U.S. patent application number 12/076827 was filed with the patent office on 2009-11-05 for human phospholipases.
This patent application is currently assigned to INCYTE CORPORATION. Invention is credited to Debopriya Das, Ameena R. Gandhi, April J. A. Hafalia, Jennifer L. Hillman, Farrah A. Khan, Preeti Lal, Danniel B. Nguyen, Jennifer L. Policky, Y. Tom Tang, Catherine M. Tribouley, Narinder Walia, Monique G. Yao, Henry Yue.
Application Number | 20090274680 12/076827 |
Document ID | / |
Family ID | 27392110 |
Filed Date | 2009-11-05 |
United States Patent
Application |
20090274680 |
Kind Code |
A1 |
Lal; Preeti ; et
al. |
November 5, 2009 |
Human Phospholipases
Abstract
The invention provides human lipid metabolism enzymes (LME) and
polynucleotides which identify and encode LME. The invention also
provides expression vectors, host cells, antibodies, agonists, and
antagonists. The invention also provides methods for diagnosing,
treating, or preventing disorders associated with aberrant
expression of LME.
Inventors: |
Lal; Preeti; (Santa Clara,
CA) ; Yao; Monique G.; (Mountain View, CA) ;
Yue; Henry; (Sunnyvale, CA) ; Gandhi; Ameena R.;
(San Francisco, CA) ; Khan; Farrah A.; (Des
Plaines, IL) ; Tang; Y. Tom; (San Jose, CA) ;
Nguyen; Danniel B.; (San Jose, CA) ; Policky;
Jennifer L.; (San Jose, CA) ; Das; Debopriya;
(Mountain View, CA) ; Hillman; Jennifer L.;
(Mountain View, CA) ; Walia; Narinder; (San
Leandro, CA) ; Hafalia; April J. A.; (Dale City,
CA) ; Tribouley; Catherine M.; (San Francisco,
CA) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
INCYTE CORPORATION
|
Family ID: |
27392110 |
Appl. No.: |
12/076827 |
Filed: |
March 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11275596 |
Jan 18, 2006 |
7368270 |
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12076827 |
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10220380 |
Aug 28, 2002 |
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PCT/US01/06771 |
Feb 28, 2001 |
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11275596 |
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60186480 |
Mar 2, 2000 |
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60190415 |
Mar 17, 2000 |
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60198437 |
Apr 19, 2000 |
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Current U.S.
Class: |
424/94.6 ;
435/196; 435/69.1; 514/1.1; 530/350; 530/387.9 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 37/08 20180101; A61K 38/00 20130101; C12N 15/52 20130101; A61P
9/00 20180101; A61P 9/10 20180101; A61P 37/02 20180101; C07K 16/40
20130101; A61P 3/00 20180101; A61P 29/00 20180101; A61P 31/04
20180101; C12Q 1/6883 20130101; A61P 17/00 20180101; A61P 25/16
20180101; A61P 25/28 20180101; A61P 1/00 20180101; A61P 7/04
20180101; A61P 43/00 20180101; A61P 37/06 20180101; A61P 31/12
20180101; A61P 11/06 20180101; A61P 25/00 20180101; C07K 14/47
20130101 |
Class at
Publication: |
424/94.6 ;
530/350; 514/12; 435/196; 435/69.1; 530/387.9 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C07K 14/00 20060101 C07K014/00; A61K 38/16 20060101
A61K038/16; C12N 9/16 20060101 C12N009/16; C12P 21/00 20060101
C12P021/00; C07K 16/00 20060101 C07K016/00; A61P 35/00 20060101
A61P035/00; A61P 29/00 20060101 A61P029/00; A61P 37/06 20060101
A61P037/06; A61P 9/00 20060101 A61P009/00; A61P 1/00 20060101
A61P001/00 |
Claims
1.-28. (canceled)
29. A substantially purified polypeptide comprising an amino acid
sequence of SEQ ID NO:5.
30. The polypeptide of claim 29, further comprising a signal
sequence.
31. The polypeptide of claim 29, further comprising a label.
32. The polypeptide of claim 29, linked to a heterologous
polypeptide.
33. A composition comprising the polypeptide of claim 29 and a
pharmaceutically acceptable excipient.
34. A substantially purified polypeptide variant of SEQ ID NO:5,
wherein the variant differs from SEQ ID NO:5 at one or more amino
acids selected from the group consisting of: T72, S14, S16, S97,
S144, T175, S206, S258, S272, S287, T320, T47, T68, S121, T124,
T348, N50, N58, N66, and N357.
35. A substantially purified polypeptide fragment of SEQ ID NO:5,
wherein the fragment is selected from the group consisting of:
amino acids P188-H205, L211-P231, N64-1335, L39-V333, M1-A18,
H31-D319, N.sub.102-1116, D146-S160, Y223-C233, S59-F78, V104-H119,
D147-E165, C246-E261, P325-K340, and a combination thereof.
36. A substantially purified polypeptide variant having at least
95% identity to SEQ ID NO:5, wherein the variant differs from SEQ
ID NO:5 at one or more amino acids selected from the group
consisting or: T348, N357, P188-H205, L211-P231, N64-1335,
L39-V333, M1-A18, H31-D319, N.sub.102-1116, D146-S160, Y223-C233,
S59-F78, V104-H119, D147-E165, C246-E261, and P325-K340.
37. A substantially purified polypeptide consisting of an amino
acid sequence of SEQ ID NO:5.
38. An isolated polypeptide comprising an amino acid sequence
having at least about 95% sequence identity to an amino acid
sequence of SEQ ID NO:5, wherein the polypeptide has phospholipase
activity.
39. The isolated polypeptide of claim 38 comprising a fragment of
the amino acid sequence of SEQ ID NO:5.
40. A composition comprising the polypeptide of claim 38 and a
pharmaceutically acceptable excipient.
41. A method for producing a polypeptide comprising the amino acid
sequence of SEQ ID NO:5 and fragments thereof, the method
comprising the steps of: a) culturing a host cell containing an
expression vector containing at least a fragment of a
polynucleotide encoding the amino acid sequence of SEQ ID NO:5
under conditions suitable for the expression of the polypeptide;
and b) recovering the polypeptide from the host cell culture.
42. A purified antibody which specifically binds to the polypeptide
of claim 29.
43. A purified antibody which specifically binds to the polypeptide
fragment of claim 35.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No.
11/275,596, filed Jan. 18, 2006, which is a divisional of U.S.
application Ser. No. 10/220,380, which is the National Stage of
PCT/US01/06771, filed Feb. 28, 2001, and published as WO 01/64907,
which claims the benefit of priority from U.S. Provisional
Application Nos. 60/186,480, filed Mar. 2, 2000, 60/190,415, filed
Mar. 17, 2000, and 60/198,437, filed Apr. 19, 2000. The contents of
these applications are incorporated herein by reference in their
entirety.
[0002] This invention relates to nucleic acid and amino acid
sequences of lipid metabolism enzymes and to the use of these
sequences in the diagnosis, treatment, and prevention of cancer,
neurological disorders, autoimmune/inflammatory disorders,
gastrointestinal disorders, and cardiovascular disorders, and in
the assessment of the effects of exogenous compounds on the
expression of nucleic acid and amino acid sequences of lipid
metabolism enzymes.
BACKGROUND OF THE INVENTION
[0003] Lipids are water-insoluble, oily or greasy substances that
are soluble in nonpolar solvents such as chloroform or ether.
Neutral fats (triacylglycerols) serve as major fuels and energy
stores. Polar lipids, such as phospholipids, sphingolipids,
glycolipids, and cholesterol, are key structural components of cell
membranes. (Lipid metabolism is reviewed in Stryer, L. (1995)
Biochemistry, W. H. Freeman and Company, New York N.Y.; Lehninger,
A. (1982) Principles of Biochemistry, Worth Publishers, Inc. New
York N.Y.; and ExPASy "Biochemical Pathways" index of Boehringer
Mannheim online publication.
[0004] Fatty acids are long-chain organic acids with a single
carboxyl group and a long non-polar hydrocarbon tail. Long-chain
fatty acids are essential components of glycolipids, phospholipids,
and cholesterol, which are building blocks for biological
membranes, and of triglycerides, which are biological fuel
molecules. Long-chain fatty acids are also substrates for
eicosanoid production, and are important in the functional
modification of certain complex carbohydrates and proteins.
16-carbon and 18-carbon fatty acids are the most common. Fatty acid
synthesis occurs in the cytoplasm. In the first step,
acetyl-Coenzyme A (CoA) carboxylase (ACC) synthesizes malonyl-CoA
from acetyl-CoA and bicarbonate. The enzymes which catalyze the
remaining reactions are covalently linked into a single polypeptide
chain, referred to as the multifunctional enzyme fatty acid
synthase (FAS). FAS catalyzes the synthesis of palmitate from
acetyl-CoA and malonyl-CoA. FAS contains acetyl transferase,
malonyl transferase, .beta.-ketoacetyl synthase, acyl carrier
protein, .beta.-ketoacyl reductase, dehydratase, enoyl reductase,
and thioesterase activities. The final product of the FAS reaction
is the 16-carbon fatty acid palmitate. Further elongation, as well
as unsaturation, of palmitate by accessory enzymes of the ER
produces the variety of long chain fatty acids required by the
individual cell. These enzymes include a NADH-cytochrome b.sub.5
reductase, cytochrome b.sub.5, and a desaturase.
