Lys Ser Leu Ala
Leu Glu Trp Ala Cys Ser Gly Ile Arg 185 190 195 Ile Asn Cys Val Ala
Pro Gly Val Ile Tyr Ser Gln Thr Ala Val 200 205 210 Glu Asn Tyr Gly
Ser Trp Gly Gln Ser Phe Phe Glu Gly Ser Phe 215 220 225 Gln Lys Ile
Pro Ala Lys Arg Ile Gly Val Pro Glu Glu Val Ser 230 235 240 Ser Val
Val Cys Phe Leu Leu Ser Pro Ala Ala Ser Phe Ile Thr 245 250 255 Gly
Gln Ser Val Asp Val Asp Gly Gly Arg Ser Leu Tyr Thr His 260 265 270
Ser Tyr Glu Val Pro Asp His Asp Asn Trp Pro Lys Gly Ala Gly 275 280
285 Asp Leu Ser Val Val Lys Lys Met Lys Glu Thr Phe Lys Glu Lys 290
295 300 Ala Lys Leu 2 1210 DNA Homo sapiens misc_feature Incyte ID
No 1995961 2 nncctgagac ccagaagggc ctgcgctcag ctgctctggc accccgctgc
agggatggcc 60 tcctgggcta agggcaggag ctacctggcg cctggtttgc
tgcagggcca agtggccatc 120 gtcaccggcg gggccacggg catcggaaaa
gccatcgtga aggagctcct ggagctgggg 180 agtaatgtgg tcattgcatc
ccgtaagttg gagagattga agtctgcggc agatgaactg 240 caggccaacc
tacctcccac aaagcaggca cgagtcattc ccatacaatg caacatccgg 300
aatgaggagg aggtgaataa tttggtcaaa tctaccttag atacttttgg taagatcaat
360 ttcttggtga acaatggagg aggccagttt ctttcccctg ctgaacacat
cagttctaag 420 ggatggcacg ctgtgcttga gaccaacctg acgggtacct
tctacatgtg caaagcagtt 480 tacagctcct ggatgaaaga gcatggagga
tctatcgtca atatcattgt ccctactaaa 540 gctggatttc cattagctgt
gcattctgga gctgcaagag caggtgttta caacctcacc 600 aaatctttag
ctttggaatg ggcctgcagt ggaatacgga tcaattgtgt tgcccctgga 660
gttatttatt cccagactgc tgtggagaac tatggttcct ggggacaaag cttctttgaa
720 gggtcttttc agaaaatccc cgctaaacga attggtgttc ctgaggaggt
ctcctctgtg 780 gtctgcttcc tactgtctcc tgcagcttcc ttcatcactg
gacagtcggt ggatgtggat 840 gggggccgga gtctctatac tcactcgtat
gaggtaccag atcatgacaa ctggcccaag 900 ggagcagggg acctttctgt
tgtcaaaaag atgaaggaga cctttaagga gaaagctaag 960 ctctgagctg
aggaaacaag gtgtcctcca tcccccagtg ccttcacatc ttgaggatat 1020
gcttctgtac tttttaaaag cttatagttg gtatggaaaa catttttctt atttttaagt
1080 gttattaatt atatctatgg aaaaactatt cctgaaatat atacagtctt
atgtcgcaat 1140 cagagtcttt taacctatga tttaagaatg tataagtaac
agagattaac atattttaat 1200 gactttactc 1210 3 301 PRT Homo sapiens
misc_feature Incyte ID No 2595635 3 Met Ala Lys Ser Leu Leu Lys Thr
Ala Ser Leu Ser Gly Arg Thr 1 5 10 15 Lys Leu Leu His Gln Thr Gly
Leu Ser Leu Tyr Ser Thr Ser His 20 25 30 Gly Phe Tyr Glu Glu Glu
Val Lys Lys Thr Leu Gln Gln Phe Pro 35 40 45 Gly Gly Ser Ile Asp