[0005] Triacylglycerols, also known as triglycerides and neutral
fats, are major energy stores in animals. Triacylglycerols are
esters of glycerol with three fatty acid chains.
Glycerol-3-phosphate is produced from dihydroxyacetone phosphate by
the enzyme glycerol phosphate dehydrogenase or from glycerol by
glycerol kinase. Fatty acid-CoA's are produced from fatty acids by
fatty acyl-CoA synthetases. Glyercol-3-phosphate is acylated with
two fatty acyl-CoA's by the enzyme glycerol phosphate
acyltransferase to give phosphatidate. Phosphatidate phosphatase
converts phosphatidate to diacylglycerol, which is subsequently
acylated to a triacylglyercol by the enzyme diglyceride
acyltransferase. Phosphatidate phosphatase and diglyceride
acyltransferase form a triacylglyerol synthetase complex bound to
the ER membrane.
[0006] A major class of phospholipids are the phosphoglycerides,
which are composed of a glycerol backbone, two fatty acid chains,
and a phosphorylated alcohol. Phosphoglycerides are components of
cell membranes. Principal phosphoglycerides are phosphatidyl
choline, phosphatidyl ethanolamine, phosphatidyl serine,
phosphatidyl inositol, and diphosphatidyl glycerol. Many enzymes
involved in phosphoglyceride synthesis are associated with
membranes (Meyers, R. A. (1995) Molecular Biology and
Biotechnology, VCH Publishers Inc., New York N.Y., pp. 494-501).
Phosphatidate is converted to CDP-diacylglycerol by the enzyme
phosphatidate cytidylyltransferase (ExPASy ENZYME EC 2.7.7.41).
Transfer of the diacylglycerol group from CDP-diacylglycerol to
serine to yield phosphatidyl serine, or to inositol to yield
phosphatidyl inositol, is catalyzed by the enzymes
CDP-diacylglycerol-serine O-phosphatidyltransferase and
CDP-diacylglycerol-inositol 3-phosphatidyltransferase, respectively
(ExPASy ENZYME EC 2.7.8.8; ExPASy ENZYME EC 2.7.8.11). The enzyme
phosphatidyl serine decarboxylase catalyzes the conversion of
phosphatidyl serine to phosphatidyl ethanolamine, using a pyruvate
cofactor (Voelker, D. R. (1997) Biochim. Biophys. Acta
1348:236-244). Phosphatidyl choline is formed using diet-derived
choline by the reaction of CDP-choline with 1,2-diacylglycerol,
catalyzed by diacylglycerol cholinephosphotransferase (ExPASy
ENZYME 2.7.8.2).
[0007] Cholesterol, composed of four fused hydrocarbon rings with
an alcohol at one end, moderates the fluidity of membranes in which
it is incorporated. In addition, cholesterol is used in the
synthesis of steroid hormones such as cortisol, progesterone,
estrogen, and testosterone. Bile salts derived from cholesterol
facilitate the digestion of lipids. Cholesterol in the skin forms a
barrier that prevents excess water evaporation from the body.
Farnesyl and geranylgeranyl groups, which are derived from
cholesterol biosynthesis intermediates, are post-translationally
added to signal transduction proteins such as Ras and
protein-targeting proteins such as Rab. These modifications are
important for the activities of these proteins (Guyton, A. C.
(1991) Textbook of Medical Physiology, W.B. Saunders Company,
Philadelphia Pa., pp. 760-763; Stryer, supra, pp. 279-280, 691-702,
934). Mammals obtain cholesterol derived from both de novo
biosynthesis and the diet.
[0008] Sphingolipids are an important class of membrane lipids that
contain sphingosine, a long chain amino alcohol. They are composed
of one long-chain fatty acid, one polar head alcohol, and
sphingosine or sphingosine derivatives. The three classes of
sphingolipids are sphingomyelins, cerebrosides, and gangliosides.
Sphingomyelins, which contain phosphocholine or phosphoethanolamine
as their head group, are abundant in the myelin sheath surrounding
nerve cells. Galactocerebrosides, which contain a glucose or
galactose head group, are characteristic of the brain. Other
cerebrosides are found in non-neural tissues. Gangliosides, whose
head groups contain multiple sugar units, are abundant in the
brain, but are also found in non-neural tissues.
[0009] Eicosanoids, including prostaglandins, prostacyclin,
thromboxanes, and leukotrienes, are 20-carbon molecules derived
from fatty acids. Eicosanoids are signaling molecules which have
roles in pain, fever, and inflammation. The precursor of all
eicosanoids is arachidonate, which is generated from phospholipids
by phospholipase A.sub.2 and from diacylglycerols by diacylglycerol
lipase. Leukotrienes are produced from arachidonate by the action
of lipoxygenases.
[0010] Within cells, fatty acids are transported by cytoplasmic
fatty acid binding proteins (Online Mendelian Inheritance in Man
(OMIM) *134650 Fatty Acid-Binding Protein 1, Liver; FABP1).
Diazepam binding inhibitor (DBI), also known as endozepine and acyl
CoA-binding protein, is an endogenous .gamma.-aminobutyric acid
(GABA) receptor ligand which is thought to down-regulate the
effects of GABA. DBI binds medium- and long-chain acyl-CoA esters
with very high affinity and may function as an intracellular
carrier of acyl-CoA esters (OMIM *125950 Diazepam Binding
Inhibitor; DBI; PROSITE PDOC00686 Acyl-CoA-binding protein
signature).
[0011] Fat stored in liver and adipose triglycerides may be
released by hydrolysis and transported in the blood. Free fatty
acids are transported in the blood by albumin. Triacylglycerols and
cholesterol esters in the blood are transported in lipoprotein
particles. The particles consist of a core of hydrophobic lipids
surrounded by a shell of polar lipids and apolipoproteins. The
protein components serve in the solubilization of hydrophobic
lipids and also contain cell-targeting signals. Lipoproteins
include chylomicrons, chylomicron remnants, very-low-density
lipoproteins (VLDL), intermediate-density lipoproteins (IDL),
low-density lipoproteins (LDL), and high-density lipoproteins
(HDL). There is a strong inverse correlation between the levels of
plasma HDL and risk of premature coronary heart disease.
[0012] Mitochondrial and peroxisomal beta-oxidation enzymes degrade
saturated and unsaturated fatty acids by sequential removal of
two-carbon units from 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. The
pathways of mitochondrial 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 P. P. Van
Veldhoven (1993) Biochimie 75:147-158). Enzymes involved in
beta-oxidation include acyl CoA synthetase, carnitine
acyltransferase, acyl CoA dehydrogenases, enoyl CoA hydratases,
L-3-hydroxyacyl CoA dehydrogenase, .beta.-ketothiolase, 2,4-dienoyl
CoA reductase, and isomerase.
[0013] Three classes of lipid metabolism enzymes are discussed in
further detail. The three classes are lipases, phospholipases and
lipoxygenases.
Lipases
[0014] Triglycerides are hydrolyzed to fatty acids and glycerol by
lipases. Adipocytes contain lipases that break down stored
triacylglycerols, releasing fatty acids for export to other tissues
where they are required as fuel. Lipases are widely distributed in
animals, plants, and prokaryotes. Triglyceride lipases (ExPASy
ENZYME EC 3.1.1.3), also known as triacylglycerol lipases and
tributyrases, hydrolyze the ester bond of triglycerides. In higher
vertebrates there are at least three tissue-specific isozymes
including gastric, hepatic, and pancreatic lipases. These three
types of lipases are structurally closely related to each other as
well as to lipoprotein lipase. The most conserved region in
gastric, hepatic, and pancreatic lipases is centered around a
serine residue which is also present in lipases of prokaryotic
origin. Mutation in the serine residue renders the enzymes
inactive. Gastric, hepatic, and pancreatic lipases hydrolyze
lipoprotein triglycerides and phospholipids. Gastric lipases in the
intestine aid in the digestion and absorption of dietary fats.
Hepatic lipases are bound to and act at the endothelial surfaces of
hepatic tissues. Hepatic lipases also play a major role in the
regulation of plasma lipids. Pancreatic lipase requires a small
protein factor, colipase, for efficient dietary lipid hydrolysis.
Colipase binds to the C-terminal, non-catalytic domain of lipase,
thereby stabilizing an active conformation and considerably
increasing the overall hydrophobic binding site. Deficiencies of
these enzymes have been identified in man, and all are associated
with pathologic levels of circulating lipoprotein particles
(Gargouri, Y. et al. (1989) Biochim. Biophys. Acta 1006:255-271;
Connelly, P. W. (1999) Clin. Chim. Acta 286:243-255; van Tilbeurgh,
H. et al. (1999) Biochim Biophys Acta 1441:173-184).
[0015] Lipoprotein lipases (ExPASy ENZYME EC 3.1.1.34), also known
as clearing factor lipases, diglyceride lipases, or diacylglycerol
lipases, hydrolyze triglycerides and phospholipids present in
circulating plasma lipoproteins, including chylomicrons, very low
and intermediate density lipoproteins, and high-density
lipoproteins (HDL). Together with pancreatic and hepatic lipases,
lipoprotein lipases (LPL) share a high degree of primary sequence
homology. Both lipoprotein lipases and hepatic lipases are anchored
to the capillary endothelium via glycosaminoglycans and can be
released by intravenous administration of heparin. LPLs are
primarily synthesized by adipocytes, muscle cells, and macrophages.