Leu Gln Lys Glu Asp Asn Gly Ile Gly Ile 50 55 60 Leu Thr Leu Asn
Asn Pro Ser Arg Met Asn Ala Phe Ser Gly Val 65 70 75 Met Met Leu
Gln Leu Leu Glu Lys Val Ile Glu Leu Glu Asn Trp 80 85 90 Thr Glu
Gly Lys Gly Leu Ile Val Arg Gly Ala Lys Asn Thr Phe 95 100 105 Ser
Ser Gly Ser Asp Leu Asn Ala Val Lys Ser Leu Gly Thr Pro 110 115 120
Glu Asp Gly Met Ala Val Cys Met Phe Met Gln Asn Thr Leu Thr 125 130
135 Arg Phe Met Arg Leu Pro Leu Ile Ser Val Ala Leu Val Gln Gly 140
145 150 Trp Ala Leu Gly Gly Gly Ala Glu Phe Thr Thr Ala Cys Asp Phe
155 160 165 Arg Leu Met Thr Pro Glu Ser Lys Ile Arg Phe Val His Lys
Glu 170 175 180 Met Gly Ile Ile Pro Ser Trp Gly Gly Thr Thr Arg Leu
Val Glu 185 190 195 Ile Ile Gly Ser Arg Gln Ala Leu Lys Val Leu Ser
Gly Ala Leu 200 205 210 Lys Leu Asp Ser Lys Asn Ala Leu Asn Ile Gly
Met Val Glu Glu 215 220 225 Val Leu Gln Ser Ser Asp Glu Thr Lys Ser
Leu Glu Glu Ala Gln 230 235 240 Glu Trp Leu Lys Gln Phe Ile Gln Gly
Pro Pro Glu Val Ile Arg 245 250 255 Ala Leu Lys Lys Ser Val Cys Ser
Gly Arg Glu Leu Tyr Leu Glu 260 265 270 Glu Ala Leu Gln Asn Glu Arg
Asp Leu Leu Gly Thr Val Trp Gly 275 280 285 Gly Pro Ala Asn Leu Glu
Ala Ile Ala Lys Lys Gly Lys Phe Asn 290 295 300 Lys 4 2714 DNA Homo
sapiens misc_feature Incyte ID No 2595635 4 naagcacttc ctgtctgcgg
catacaaaat gtatggcacg gaattttaag cttactgagc 60 tttataaaca
cgtcacattc acacattcaa gacacacact ggatattcgg ataaaaacaa 120
acaaacaaaa aacaggctaa atacccattc ccctcaataa cttggataag atacctaaaa
180 aaggtcgact tcggtacctt tctgtcttct cccctctcgc tatttgccta
cactggcttc 240 ctcacctcca ctttttctca cgtttatctg agcgaaaaca
agcacggttc ggcagcctcc 300 tttcccagcc ctacctttgt gctgcaaaag
cgaaaattca aaagccaagt acaataggag 360 accgcccacc ctggctccct
cgtgacacga gggagcgcga agcggagggc gcctcgcggc 420 aggagcggga
tttccggggt cacgggaacc ggcaggggaa cgggataaag ttcctggaga 480
aaggaaagga gagcgtggga tagtaaaaga gaagacgcgg agaagaggag aggacctaca
540 agaacggagg acaggggcgc acgatggtcc cggggggagc ggaaacaaag
gcacgcaaaa 600 cggaaaagcg tgtgtagggg agcggaaaag gaagtcacca
ccgtggcctg cgacgaaatg 660 gcgaaaagtc ttttgaagac agcctctctg
tctggaagga caaaattgct acatcaaaca 720 ggattgtcac tttatagtac
atcccatgga ttttatgagg aagaagtgaa aaaaacactt 780 cagcagtttc
ctggtggatc cattgacctt cagaaggaag acaatggcat tggcattctt 840
actctgaaca atccaagtag aatgaatgcc ttttcaggtg ttatgatgct acaacttctg
900 gaaaaagtaa ttgaattgga aaattggaca gaggggaaag gcctcattgt
ccgtggggca 960 aaaaatactt tctcttcagg atctgatctg aatgctgtga
aatcactagg aactccagag 1020 gatggaatgg ccgtatgcat gttcatgcaa
aacaccttaa caagatttat gagacttcct 1080 ttaataagtg ttgcgctggt
tcaaggttgg gcattgggtg gaggagcaga atttactaca 1140 gcatgtgatt
tcaggttaat gactccagag agtaagatca gattcgtcca caaagagatg 1200
ggcataatac caagctgggg tggcaccacc cggctagttg aaataatcgg aagtagacaa
1260 gctctcaaag tgttgagtgg ggcccttaaa ctggattcaa aaaatgctct
aaacatagga 1320 atggttgaag aggtcttgca gtcttcagat gaaactaaat
ctctagaaga ggcacaagaa 1380 tggctaaagc aattcatcca agggccaccg
gaagtaatta gagctttgaa aaaatctgtt 1440 tgttcaggca gagagctata
tttggaggaa gcattacaga acgaaagaga tcttttagga 1500 acagtttggg
gtgggcctgc aaatttagag gctattgcta agaaaggaaa atttaataaa 1560
taattggttt ttcgtgtgga tgtactccaa gtaaagctcc agtgactaat atgtataaat
1620 gttaaatgat attaaatatg aacatcagaa ttactttgaa ggctactatt
aatatgcaga 1680 cttactttta atcatttgaa tatctgaact catttacctc
atttcttgcc aattactcac 1740 ttgggtattt actgcgtaat ctggaacatt
tagctaaaat atacactttt ggcttaaaaa 1800 ttattgctgt caattccaat
aataattctt agcttataac caaagagcag tgtttaaaag 1860 gagagcttct
atacaaaacc tattcctggc gttacttttc atacaatttt tgttctgttt 1920
tacctggaaa taatttacca aaataactga gtgttgctgc taaagaacaa aagtggggag
1980 gtatcaggga acaagaaaac aagaaagggt atgatcaatc attttcttct
gctccaaaca 2040 gctggagtaa aattcatggg aaatggccct tcatttaaaa
aaagatgtac ctcactaccc 2100 actacaaatt tggaactttg ttcttttcaa
taattagttt tctattgtaa attacctact 2160 aaacagtggt agccatgaca
tggaaagtca actgattcta caattggaca ttcatttgtg 2220 tgccctggaa
tttccaacta gtaataaaca actactgttg atgtagtttt aaaccacttg 2280
aagggactca tgaagcatcc tgcaacataa atttgcattt ttacatcaga tttctttttt
2340 ttcctgaaaa acaactaacc ttctaacaac tatctttcaa aagtaaatgt
aataaaaatg 2400 cacaacataa aatgtttatg atcccagcaa tacacttttt
aaaaaatgtg aaagtcaaag 2460 aattaagttc tagttctgac tcatcacaag
aggtcaaaag tatttgctac tgtaacattc 2520 aattcacatt tgagaatcat
ggtaaaaata acttgtattt gccttaccat catgatccta 2580 ctgttgagtt
aggaaaatat ggttagacag actcacatta ctttttttca gaggtaaact 2640
ctagattact gtgtcaaccc aatactattt ggccatagat gtaaaaacta ccaaataaaa
2700 gtggattttg tgtc 2714 5 295 PRT Homo sapiens misc_feature
GenBank ID No g730864 5 Met Asp Thr Met Asn Thr Ala Asn Thr Leu Asp
Gly Lys Phe Val 1 5 10 15 Thr Glu Gly Ser Trp Arg Pro Asp Leu Phe