Catalytic activities of LPLs are activated by apolipoprotein C--II
and are inhibited by high ionic strength. conditions such as 1 M
NaCl. LPL deficiencies in humans contribute to metabolic diseases
such as hypertriglyceridemia, HDL2 deficiency, and obesity
(Jackson, R. L. (1983) in The Enzymes (Boyer, P. D., ed.) Vol. XVI,
pp. 141-186, Academic Press, New York N.Y.; Eckel, R. H. (1989) New
Engl. J. Med. 320:1060-1068).
Phospholipases
[0016] Phospholipases, a group of enzymes that catalyze the
hydrolysis of membrane phospholipids, are classified according to
the bond cleaved in a phospholipid. They are classified into PLA1,
PLA2, PLB, PLC, and PLD families. Phospholipases are involved in
many inflammatory reactions by making arachidonate available for
eicosanoid biosynthesis. More specifically, aracbidonic acid is
processed into bioactive lipid mediators of inflammation such as
lyso-platelet-activating factor and eicosanoids. The synthesis of
arachidonic acid from membrane phospholipids is the rate-limiting
step in the biosynthesis of the four major classes of eicosanoids
(prostaglandins, prostacyclins, thromboxanes and leukotrienes)
which are involved in pain, fever, and inflammation (Kaiser, E. et
al. (1990) Clin. Biochem. 23:349-370). Furthermore, leukotriene-B4
is known to function in a feedback loop which further increases
PLA2 activity (Wijkander, J. et al. (1995) J. Biol. Chem.
270:26543-26549).
[0017] The secretory phospholipase A.sub.2 (PLA2) superfamily
comprises a number of heterogeneous enzymes whose common feature is
to hydrolyze the sn-2 fatty acid acyl ester bond of
phosphoglycerides. Hydrolysis of the glycerophospholipids releases
free fatty acids and lysophospholipids. PLA2 activity generates
precursors for the biosynthesis of biologically active lipids,
hydroxy fatty acids, and platelet-activating factor. PLA2s were
first described as components of snake venoms, and were later
characterized in numerous species. PLA2s have traditionally been
classified into several major groups and subgroups based on their
amino acid sequences, divalent cation requirements, and location of
disulfide bonds. The PLA2s of Groups I, II, and III consist of low
molecular weight, secreted, Ca.sup.2+-dependent proteins. Group IV
PLA2s are primarily 85-kDa, Ca.sup.2+-dependent cytosolic
phospholipases. Finally, a number of Ca.sup.2+-independent PLA2s
have been described, which comprise Group V (Davidson, F. F. and E.
A. Dennis (1990) J. Mol. Evol. 31:228-238; and Dennis, E. F. (1994)
J. Biol. Chem. 269:13057-13060).
[0018] The first PLA2s to be extensively characterized were the
Group I, II, and III PLA2s found in snake and bee venoms. These
venom PLA2s share many features with mammalian PLA2s including a
common catalytic mechanism, the same Ca.sup.2+ requirement, and
conserved primary and tertiary structures. In addition to their
role in the digestion of prey, the venom PLA2s display neurotoxic,
myotoxic, anticoagulant, and proinflammatory effects in mammalian
tissues. This diversity of pathophysiological effects is due to the
presence of specific, high affinity receptors for these enzymes on
various cells and tissues (Lambeau, G. et al. (1995) J. Biol. Chem.
270:5534-5540).
[0019] PLA2s from Groups I, IIA, IIC, and V have been described in
mammalian and avian cells, and were originally characterized by
tissue distribution, although the distinction is no longer
absolute. Thus, Group I PLA2s were found in the pancreas, Group IIA
and IIC were derived from inflammation-associated tissues (e.g.,
the synovium), and Group V were from cardiac tissue. The pancreatic
PLA2s function in the digestion of dietary lipids and have been
proposed to play a role in cell proliferation, smooth muscle
contraction, and acute lung injury. The Group II inflammatory PLA2s
are potent mediators of inflammatory processes and are highly
expressed in serum and synovial fluids of patients with
inflammatory disorders. These Group II PLA2s are found in most
human cell types assayed and are expressed in diverse pathological
processes such as septic shock, intestinal cancers, rheumatoid
arthritis, and epidermal hyperplasia. A Group V PLA2 has been
cloned from brain tissue and is strongly expressed in heart tissue.
A human PLA2 was recently cloned from fetal lung, and based on its
structural properties, appears to be the first member of a new
group of mammalian PLA2s, referred to as Group X. Other PLA2s have
been cloned from various human tissues and cell lines, suggesting a
large diversity of PLA2s (Chen, J. et al. (1994) J. Biol. Chem.
269:2365-2368; Kennedy, B. P. et al. (1995) J. Biol. Chem.
270:22378-22385; Komada, M. et al. (1990) Biochem. Biophys. Res.
Commun. 168:1059-1065; Cupillard, L. et al. (1997) J. Biol. Chem.
272:15745-15752; and Nalefski, E. A. et al. (1994) J. Biol. Chem.
269:18239-18249).
[0020] Phospholipases B (PLB) (ExPASy ENZYME EC 3.1.1.5), also
known as lysophospholipase, lecithinase B, or lysolecithinase are
widely distributed enzymes that metabolize intracellular lipids,
and occur in numerous isoforms. Small isoforms, approximately 15-30
kD, function as hydrolases; large isoforms, those exceeding 60 kD,
function both as hydrolases and transacylases. A particular
substrate for PLBs, lysophosphatidylcholine, causes lysis of cell
membranes when it is formed or imported into a cell. PLBs are
regulated by lipid factors including acylcarnitine, arachidonic
acid, and phosphatidic acid. These lipid factors are signaling
molecules important in numerous pathways, including the
inflammatory response (Anderson, R. et al. (1994) Toxicol. Appl.
Pharmacol. 125:176-183; Selle, H. et al. (1993); Eur. J. Biochem.
212:411-416).
[0021] Phospholipase C (PLC) (ExPASy ENZYME EC 3.1.4.10) plays an
important role in transmembrane signal transduction. Many
extracellular signaling molecules including hormones, growth
factors, neurotransmitters, and immunoglobulins bind to their
respective cell surface receptors and activate PLCs. The role of an
activated PLC is to catalyze the hydrolysis of
phosphatidyl-inositol-4,5-bisphosphate (PIP2), a minor component of
the plasma membrane, to produce diacylglycerol and inositol
1,4,5-trisphosphate (IP3). In their respective biochemical
pathways, IP3 and diacylglycerol serve as second messengers and
trigger a series of intracellular responses. IP3 induces the
release of Ca.sup.2+ from internal cellular storage, and
diacylglycerol activates protein kinase C (PKC). Both pathways are
part of transmembrane signal transduction mechanisms which regulate
cellular processes which include secretion, neural activity,
metabolism, and proliferation.
[0022] Several distinct isoforms of PLC have been identified and
are categorized as PLC-beta, PLC-gamma, and PLC-delta. Subtypes are
designated by adding Arabic numbers after the Greek letters, e.g.
PLC-.beta.-1. PLCs have a molecular mass of 62-68 kDa, and their
amino acid sequences show two regions of significant similarity.
The first region designated X has about 170 amino acids, and the
second or Y region contains about 260 amino acids.
[0023] The catalytic activities of the three isoforms of PLC are
dependent upon Ca.sup.2+. It has been suggested that the binding
sites for Ca.sup.2+ in the PLCs are located in the Y-region, one of
two conserved regions. The hydrolysis of common inositol-containing
phospholipids, such as phosphatidylinositol (PI),
phosphatidylinositol 4-monophosphate (PIP), and
phosphatidylinositol 4,5-bisphosphate. (PIP2), by any of the
isoforms yields cyclic and noncyclic inositol phosphates (Rhee, S.
G. and Y. S. Bae (1997) J. Biol. Chem. 272:15045-15048).
[0024] All mammalian PLCs contain a pleckstrin homology (PH) domain
which is about 100 amino acids in length and is composed of two
antiparallel beta sheets flanked by an amphipathic alpha helix. PH
domains target PLCs to the membrane surface by interacting with
either the beta/gamma subunits of G proteins or PIP2 (PROSITE
PDOC50003).
[0025] Phospholipase D (PLD) (ExPASy ENZYME EC 3.1.4.4), also known
as lecithinase D, lipophosphodiesterase II, and choline phosphatase
catalyzes the hydrolysis of phosphatidylcholine and other
phospholipids to generate phosphatidic acid. PLD plays an important
role in membrane vesicle trafficking, cytoskeletal dynamics, and
transmembrane signal transduction. In addition, the activation of
PLD is involved in cell differentiation and growth (reviewed in
Liscovitch, M. (2000) Biochem. J. 345:401-415).
[0026] PLD is activated in mammalian cells in response to diverse
stimuli that include hormones, neurotransmitters, growth factors,
cytokines, activators of protein kinase C, and agonists binding to
G-protein-coupled receptors. At least two forms of mammalian PLD,
PLD1 and PLD2, have been identified. PLD1 is activated by protein
kinase C alpha and by the small GTPases ARF and RhoA. (Houle, M. G.
and S. Bourgoin (1999) Biochim. Biophys. Acta 1439:135-149). PLD2
can be selectively activated by unsaturated fatty acids such as
oleate (Kim, J. H. (1999) FEBS Lett. 454:42-46).
Lipoxygenases
[0027] Lipoxygenases (ExPASy ENZYME EC 1.13.11.12) are non-heme
iron-containing enzymes that catalyze the dioxygenation of certain
polyunsaturated fatty acids such as lipoproteins. Lipoxygenases are
found widely in plants, fungi, and animals. Several different
lipoxygenase enzymes are known, each having a characteristic
oxidation action. In animals, there are specific lipoxygenases that
catalyze the dioxygenation of arachidonic acid at the carbon-3, 5,
8, 11, 12, and 15 positions. These enzymes are named after the
position of arachidonic acid that they dioxygenate. Lipoxygenases
have a single polypeptide chain with a molecular mass of
.about.75-80 kDa in animals. The proteins have an N-terminal-barrel
domain and a larger catalytic domain containing a single atom of
non-heme iron. Oxidation of the ferric enzyme to an active form is
required for catalysis (Yamamoto, S. (1992) Biochim. Biophys. Acta
1128:117-131; Brash, A. R. (1999) J. Biol. Chem. 274:23679-23682).
A variety of lipoxygenase inhibitors exist and are classified into
five major categories according to their mechanism of inhibition.
These include antioxidants, iron chelators, substrate analogues,
lipoxygenase-activating protein inhibitors, and, finally, epidermal
growth factor-receptor inhibitors.
[0028] 3-Lipoxygenase, also known as e-LOX-3 or Aloxe3 has recently
been cloned from murine epidermis. Aloxe3 resides on mouse
chromosome 11, and the deduced amino acid sequence for Aloxe3 is
54% identical to the 12-lipoxygenase sequences (Kinzig, A. (1999)
Genomics 58:158-164).
[0029] 5-Lipoxygenase (5-LOX, ExPASy ENZYME EC 1.13.11.34), also
known as arachidonate: oxygen 5-oxidoreductase, is found primarily
in white blood cells, macrophages, and mast cells. 5-LOX converts
arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid
(5-HPETE) and then to leukotriene (LTA4
(5,6-oxido-7,9,11,14-eicosatetraenoic acid)). Subsequent conversion
of leukotriene A4 by leukotriene A4 hydrolase yields the potent
neutrophil chemoattractant leukotriene B4. Alternatively,
conjugation of LTA4 with glutathione by leukotriene C4 synthase
plus downstream metabolism leads to the cysteinyl leukotrienes that
influence airway reactivity and mucus secretion, especially in
asthmatics. Most lipoxygenases require no other cofactors or
proteins for activity. In contrast, the mammalian 5-LOX requires
calcium and ATP, and is activated in the presence of a 5-LOX
activating protein (FLAP). FLAP itself binds to arachidonic acid
and supplies 5-LOX with substrate (Lewis, R. A. et al. (1990) New
Engl. J. Med. 323:645-655). The expression levels of 5-LOX and FLAP
are found to be increased in the lungs of patients with plexogenic
(primary) pulmonary hypertension (Wright, L. et al. (1998) Am. J.
Respir. Crit. Care Med. 157:219-229).
[0030] 12-Lipoxygenase (12-LOX, ExPASy ENZYME: EC 1.13.11.31)
oxygenates arachidonic acid to form 12-hydroperoxyeicosatetraenoic
acid (12-HPETE). Mammalian 12-lipoxygenases are named after the
prototypical tissues of their occurrence (hence, the leukocyte,
platelet, or epidermal types). Platelet-type 12-LOX has been found
to be the predominant isoform in epidermal skin specimens and
epidermoid cells. Leukocyte 12-LOX was first characterized
extensively from porcine leukocytes and was found to have a rather
broad distribution in mammalian tissues by immunochemical assays.
Besides tissue distribution, the leukocyte 12-LOX is distinguished
from the platelet-type enzyme by its ability to form 15-HPETE, in
addition to 12-PETE from arachidonic acid substrate. Leukocyte
12-LOX is highly related to 15-lipoxygenase (15-LOX) in that both
are dual specificity lipoxygenases, and they are about 85%
identical in primary structure in higher mammals. Leukocyte 12-LOX
is found in tracheal epithelium, leukocytes, and macrophages
(Conrad, D. J. (1999) Clin. Rev. Allergy Immunol. 17:71-89).
[0031] 15-Lipoxygenase (15-LOX; ExPASy ENZYME:EC 1.13.11.33) is
found inhuman reticulocytes, airway epithelium, and eosinophils.
15-LOX has been detected in atherosclerotic lesions in mammals,
specifically rabbit and man. The enzyme, in addition to its role in
oxidative modification of lipoproteins, is important in the
inflammatory reaction in atherosclerotic lesions. 15-LOX has been
shown to be induced in human monocytes by the cytokine IL-4, which
is known to be implicated in the inflammatory process (Kuhn, H. and
S. Borngraber (1999) Adv. Exp. Med. Biol. 447:5-28).
Disease Correlation
[0032] Lipid metabolism is involved in human diseases and
disorders. In the arterial disease atherosclerosis, fatty lesions
form on the inside of the arterial wall. These lesions promote the
loss of arterial flexibility and the formation of blood clots
(Guyton, supra). In Tay-Sachs disease, the GM.sub.2 ganglioside (a
sphingolipid) accumulates in lysosomes of the central nervous
system due to a lack of the enzyme N-acetylhexosaminidase. Patients
suffer nervous system degeneration leading to early death (Fauci,
A. S. et al. (1998) Harrison's Principles of Internal Medicine,
McGraw-Hill, New York N.Y., p. 2171). The Niemann-Pick diseases are
caused by defects in lipid metabolism. Niemann-Pick diseases types
A and B are caused by accumulation of sphingomyelin (a
sphingolipid) and other lipids in the central nervous system due to
a defect in the enzyme sphingomyelinase, leading to
neurodegeneration and lung disease. Niemann-Pick disease type C
results from a defect in cholesterol transport, leading to the
accumulation of sphingomyelin and cholesterol in lysosomes and a
secondary reduction in sphingomyelinase activity. Neurological
symptoms such as grand mal seizures, ataxia, and loss of previously
learned speech, manifest 1-2 years after birth. A mutation in the
NPC protein, which contains a putative cholesterol-sensing domain,
was found in a mouse model of Niemann-Pick disease type C (Fauci,
supra, p. 2175; Loftus, S. K. et al. (1997) Science
277:232-235).
[0033] PLAs are implicated in a variety of disease processes. For
example, PLAs are found in the pancreas, in cardiac tissue, and in
inflammation-associated tissues. Pancreatic PLAs function in the
digestion of dietary lipids and have been proposed to play a role
in cell proliferation, smooth muscle contraction, and acute lung
injury. Inflammatory PLAs are potent mediators of inflammatory
processes and are highly expressed in serum and synovial fluids of
patients with inflammatory disorders. Additionally, inflammatory
PLAs are found in most human cell types and are expressed in
diverse pathological processes such as septic shock, intestinal
cancers, rheumatoid arthritis, and epidermal hyperplasia.
[0034] The role of PLBs in human tissues has been investigated in
various research studies. Hydrolysis of lysophosphatidylcholine by
PLBs causes lysis in erythrocyte membranes (Selle, supra).
Similarly, Endresen, M. J. et al. (1993; Scand. J. Clin. Invest.
53:733-739) reported that the increased hydrolysis of
lysophosphatidylcholine by PLB in pre-eclamptic women causes
release of free fatty acids into the sera. In renal studies, PLB
was shown to protect Na.sup.+, K.sup.+-ATPase from the cytotoxic
and cytolytic effects of cyclosporin A (Anderson, supra).
[0035] Lipases, phospholipases, and lipoxygenases are thought to
contribute to complex diseases, such as atherosclerosis, obesity,
arthritis, asthma, and cancer, as well as to single gene defects,
such as Wolman's disease and Type I hyperlipoproteinemia.
[0036] The discovery of new lipid metabolism enzymes and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer, neurological disorders,
autoimmune/inflammatory disorders, gastrointestinal disorders, and
cardiovascular disorders, and in the assessment of the effects of
exogenous compounds on the expression of nucleic acid and amino
acid sequences of lipid metabolism enzymes.
SUMMARY OF THE INVENTION
[0037] The invention features purified polypeptides, lipid
metabolism enzymes, referred to collectively as "LME" and
individually as "LME-1," "LME-2," "LME-3," "LME-4," "LME-5," and
"LME-6." In one aspect, the invention provides an isolated
polypeptide comprising an amino acid sequence selected from the
group consisting of a) an amino acid sequence selected from the
group consisting of SEQ ID NO: 1-6, b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-6,
c) a biologically active fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6, and d) an
immunogenic fragment of an amino acid sequence selected from the
group consisting of SEQ ID NO:1-6. In one alternative, the
invention provides an isolated polypeptide comprising the amino
acid sequence of SEQ ID NO:1-6.
[0038] The invention further provides an isolated polynucleotide
encoding a polypeptide comprising an amino acid sequence selected
from the group consisting of a) an amino acid sequence selected
from the group consisting of SEQ ID NO:1-6, b) a naturally
occurring amino acid sequence having at least 90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-6, c) a biologically active fragment of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, and
d) an immunogenic fragment of an amino acid sequence selected from
the group consisting of SEQ ID NO:1-6. In one alternative, the
polynucleotide encodes a polypeptide selected from the group
consisting of SEQ ID NO:1-6. In another alternative, the
polynucleotide is selected from the group consisting of SEQ ID
NO:7-12.