Lys Gly Lys Val Ala 20 25 30 Phe Val Thr Gly Gly Ala Gly Thr Ile
Cys Arg Val Gln Thr Glu 35 40 45 Ala Leu Val Leu Leu Gly Cys Lys
Ala Ala Ile Val Gly Arg Asp 50 55 60 Gln Glu Arg Thr Glu Gln Ala
Ala Lys Gly Ile Ser Gln Leu Ala 65 70 75 Lys Asp Lys Asp Ala Val
Leu Ala Ile Ala Asn Val Asp Val Arg 80 85 90 Asn Phe Glu Gln Val
Glu Asn Ala Val Lys Lys Thr Val Glu Lys 95 100 105 Phe Gly Lys Ile
Asp Phe Val Ile Ala Gly Ala Ala Gly Asn Phe 110 115 120 Val Cys Asp
Phe Ala Asn Leu Ser Pro Asn Ala Phe Lys Ser Val 125 130 135 Val Asp
Ile Asp Leu Leu Gly Ser Phe Asn Thr Ala Lys Ala Cys 140 145 150 Leu
Lys Glu Leu Lys Lys Ser Lys Gly Ser Ile Leu Phe Val Ser 155 160 165
Ala Thr Phe His Tyr Tyr Gly Val Pro Phe Gln Gly His Val Gly 170 175
180 Ala Ala Lys Ala Gly Ile Asp Ala Leu Ala Lys Asn Leu Ala Val 185
190 195 Glu Leu Gly Pro Leu Gly Ile Arg Ser Asn Cys Ile Ala Pro Gly
200 205 210 Ala Ile Asp Asn Thr Glu Gly Leu Lys Arg Leu Ala Gly Lys
Lys 215 220 225 Tyr Lys Glu Lys Ala Leu Ala Lys Ile Pro Leu Gln Arg
Leu Gly 230 235 240 Ser Thr Arg Asp Ile Ala Glu Ser Thr Val Tyr Ile
Phe Ser Pro 245 250 255 Ala Ala Ser Tyr Val Thr Gly Thr Val Leu Val
Val Asp Gly Gly 260 265 270 Met Trp His Leu Gly Thr Tyr Phe Gly His
Glu Leu Tyr Pro Glu 275 280 285 Ala Leu Ile Lys Ser Met Thr Ser Lys
Leu 290 295 6 335 PRT Homo sapiens misc_feature GenBank ID No
g602703 6 Met Lys Leu Pro Ala Arg Val Phe Phe Thr Leu Gly Ser Arg
Leu 1 5 10 15 Pro Cys Gly Leu Ala Pro Arg Arg Phe Phe Ser Tyr Gly
Thr Lys 20 25 30 Ile Leu Tyr Gln Asn Thr Glu Ala Leu Gln Ser Lys
Phe Phe Ser 35 40 45 Pro Leu Gln Lys Ala Met Leu Pro Pro Asn Ser
Phe Gln Gly Lys 50 55 60 Val Ala Phe Ile Thr Gly Gly Gly Thr Gly
Leu Gly Lys Gly Met 65 70 75 Thr Thr Leu Leu Ser Ser Leu Gly Ala
Gln Cys Val Ile Ala Ser 80 85 90 Arg Lys Met Asp Val Leu Lys Ala
Thr Ala Glu Gln Ile Ser Ser 95 100 105 Gln Thr Gly Asn Lys Val His
Ala Ile Gln Cys Asp Val Arg Asp 110 115 120 Pro Asp Met Val Gln Asn
Thr Val Ser Glu Leu Ile Lys Val Ala 125 130 135 Gly His Pro Asn Ile
Val Ile Asn Asn Ala Ala Gly Asn Phe Ile 140 145 150 Ser Pro Thr Glu
Arg Leu Ser Pro Asn Ala Trp Lys Thr Ile Thr 155 160 165 Asp Ile Val
Leu Asn Gly Thr Ala Phe Val Thr Leu Glu Ile Gly 170 175 180 Lys Gln
Leu Ile Lys Ala Gln Lys Gly Ala Ala Phe Leu Ser Ile 185 190 195 Thr
Thr Ile Tyr Ala Glu Thr Gly Ser Gly Phe Val Val Pro Ser 200 205 210