[0039] Additionally, the invention provides a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding a polypeptide comprising an amino acid
sequence selected from the group consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6. In one
alternative, the invention provides a cell transformed with the
recombinant polynucleotide. In another alternative, the invention
provides a transgenic organism comprising the recombinant
polynucleotide.
[0040] The invention also provides a method for producing a
polypeptide comprising an amino acid sequence selected from the
group consisting of a) an amino acid sequence selected from the
group consisting of SEQ ID NO:1-6, b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-6,
c) a biologically active fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6, and d) an
immunogenic fragment of an amino acid sequence selected from the
group consisting of SEQ ID NO:1-6. The method comprises a)
culturing a cell under conditions suitable for expression of the
polypeptide, wherein said cell is transformed with a recombinant
polynucleotide comprising a promoter sequence operably linked to a
polynucleotide encoding the polypeptide, and b) recovering the
polypeptide so expressed.
[0041] Additionally, the invention provides an isolated antibody
which specifically binds to a polypeptide comprising an amino acid
sequence selected from the group consisting of a) an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, b) a
naturally occurring amino acid sequence having at least 90%
sequence identity to an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6, c) a biologically active fragment of
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, and d) an immunogenic fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6. The invention
further provides an isolated polynucleotide comprising a
polynucleotide sequence selected from the group consisting of a) a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:7-12, b) a naturally occurring polynucleotide sequence having
at least 90% sequence identity to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:7-12, c) a
polynucleotide sequence complementary to a), d) a polynucleotide
sequence complementary to b), and e) an RNA equivalent of a)-d). In
one alternative, the polynucleotide comprises at least 60
contiguous nucleotides.
[0042] Additionally, the invention provides a method for detecting
a target polynucleotide in a sample, said target polynucleotide
having a sequence of a polynucleotide comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO:7-12, b) a
naturally occurring polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the
group consisting of SEQ ID NO:7-12, c) a polynucleotide sequence
complementary to a), d) a polynucleotide sequence complementary to
b), and e) an RNA equivalent of a)-d). The method comprises 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. In one alternative, the probe
comprises at least 60 contiguous nucleotides.
[0043] The invention further provides a method for detecting a
target polynucleotide in a sample, said target polynucleotide
having a sequence of a polynucleotide comprising a polynucleotide
sequence selected from the group consisting of a) a polynucleotide
sequence selected from the group consisting of SEQ ID NO:7-12, b) a
naturally occurring polynucleotide sequence having at least 90%
sequence identity to a polynucleotide sequence selected from the
group consisting of SEQ ID NO:7-12, c) a polynucleotide sequence
complementary to a), d) a polynucleotide sequence complementary to
b), and e) an RNA equivalent of a)-d). The method comprises 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.
[0044] The invention further provides a composition comprising an
effective amount of a polypeptide comprising an amino acid sequence
selected from the group consisting of a) an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6, b) a naturally
occurring amino acid sequence having at least 90% sequence identity
to an amino acid sequence selected from the group consisting of SEQ
ID NO:1-6, c) a biologically active fragment of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, and
d) an immunogenic fragment of an amino acid sequence selected from
the group consisting of SEQ ID NO:1-6, and a pharmaceutically
acceptable excipient. In one embodiment, the composition comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO:1-6. The invention additionally provides a method of treating a
disease or condition associated with decreased expression of
functional LME, comprising administering to a patient in need of
such treatment the composition. The invention also provides a
method for screening a compound for effectiveness as an agonist of
a polypeptide comprising an amino acid sequence selected from the
group consisting of a) an amino acid sequence selected from the
group consisting of SEQ ID NO:1-6, b) a naturally occurring amino
acid sequence having at least 90% sequence identity to an amino
acid sequence selected from the group consisting of SEQ ID NO:1-6,
c) a biologically active fragment of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-6, and d) an
immunogenic fragment of an amino acid sequence selected from the
group consisting of SEQ ID NO:1-6. The method comprises a) exposing
a sample comprising the polypeptide to a compound, and b) detecting
agonist activity in the sample. In one alternative, the invention
provides a composition comprising an agonist compound identified by
the method and a pharmaceutically acceptable excipient. In another
alternative, the invention provides a method of treating a disease
or condition associated with decreased expression of functional
LME, comprising administering to a patient in need of such
treatment the composition.
[0045] Additionally, the invention provides a method for screening
a compound for effectiveness as an antagonist of a polypeptide
comprising an amino acid sequence selected from the group
consisting of a) an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6, b) a naturally occurring amino acid
sequence having at least 90% sequence identity to an amino acid
sequence selected from the group consisting of SEQ ID NO:1-6, c) a
biologically active fragment of an amino acid sequence selected
from the group consisting of SEQ ID NO:1-6, and d) an immunogenic
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6. The method comprises a) exposing a
sample comprising the polypeptide to a compound, and b) detecting
antagonist activity in the sample. In one alternative, the
invention provides a composition comprising an antagonist compound
identified by the method and a pharmaceutically acceptable
excipient. In another alternative, the invention provides a method
of treating a disease or condition associated with overexpression
of functional LME, comprising administering to a patient in need of
such treatment the composition.
[0046] The invention further provides a method of screening for a
compound that specifically binds to a polypeptide comprising an
amino acid sequence selected from the group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, b) a naturally occurring amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NO:1-6, c) a biologically active
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-6. The method comprises a) combining the polypeptide with at
least one test compound under suitable conditions, and b) detecting
binding of the polypeptide to the test compound, thereby
identifying a compound that specifically binds to the
polypeptide.
[0047] The invention further provides a method of screening for a
compound that modulates the activity of a polypeptide comprising an
amino acid sequence selected from the group consisting of a) an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-6, b) a naturally occurring amino acid sequence having at
least 90% sequence identity to an amino acid sequence selected from
the group consisting of SEQ ID NO:1-6, c) a biologically active
fragment of an amino acid sequence selected from the group
consisting of SEQ ID NO:1-6, and d) an immunogenic fragment of an
amino acid sequence selected from the group consisting of SEQ ID
NO:1-6. The method comprises a) combining the polypeptide with at
least one test compound under conditions permissive for the
activity of the polypeptide, b) assessing the activity of the
polypeptide in the presence of the test compound, and c) comparing
the activity of the polypeptide in the presence of the test
compound with the activity of the polypeptide in the absence of the
test compound, wherein a change in the activity of the polypeptide
in the presence of the test compound is indicative of a compound
that modulates the activity of the polypeptide.
[0048] The invention further provides a method for screening a
compound for effectiveness in altering expression of a target
polynucleotide, wherein said target polynucleotide comprises a
sequence selected from the group consisting of SEQ ID NO:7-12, the
method comprising a) exposing a sample comprising the target
polynucleotide to a compound, and b) detecting altered expression
of the target polynucleotide.
[0049] The invention further provides a method for assessing
toxicity of a test compound, said 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 comprising a polynucleotide sequence selected from
the group consisting of i) a polynucleotide sequence selected from
the group consisting of SEQ ID NO:7-12, ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:7-12, iii) a polynucleotide sequence complementary to i), iv)
a polynucleotide sequence complementary to ii), and v) an RNA
equivalent of i)-iv). Hybridization occurs 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 selected from
the group consisting of i) a polynucleotide sequence selected from
the group consisting of SEQ ID NO: 7-12, ii) a naturally occurring
polynucleotide sequence having at least 90% sequence identity to a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:7-12, iii) a polynucleotide sequence complementary to i), iv)
a polynucleotide sequence complementary to ii), and v) an RNA
equivalent of i)-iv). Alternatively, the target polynucleotide
comprises a fragment of a polynucleotide sequence selected from the
group consisting of i)-v) above; 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.
BRIEF DESCRIPTION OF THE TABLES
[0050] Table 1 summarizes the nomenclature for the full length
polynucleotide and polypeptide sequences of the present
invention.
[0051] Table 2 shows the GenBank identification number and
annotation of the nearest GenBank homolog for polypeptides of the
invention. The probability score for the match between each
polypeptide and its GenBank homolog is also shown.
[0052] Table 3 shows structural features of polypeptide sequences
of the invention, including predicted motifs and domains, along
with the methods, algorithms, and searchable databases used for
analysis of the polypeptides.
[0053] Table 4 lists the cDNA fragments which were used to assemble
polynucleotide sequences of the invention, along with selected
fragments of the polynucleotide sequences.
[0054] Table 5 shows the representative cDNA library for
polynucleotides of the invention.
[0055] Table 6 provides an appendix which describes the tissues and
vectors used for construction of the cDNA libraries shown in Table
5.
[0056] Table 7 shows the tools, programs, and algorithms used to
analyze the polynucleotides and polypeptides of the invention,
along with applicable descriptions, references, and threshold
parameters.
DETAILED DESCRIPTION OF THE INVENTION
[0057] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular machines, materials and methods
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.
[0058] 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.
[0059] 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 machines, materials, and methods similar or equivalent to those
described herein can be used to practice or test the present
invention, the preferred machines, materials and methods are now
described. All publications mentioned herein are cited for the
purpose of describing and disclosing the cell lines, protocols,
reagents and vectors which are reported in the publications and
which might be used in connection with the invention. Nothing
herein is to be construed as an admission that the invention is not
entitled to antedate such disclosure by virtue of prior
invention.
DEFINITIONS
[0060] "LME" refers to the amino acid sequences of substantially
purified LME obtained from any species, particularly a mammalian
species, including bovine, ovine, porcine, murine, equine, and
human, and from any source, whether natural, synthetic,
semi-synthetic, or recombinant.
[0061] The term "agonist" refers to a molecule which intensifies or
mimics the biological activity of LME. Agonists may include
proteins, nucleic acids, carbohydrates, small molecules, or any
other compound or composition which modulates the activity of LME
either by directly interacting with LME or by acting on components
of the biological pathway in which LME participates.
[0062] An "allelic variant" is an alternative form of the gene
encoding LME. Allelic variants 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. A gene may have none, one, or many allelic variants of
its naturally occurring form. Common mutational changes which give
rise to allelic variants 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.
[0063] "Altered" nucleic acid sequences encoding LME include those
sequences with deletions, insertions, or substitutions of different
nucleotides, resulting in a polypeptide the same as LME or a
polypeptide with at least one functional characteristic of LME.
Included within this definition are polymorphisms which may or may
not be readily detectable using a particular oligonucleotide probe
of the polynucleotide encoding LME, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
LME. 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 LME. 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 LME is retained. For example, negatively charged amino acids may
include aspartic acid and glutamic acid, and positively charged
amino acids may include lysine and arginine. Amino acids with
uncharged polar side chains having similar hydrophilicity values
may include: asparagine and glutamine; and serine and threonin.
Amino acids with uncharged side chains having similar
hydrophilicity values may include: leucine, isoleucine, and valine;
glycine and alanine; and phenylalanine and tyrosine.
[0064] The terms "amino acid" and "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide, or protein sequence, or a
fragment of any of these, and to naturally occurring or synthetic
molecules. Where "amino acid sequence" is recited to refer to a
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.
[0065] "Amplification" relates to the production of additional
copies of a nucleic acid sequence. Amplification is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art.
[0066] The term "antagonist" refers to a molecule which inhibits or
attenuates the biological activity of LME. Antagonists may include
proteins such as antibodies, nucleic acids, carbohydrates, small
molecules, or any other compound or composition which modulates the
activity of LME either by directly interacting with LME or by
acting on components of the biological pathway in which LME
participates.
[0067] The term "antibody" refers to intact immunoglobulin
molecules as well as to fragments thereof, such as Fab, F
(ab').sub.2, and Fv fragments, which are capable of binding an
epitopic determinant. Antibodies that bind LME 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.
[0068] The term "antigenic determinant" refers to that region 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 (particular 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.
[0069] The term "antisense" refers to any composition capable of
base-pairing with the "sense" (coding) strand of a specific nucleic
acid sequence. Antisense compositions may include DNA; RNA; peptide
nucleic acid (PNA); oligonucleotides having modified backbone
linkages such as phosphorothioates, methylphosphonates, or
benzylphosphonates; oligonucleotides having modified sugar groups
such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or
oligonucleotides having modified bases such as 5-methyl cytosine,
2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine. Antisense molecules
may be produced by any method including chemical synthesis or
transcription. Once introduced into a cell, the complementary
antisense molecule base-pairs with a naturally occurring nucleic
acid sequence produced by the cell to form duplexes which block
either transcription or translation. The designation "negative" or
"minus" can refer to the antisense strand, and the designation
"positive" or "plus" can refer to the sense strand of a reference
DNA molecule.
[0070] The term "biologically active" refers to a protein having
structural, regulatory, or biochemical functions of a naturally
occurring molecule. Likewise, "immunologically active" or
"immunogenic" refers to the capability of the natural, recombinant,
or synthetic LME, or of any oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to
bind with specific antibodies. "Complementary" describes the
relationship between two single-stranded nucleic acid sequences
that anneal by base-pairing. For example, 5'-AGT-3' pairs with its
complement, 3'-TCA-5'.
[0071] A "composition comprising a given polynucleotide sequence"
and a "composition comprising a given amino acid sequence" refer
broadly to any composition containing the given polynucleotide or
amino acid sequence. The composition may comprise a dry formulation
or an aqueous solution. Compositions comprising polynucleotide
sequences encoding LME or fragments of LME 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.,
sodium dodecyl sulfate; SDS), and other components (e.g.,
Denhardt's solution, dry milk, salmon sperm DNA, etc.).
[0072] "Consensus sequence" refers to a nucleic acid sequence which
has been subjected to repeated DNA sequence analysis to resolve
uncalled bases, extended using the XL-PCR kit (Applied Biosystems,
Foster City Calif.) in the 5' and/or the 3' direction, and
resequenced, or which has been assembled from one or more
overlapping cDNA, EST, or genomic DNA fragments using a computer
program for fragment assembly, such as the GEL VIEW fragment
assembly system (GCG, Madison Wis.) or Phrap (University of
Washington, Seattle Wash.). Some sequences have been both extended
and assembled to produce the consensus sequence. "Conservative
amino acid substitutions" are those substitutions that are
predicted to least interfere with the properties of the original
protein, i.e., the structure and especially the function of the
protein is conserved and not significantly changed by such
substitutions. The table below shows amino acids which may be
substituted for an original amino acid in a protein and which are
regarded as conservative amino acid substitutions.
TABLE-US-00001 Original Residue Conservative Substitution Ala Gly,
Ser Arg His, Lys Asn Asp, Gln, His Asp Asn, Glu Cys Ala, Ser Gln
Asn, Glu, His Glu Asp, Gln, His Gly Ala His Asn, Arg, Gin, Glu Ile
Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe His, Met,
Leu, Trp, Tyr Ser Cys, Thr Thr Ser, Val Trp Phe, Tyr Tyr His, Phe,
Trp Val Ile, Leu, Thr
[0073] Conservative amino acid substitutions generally maintain (a)
the structure of the polypeptide backbone in the area of the
substitution, for example, as a beta sheet or alpha helical
conformation, (b) the charge or hydrophobicity of the molecule at
the site of the substitution, and/or (c) the bulk of the side
chain.
[0074] A "deletion" refers to a change in the amino acid or
nucleotide sequence that results in the absence of one or more
amino acid residues or nucleotides. The term "derivative" refers to
a chemically modified polynucleotide or polypeptide. Chemical
modifications of a polynucleotide can include, for example,
replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino
group. A derivative polynucleotide encodes a polypeptide which
retains at least one biological or immunological function of the
natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any similar process that retains at
least one biological or immunological function of the polypeptide
from which it was derived.
[0075] A "detectable label" refers to a reporter molecule or enzyme
that is capable of generating a measurable signal and is covalently
or noncovalently joined to a polynucleotide or polypeptide.
[0076] A "fragment" is a unique portion of LME or the
polynucleotide encoding LME which is identical in sequence to but
shorter in length than the parent sequence. A fragment may comprise
up to the entire length of the defined sequence, minus one
nucleotide/amino acid residue. For example, a fragment may comprise
from 5 to 1000 contiguous nucleotides or amino acid residues. A
fragment used as a probe, primer, antigen, therapeutic molecule, or
for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40,
50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or
amino acid residues in length. Fragments may be preferentially
selected from certain regions of a molecule. For example, a
polypeptide fragment may comprise a certain length of contiguous
amino acids selected from the first 250 or 500 amino acids (or
first 25% or 50%) of a polypeptide as shown in a certain defined
sequence. Clearly these lengths are exemplary, and any length that
is supported by the specification, including the Sequence Listing,
tables, and figures, may be encompassed by the present
embodiments.
[0077] A fragment of SEQ ID NO:7-12 comprises a region of unique
polynucleotide sequence that specifically identifies SEQ ID
NO:7-12, for example, as distinct from any other sequence in the
genome from which the fragment was obtained. A fragment of SEQ ID
NO:7-12 is useful, for example, in hybridization and amplification
technologies and in analogous methods that distinguish SEQ ID
NO:7-12 from related polynucleotide sequences. The precise length
of a fragment of SEQ ID NO:7-12 and the region of SEQ ID NO:7-12 to
which the fragment corresponds are routinely determinable by one of
ordinary skill in the art based on the intended purpose for the
fragment.
[0078] A fragment of SEQ ID NO:1-6 is encoded by a fragment of SEQ
ID NO:7-12. A fragment of SEQ ID NO:1-6 comprises a region of
unique amino acid sequence that specifically identifies SEQ ID
NO:1-6. For example, a fragment of SEQ ID NO:1-6 is useful as an
immunogenic peptide for the development of antibodies that
specifically recognize SEQ ID NO:1-6. The precise length of a
fragment of SEQ ID NO:1-6 and the region of SEQ ID NO:1-6 to which
the fragment corresponds are routinely determinable by one of
ordinary skill in the art based on the intended purpose for the
fragment.
[0079] A "full length" polynucleotide sequence is one containing at
least a translation initiation codon (e.g., methionine) followed by
an open reading frame and a translation termination codon. A "full
length" polynucleotide sequence encodes a "full length" polypeptide
sequence.
[0080] "Homology" refers to sequence similarity or,
interchangeably, sequence identity, between two or more
polynucleotide sequences or two or more polypeptide sequences.
[0081] The terms "percent identity" and "% identity," as applied to
polynucleotide sequences, refer to the percentage of residue
matches between at least two polynucleotide sequences aligned using
a standardized algorithm. Such an algorithm may insert, in a
standardized and reproducible way, gaps in the sequences being
compared in order to optimize alignment between two sequences, and
therefore achieve a more meaningful comparison of the two
sequences.