Ala Ser Ala Lys Ala Gly Val Glu Ala Met Ser Lys Ser Leu Ala 215 220
225 Ala Glu Trp Gly Lys Tyr Gly Met Arg Phe Asn Val Ile Gln Pro 230
235 240 Gly Pro Ile Lys Thr Lys Gly Ala Phe Ser Arg Leu Asp Pro Thr
245 250 255 Gly Thr Phe Glu Lys Glu Met Ile Gly Arg Ile Pro Cys Gly
Arg 260 265 270 Leu Gly Thr Val Glu Glu Leu Ala Asn Leu Ala Ala Phe
Leu Cys 275 280 285 Ser Asp Tyr Ala Ser Trp Ile Asn Gly Ala Val Ile
Lys Phe Asp 290 295 300 Gly Gly Glu Glu Val Leu Ile Ser Gly Glu Phe
Asn Asp Leu Arg 305 310 315 Lys Val Thr Lys Glu Gln Trp Asp Thr Ile
Glu Glu Leu Ile Arg 320 325 330 Lys Thr Lys Gly Ser 335 7 335 PRT
Homo sapiens misc_feature GenBank ID No g111287 7 Met Ala Leu Leu
Ala Arg Ala Phe Phe Ala Gly Val Ser Arg Leu 1 5 10 15 Pro Cys Asp
Pro Gly Pro Gln Arg Phe Phe Ser Phe Gly Thr Lys 20 25 30 Thr Leu
Tyr Gln Ser Ile Asp Ala Pro Gln Ser Lys Phe Phe Pro 35 40 45 Pro
Ile Leu Lys Pro Met Leu Pro Pro Asn Ala Phe Gln Gly Lys 50 55 60
Val Ala Phe Ile Thr Gly Gly Gly Thr Gly Leu Gly Lys Ala Met 65 70
75 Thr Thr Phe Leu Ser Ser Leu Gly Ala Gln Cys Val Ile Ala Ser 80
85 90 Arg Asn Ile Asp Val Leu Lys Ala Thr Ala Glu Glu Ile Thr Ser
95 100 105 Lys Thr Gly Asn Lys Val Tyr Ala Ile Arg Cys Asp Val Arg
Asp 110 115 120 Pro Asp Met Val His Asn Thr Val Leu Glu Leu Ile Lys
Val Ala 125 130 135 Gly His Pro Asp Val Val Ile Asn Asn Ala Ala Gly
Asn Phe Ile 140 145 150 Ser Pro Ser Glu Arg Leu Ser Pro Asn Gly Trp
Lys Thr Ile Thr 155 160 165 Asp Ile Val Leu Asn Gly Thr Ala Tyr Val
Thr Ile Glu Ile Gly 170 175 180 Lys Gln Leu Ile Lys Ala Gln Lys Gly
Ala Ala Phe Leu Ala Ile 185 190 195 Thr Thr Ile Tyr Ala Glu Ser Gly
Ser Gly Phe Val Met Pro Ser 200 205 210 Ser Ser Ala Lys Ser Gly Val
Glu Ala Met Asn Lys Ser Leu Ala 215 220 225 Ala Glu Trp Gly Arg Tyr
Gly Met Arg Phe Asn Ile Ile Gln Pro 230 235 240 Gly Pro Ile Lys Thr
Lys Gly Ala Phe Ser Arg Leu Asp Pro Thr 245 250 255 Gly Lys Phe Glu
Lys Asp Met Ile Glu Arg Ile Pro Cys Gly Arg 260 265 270 Leu Gly Thr
Val Glu Glu Leu Ala Asn Leu Ala Thr Phe Leu Cys 275 280 285 Ser Asp
Tyr Ala Ser Trp Ile Asn Gly Ala Val Ile Arg Phe Asp 290 295 300 Gly
Gly Glu Glu Val Phe Leu Ser Gly Glu Phe Asn Ser Leu Lys 305 310 315
Lys Val Thr Lys Glu Glu Trp Asp Val Ile Glu Gly Leu Ile Arg 320 325
330 Lys Thr Lys Gly Ser 335 8 339 PRT Homo sapiens misc_feature
GenBank ID No g780241 8 Met Ala Ala Ala Val Ala Ala Ala Pro Gly Ala