[0082] Percent identity between polynucleotide sequences may be
determined using the default parameters of the CLUSTAL V algorithm
as incorporated into the MEGALIGN version 3.12e sequence alignment
program. This program is part of the LASERGENE software package, a
suite of molecular biological analysis programs (DNASTAR, Madison
Wis.). CLUSTAL V is described in Higgins, D. G. and P. M. Sharp
(1989) CABIOS 5:151-153 and in Higgins, D. G. et al. (1992) CABIOS
8:189-191. For pairwise alignments of polynucleotide sequences, the
default parameters are set as follows: Ktuple=2, gap penalty=5,
window=4, and "diagonals saved"=4. The "weighted" residue weight
table is selected as the default. Percent identity is reported by
CLUSTAL V as the "percent similarity" between aligned
polynucleotide sequences.
[0083] Alternatively, a suite of commonly used and freely available
sequence comparison algorithms is provided by the National Center
for Biotechnology Information (NCBI) Basic Local Alignment Search
Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol.
215:403-410), which is available from several sources, including
the NCBI, Bethesda, Md., and on the Internet at
http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST software suite
includes various sequence analysis programs including "blastn,"
that is used to align a known polynucleotide sequence with other
polynucleotide sequences from a variety of databases. Also
available is a tool called "BLAST 2 Sequences" that is used for
direct pairwise comparison of two nucleotide sequences. "BLAST 2
Sequences" can be accessed and used interactively on the internet.
The "BLAST 2 Sequences" tool can be used for both blastn and blastp
(discussed below). BLAST programs are commonly used with gap and
other parameters set to default settings. For example, to compare
two nucleotide sequences, one may use blastn with the "BLAST 2
Sequences" tool Version 2.0.12 (Apr. 21, 2000) set at default
parameters. Such default parameters may be, for example:
[0084] Matrix: BLOSUM62
[0085] Reward for match: 1
[0086] Penalty for mismatch: -2
[0087] Open Gap: 5 and Extension Gap: 2 penalties
[0088] Gap.times.drop-off: 50
[0089] Expect: 10
[0090] Word Size: 11
[0091] Filter: on
[0092] Percent identity may be measured over the length of an
entire defined sequence, for example, as defined by a particular
SEQ ID number, or may be measured over a shorter length, for
example, over the length of a fragment taken from a larger, defined
sequence, for instance, a fragment of at least 20, at least 30, at
least 40, at least 50, at least 70, at least 100, or at least 200
contiguous nucleotides. Such lengths are exemplary only, and it is
understood that any fragment length supported by the sequences
shown herein, in the tables, figures, or Sequence Listing, may be
used to describe a length over which percentage identity may be
measured.
[0093] Nucleic acid sequences that do not show a high degree of
identity may nevertheless encode similar amino acid sequences due
to the degeneracy of the genetic code. It is understood that
changes in a nucleic acid sequence can be made using this
degeneracy to produce multiple nucleic acid sequences that all
encode substantially the same protein.
[0094] The phrases "percent identity" and "% identity," as applied
to polypeptide sequences, refer to the percentage of residue
matches between at least two polypeptide sequences aligned using a
standardized algorithm. Methods of polypeptide sequence alignment
are well-known. Some alignment methods take into account
conservative amino acid substitutions. Such conservative
substitutions, explained in more detail above, generally preserve
the charge and hydrophobicity at the site of substitution, thus
preserving the structure (and therefore function) of the
polypeptide.
[0095] Percent identity between polypeptide sequences may be
determined using the default parameters of the CLUSTAL V algorithm
as incorporated into the MEGALIGN version 3.12e sequence alignment
program (described and referenced above). For pairwise alignments
of polypeptide sequences using CLUSTAL V, the default parameters
are set as follows: Ktuple=1, gap penalty=3, window=5, and
"diagonals saved"=5. The PAM250 matrix is selected as the default
residue weight table. As with polynucleotide alignments, the
percent identity is reported by CLUSTAL V as the "percent
similarity" between aligned polypeptide sequence pairs.
Alternatively the NCBI BLAST software suite may be used. For
example, for a pairwise comparison of two polypeptide sequences,
one may use the "BLAST 2 Sequences" tool Version 2.0.12 (Apr. 21,
2000) with blastp set at default parameters.
Such default parameters may be, for example:
[0096] Matrix: BLOSUM62
[0097] Open Gap: 11 and Extension Gap: I penalties
[0098] Gap.times.drop-off: 50
[0099] Expect: 10
[0100] Word Size: 3
[0101] Filter: on
[0102] Percent identity may be measured over the length of an
entire defined polypeptide sequence, for example, as defined by a
particular SEQ ID number, or may be measured over a shorter length,
for example, over the length of a fragment taken from a larger,
defined polypeptide sequence, for instance, a fragment of at least
15, at least 20, at least 30, at least 40, at least 50, at least 70
or at least 150 contiguous residues. Such lengths are exemplary
only, and it is understood that any fragment length supported by
the sequences shown herein, in the tables, figures or Sequence
Listing, may be used to describe a length over which percentage
identity may be measured.
[0103] "Human artificial chromosomes" (HACs) are linear
microchromosomes which may contain DNA sequences of about 6 kb to
10 Mb in size and which contain all of the elements required for
chromosome replication, segregation and maintenance.
[0104] The term "humanized antibody" refers to an antibody molecule
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.
[0105] "Hybridization" refers to the process by which a
polynucleotide strand anneals with a complementary strand through
base pairing under defined hybridization conditions. Specific
hybridization is an indication that two nucleic acid sequences
share a high degree of complementarity. Specific hybridization
complexes form under permissive annealing conditions and remain
hybridized after the "washing" step(s). The washing step(s) is
particularly important in determining the stringency of the
hybridization process, with more stringent conditions allowing less
non-specific binding, i.e., binding between pairs of nucleic acid
strands that are not perfectly matched. Permissive conditions for
annealing of nucleic acid sequences are routinely determinable by
one of ordinary skill in the art and may be consistent among
hybridization experiments, whereas wash conditions may be varied
among experiments to achieve the desired stringency, and therefore
hybridization specificity. Permissive annealing conditions occur,
for example, at 68.degree. C. in the presence of about 6.times.SSC,
about 1% (w/v) SDS, and about 100 .mu.g/ml sheared, denatured
salmon sperm DNA.
[0106] Generally, stringency of hybridization is expressed, in
part, with reference to the temperature under which the wash step
is carried out. Such wash temperatures are typically selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. An equation for
calculating T.sub.m and conditions for nucleic acid hybridization
are well known and can be found in Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Press, Plainview N.Y.; specifically see volume
2, chapter 9.
[0107] High stringency conditions for hybridization between
polynucleotides of the present invention include wash conditions of
68.degree. C. in the presence of about 0.2.times.SSC and about 0.1%
SDS, for 1 hour. Alternatively, temperatures of about 65.degree.
C., 60.degree. C., 55.degree. C., or 42.degree. C. may be used. SSC
concentration may be varied from about 0.1 to 2.times.SSC, with SDS
being present at about 0.1%. Typically, blocking reagents are used
to block non-specific hybridization. Such blocking reagents
include, for instance, sheared and denatured salmon sperm DNA at
about 100-200 .mu.g/ml. Organic solvent, such as formamide at a
concentration of about 35-50% v/v, may also be used under
particular circumstances, such as for RNA: DNA hybridizations.
Useful variations on these wash conditions will be readily apparent
to those of ordinary skill in the art. Hybridization, particularly
under high stringency conditions, may be suggestive of evolutionary
similarity between the nucleotides. Such similarity is strongly
indicative of a similar role for the nucleotides and their encoded
polypeptides.
[0108] The term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of
hydrogen bonds between complementary bases. A hybridization complex
may be formed in solution (e.g., 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).
[0109] The words "insertion" and "addition" refer to changes in an
amino acid or nucleotide sequence resulting in the addition of one
or more amino acid residues or nucleotides, respectively.
[0110] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems. An "immunogenic fragment" is a polypeptide or oligopeptide
fragment of LME which is capable of eliciting an immune response
when introduced into a living organism, for example, a mammal. The
term "immunogenic fragment" also includes any polypeptide or
oligopeptide fragment of LME which is useful in any of the antibody
production methods disclosed herein or known in the art.
[0111] The term "microarray" refers to an arrangement of a
plurality of polynucleotides, polypeptides, or other chemical
compounds on a substrate.
[0112] The terms "element" and "array element" refer to a
polynucleotide, polypeptide, or other chemical compound having a
unique and defined position on a microarray.
[0113] The term "modulate" refers to a change in the activity of
LME. For example, modulation may cause an increase or a decrease in
protein activity, binding characteristics, or any other biological,
functional, or immunological properties of LME.
[0114] The phrases "nucleic acid" and "nucleic acid sequence" refer
to a nucleotide, oligonucleotide, polynucleotide, or any fragment
thereof. These phrases also refer 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.
[0115] "Operably linked" refers to the situation in which a first
nucleic acid sequence is placed in a functional relationship with a
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Operably linked
DNA sequences may be in close proximity or contiguous and, where
necessary to join two protein coding regions, in the same reading
frame.
[0116] "Peptide nucleic acid" (PNA) refers to an antisense molecule
or anti-gene agent which comprises an oligonucleotide of at least
about 5 nucleotides in length linked to a peptide backbone of amino
acid residues ending in lysine. The terminal lysine confers
solubility to the composition. PNAs preferentially bind
complementary single stranded DNA or RNA and stop transcript
elongation, and may be pegylated to extend their lifespan in the
cell.