Leu Gly Ser Leu 1 5 10 15 His Ala Gly Gly Ala Arg Leu Val Ala Ala
Cys Ser Ala Trp Leu 20 25 30 Cys Pro Gly Leu Arg Leu Pro Gly Ser
Leu Ala Gly Arg Arg Ala 35 40 45 Gly Pro Ala Ile Trp Ala Gln Gly
Trp Val Pro Ala Ala Gly Gly 50
55 60 Pro Ala Pro Lys Arg Gly Tyr Ser Ser Glu Met Lys Thr Glu Asp
65 70 75 Glu Leu Arg Val Arg His Leu Glu Glu Glu Asn Arg Gly Ile
Val 80 85 90 Val Leu Gly Ile Asn Arg Ala Tyr Gly Lys Asn Ser Leu
Ser Lys 95 100 105 Asn Leu Ile Lys Met Leu Ser Lys Ala Val Asp Ala
Leu Lys Ser 110 115 120 Asp Lys Lys Val Arg Thr Ile Ile Ile Arg Ser
Glu Val Pro Gly 125 130 135 Ile Phe Cys Ala Gly Ala Asp Leu Lys Glu
Arg Ala Lys Met Ser 140 145 150 Ser Ser Glu Val Gly Pro Phe Val Ser
Lys Ile Arg Ala Val Ile 155 160 165 Asn Asp Ile Ala Asn Leu Pro Val
Pro Thr Ile Ala Ala Ile Asp 170 175 180 Gly Leu Ala Leu Gly Gly Gly
Leu Glu Leu Ala Leu Ala Cys Asp 185 190 195 Ile Arg Val Ala Ala Ser
Ser Ala Lys Met Gly Leu Val Glu Thr 200 205 210 Lys Leu Ala Ile Ile
Pro Gly Gly Gly Gly Thr Gln Arg Leu Pro 215 220 225 Arg Ala Ile Gly
Met Ser Leu Ala Lys Glu Leu Ile Phe Ser Ala 230 235 240 Arg Val Leu
Asp Gly Lys Glu Ala Lys Ala Val Gly Leu Ile Ser 245 250 255 His Val
Leu Glu Gln Asn Gln Glu Gly Asp Ala Ala Tyr Arg Lys 260 265 270 Ala
Leu Asp Leu Ala Arg Glu Phe Leu Pro Gln Gly Pro Val Ala 275 280 285
Met Arg Val Ala Lys Leu Ala Ile Asn Gln Gly Met Glu Val Asp 290 295
300 Leu Val Thr Gly Leu Ala Ile Glu Glu Ala Cys Tyr Ala Gln Thr 305
310 315 Ile Pro Thr Lys Asp Arg Leu Glu Gly Leu Leu Ala Phe Lys Glu
320 325 330 Lys Arg Pro Pro Arg Tyr Lys Gly Glu 335 9 290 PRT Homo
sapiens misc_feature GenBank ID No g1922287 9 Met Ala Ala Leu Arg
Val Leu Leu Ser Cys Ala Arg Gly Pro Leu 1 5 10 15 Arg Pro Pro Val
Arg Cys Pro Ala Trp Arg Pro Phe Ala Ser Gly 20 25 30 Ala Asn Phe
Glu Tyr Ile Ile Ala Glu Lys Arg Gly Lys Asn Asn 35 40 45 Thr Val
Gly Leu Ile Gln Leu Asn Arg Pro Lys Ala Leu Asn Ala 50 55 60 Leu
Cys Asp Gly Leu Ile Asp Glu Leu Asn Gln Ala Leu Lys Ile 65 70 75
Phe Glu Glu Asp Pro Ala Val Gly Ala Ile Val Leu Thr Gly Gly 80 85
90 Asp Lys Ala Phe Ala Ala Gly Ala Asp Ile Lys Glu Met Gln Asn 95
100 105 Leu Ser Phe Gln Asp Cys Tyr Ser Ser Lys Phe Leu Lys His Trp
110 115 120 Asp His Leu Thr Gln Val Lys Lys Pro Val Ile Ala Ala Val
Asn 125 130 135 Gly Tyr Ala Phe Gly Gly Gly Cys Glu Leu Ala Met Met
Cys Asp 140 145 150 Ile Ile Tyr Ala Gly Glu Lys Ala Gln Phe Ala Gln
Pro Glu Ile 155 160 165 Leu Ile Gly Thr Ile Pro Gly Ala Gly Gly Thr
Gln Arg Leu Thr 170 175 180 Arg Ala Val Gly Lys Ser Leu Ala Met Glu
Met Val Leu Thr Gly 185 190 195 Asp Arg Ile Ser Ala Gln Asp Ala Lys
Gln Ala Gly Leu Val Ser 200 205 210 Lys Ile Cys Pro Val Glu Thr Leu
Val Glu Glu Ala Ile Gln Cys 215 220 225 Ala Glu Lys Ile Ala Ser Asn
Ser Lys Ile Val Val Ala Met Ala 230 235 240 Lys Glu Ser Val Asn Ala
Ala Phe Glu Met Thr Leu Thr Glu Gly 245 250 255 Ser Lys Leu Glu Lys
Lys Leu Phe Tyr Ser Thr Phe Ala Thr Asp 260 265 270 Asp Arg Lys Glu
Gly Met Thr Ala Phe Val Glu Lys Arg Lys Ala 275 280 285 Asn Phe Lys
Asp Gln 290 10 328 PRT Homo sapiens misc_feature GenBank ID No
g564065 10 Met Ala Ala Gly Ile Val Ala Ser Arg Arg Leu Arg Asp Leu
Leu 1 5 10 15 Thr Arg Arg Leu Thr Gly Ser Asn Tyr Pro Gly Leu Ser
Ile Ser 20 25 30 Leu Arg Leu Thr Gly Ser Ser Ala Gln Glu Glu Ala
Ser Gly Val 35 40 45 Ala Leu Gly Glu Ala Pro Asp His Ser Tyr Glu
Ser Leu Arg Val 50 55 60 Thr Ser Ala Gln Lys His Val Leu His Val
Gln Leu Asn Arg Pro 65 70 75 Asn Lys Arg Asn Ala Met Asn Lys Val
Phe Trp Arg Glu Met Val 80 85 90 Glu Cys Phe Asn Lys Ile Ser Arg
Asp Ala Asp Cys Arg Ala Val 95 100 105 Val Ile Ser Gly Ala Gly Lys
Met Phe Thr Ala Gly Ile Asp Leu 110 115 120 Met Asp Met Ala Ser Asp
Ile Leu Gln Pro Lys Gly Asp Asp Val 125 130 135 Ala Arg Ile Ser Trp
Tyr Leu Arg Asp Ile Ile Thr Arg Tyr Gln 140 145 150 Glu Thr Phe Asn
Val Ile Glu Arg Cys Pro Lys Pro Val Ile Ala 155 160 165 Ala Val His
Gly Gly Cys Ile Gly Gly Gly Val Asp Leu Val Thr 170 175 180 Ala Cys
Asp Ile Arg Tyr Cys Ala Gln Asp Ala Phe Phe Gln Val 185 190 195 Lys
Glu Val Asp Val Gly Leu Ala Ala Asp Val Gly Thr Leu Glu 200 205 210
Arg Leu Pro Lys Val Ile Gly Asn Gln Ser Leu Val Asn Glu Leu 215 220
225 Ala Phe Thr Ala His Lys Met Met Ala Asp Glu Ala Leu Asp Ser 230
235 240 Gly Leu Val Ser Arg Val Phe Pro Asp Lys Glu Val Met Leu Asp
245 250 255 Ala Ala Leu Pro Leu Ala Pro Glu Ile Ser Ser Lys Thr Thr
Val 260 265 270 Leu Val Gln Ser Thr Lys Val Asn Leu Leu Tyr Ser Arg
Asp His 275 280 285 Ser Val Ala Glu Ser Leu Asn Tyr Val Ala Ser Trp
Asn Met Ser 290 295 300 Met Leu Gln Thr Gln Asp Leu Val Lys Ser Val
Gln Pro Thr Thr 305 310 315 Glu Asn Lys Glu Leu Lys Thr Val Thr Phe
Ser Lys Leu 320 325
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