[0117] "Post-translational modification" of an LME may involve
lipidation, glycosylation, phosphorylation, acetylation,
racemization, proteolytic cleavage, and other modifications known
in the art. These processes may occur synthetically or
biochemically. Biochemical modifications will vary by cell type
depending on the enzymatic milieu of LME.
[0118] "Probe" refers to nucleic acid sequences encoding LME, their
complements, or fragments thereof, which are used to detect
identical, allelic or related nucleic acid sequences. Probes are
isolated oligonucleotides or polynucleotides attached to a
detectable label or reporter molecule. Typical labels include
radioactive isotopes, ligands, chemiluminescent agents, and
enzymes. "Primers" are short nucleic acids, usually DNA
oligonucleotides, which may be annealed to a target polynucleotide
by complementary base-pairing. The primer may then be extended
along the target DNA strand by a DNA polymerase enzyme. Primer
pairs can be used for amplification (and identification) of a
nucleic acid sequence, e.g., by the polymerase chain reaction
(PCR).
[0119] Probes and primers as used in the present invention
typically comprise at least 15 contiguous nucleotides of a known
sequence. In order to enhance specificity, longer probes and
primers may also be employed, such as probes and primers that
comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at
least 150 consecutive nucleotides of the disclosed nucleic acid
sequences. Probes and primers may be considerably longer than these
examples, and it is understood that any length supported by the
specification, including the tables, figures, and Sequence Listing,
may be used.
[0120] Methods for preparing and using probes and primers are
described in the references, for example Sambrook, J. et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3,
Cold Spring Harbor Press, Plainview N.Y.; Ausubel, F. M. et al.
(1987) Current Protocols in Molecular Biology, Greene Publ. Assoc.
& Wiley-Intersciences, New York N.Y.; Innis, M. et al. (1990)
PCR Protocols. A Guide to Methods and Applications, Academic Press,
San Diego Calif. PCR primer pairs can be derived from a known
sequence, for example, by using computer programs intended for that
purpose such as Primer (Version 0.5, 1991, Whitehead Institute for
Biomedical Research, Cambridge Mass.).
[0121] Oligonucleotides for use as primers are selected using
software known in the art for such purpose. For example, OLIGO 4.06
software is useful for the selection of PCR primer pairs of up to
100 nucleotides each, and for the analysis of oligonucleotides and
larger polynucleotides of up to 5,000 nucleotides from an input
polynucleotide sequence of up to 32 kilobases. Similar primer
selection programs have incorporated additional features for
expanded capabilities. For example, the PrimOU primer selection
program (available to the public from the Genome Center at
University of Texas South West Medical Center, Dallas Tex.) is
capable of choosing specific primers from megabase sequences and is
thus useful for designing primers on a genome-wide scope. The
Primer3 primer selection program (available to the public from the
Whitehead Institute/MIT Center for Genome Research, Cambridge
Mass.) allows the user to input a "mispriming library," in which
sequences to avoid as primer binding sites are user-specified.
Primer3 is useful, in particular, for the selection of
oligonucleotides for microarrays. (The source code for the latter
two primer selection programs may also be obtained from their
respective sources and modified to meet the user's specific needs.)
The PrimeGen program (available to the public from the UK Human
Genome Mapping Project Resource Centre, Cambridge UK) designs
primers based on multiple sequence alignments, thereby allowing
selection of primers that hybridize to either the most conserved or
least conserved regions of aligned nucleic acid sequences. Hence,
this program is useful for identification of both unique and
conserved oligonucleotides and polynucleotide fragments. The
oligonucleotides and polynucleotide fragments identified by any of
the above selection methods are useful in hybridization
technologies, for example, as PCR or sequencing primers, microarray
elements, or specific probes to identify fully or partially
complementary polynucleotides in a sample of nucleic acids. Methods
of oligonucleotide selection are not limited to those described
above.
[0122] A "recombinant nucleic acid" is a sequence that is not
naturally occurring or has a sequence that is made by an artificial
combination of two or more otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques such as those described in Sambrook,
supra. The term recombinant includes nucleic acids that have been
altered solely by addition, substitution, or deletion of a portion
of the nucleic acid. Frequently, a recombinant nucleic acid may
include a nucleic acid sequence operably linked to a promoter
sequence. Such a recombinant nucleic acid may be part of a vector
that is used, for example, to transform a cell.
[0123] Alternatively, such recombinant nucleic acids may be part of
a viral vector, e.g., based on a vaccinia virus, that could be use
to vaccinate a mammal wherein the recombinant nucleic acid is
expressed, inducing a protective immunological response in the
mammal.
[0124] A "regulatory element" refers to a nucleic acid sequence
usually derived from untranslated regions of a gene and includes
enhancers, promoters, introns, and 5' and 3' untranslated regions
(UTRs). Regulatory elements interact with host or viral proteins
which control transcription, translation, or RNA stability.
[0125] "Reporter molecules" are chemical or biochemical moieties
used for labeling a nucleic acid, amino acid, or antibody. Reporter
molecules include radionuclides; enzymes; fluorescent,
chemiluminescent, or chromogenic agents; substrates; cofactors;
inhibitors; magnetic particles; and other moieties known in the
art. An "RNA equivalent," in reference to a DNA sequence, is
composed of the same linear sequence of nucleotides as the
reference DNA sequence with the exception that all occurrences of
the nitrogenous base thymine are replaced with uracil, and the
sugar backbone is composed of ribose instead of deoxyribose.
[0126] The term "sample" is used in its broadest sense. A sample
suspected of containing LME, nucleic acids encoding LME, or
fragments thereof 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
substrate;
[0127] a tissue; a tissue print; etc.
[0128] The terms "specific binding" and "specifically binding"
refer to that interaction between a protein or peptide and an
agonist, an antibody, an antagonist, a small molecule, or any
natural or synthetic binding composition. The interaction is
dependent upon the presence of a particular structure of the
protein, e.g., the antigenic determinant or epitope, recognized by
the binding molecule. For example, if an antibody is specific for
epitope "A," the presence of a polypeptide comprising 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.
[0129] The term "substantially purified" refers to nucleic acid or
amino acid sequences that are removed from their natural
environment and are isolated or separated, and are at least 60%
free, preferably at least 75% free, and most preferably at least
90% free from other components with which they are naturally
associated. A "substitution" refers to the replacement of one or
more amino acid residues or nucleotides by different amino acid
residues or nucleotides, respectively.
[0130] "Substrate" refers to any suitable rigid or semi-rigid
support including membranes, filters, chips, slides, wafers,
fibers, magnetic or nonmagnetic beads, gels, tubing, plates,
polymers, microparticles and capillaries. The substrate can have a
variety of surface forms, such as wells, trenches, pins, channels
and pores, to which polynucleotides or polypeptides are bound.
[0131] A "transcript image" refers to the collective pattern of
gene expression by a particular cell type or tissue under given
conditions at a given time.
[0132] "Transformation" describes a process by which exogenous DNA
is introduced into 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, bacteriophage or viral infection,
electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed cells" includes stably transformed cells in
which the inserted DNA is capable of replication either as an
autonomously replicating plasmid or as part of the host chromosome,
as well as transiently transformed cells which express the inserted
DNA or RNA for limited periods of time.
[0133] A "transgenic organism," as used herein, is any organism,
including but not limited to animals and plants, in which one or
more of the cells of the organism contains heterologous nucleic
acid introduced by way of human intervention, such as by transgenic
techniques well known in the art. The nucleic acid is introduced
into the cell, directly or indirectly by introduction into a
precursor of the cell, by way of deliberate genetic manipulation,
such as by microinjection or by infection with a recombinant virus.
The term genetic manipulation does not include classical
cross-breeding, or in vitro fertilization, but rather is directed
to the introduction of a recombinant DNA molecule. The transgenic
organisms contemplated in accordance with the present invention
include bacteria, cyanobacteria, fungi, plants and animals. The
isolated DNA of the present invention can be introduced into the
host by methods known in the art, for example infection,
transfection, transformation or transconjugation. Techniques for
transferring the DNA of the present invention into such organisms
are widely known and provided in references such as Sambrook et al.
(1989), supra.
[0134] A "variant" of a particular nucleic acid sequence is defined
as a nucleic acid sequence having at least 40% sequence identity to
the particular nucleic acid sequence over a certain length of one
of the nucleic acid sequences using blastn with the "BLAST 2
Sequences" tool Version 2.0.9 (May 7, 1999) set at default
parameters. Such a pair of nucleic acids may show, for example, at
least 50%, at least 60%, at least 70%, at least 80%, at least 85%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, or at
least 99% or greater sequence identity over a certain defined
length. A variant may be described as, for example, an "allelic"
(as defined above), "splice," "species," or "polymorphic" variant.
A splice variant may have significant identity to a reference
molecule, but will generally have a greater or lesser number of
polynucleotides due to alternative splicing of exons during mRNA
processing. The corresponding polypeptide may possess additional
functional domains or lack domains that are present in the
reference molecule. Species variants are polynucleotide sequences
that vary from one species to another. The resulting polypeptides
will generally have significant amino acid identity relative to
each other. A polymorphic variant is a variation in the
polynucleotide sequence of a particular gene between individuals of
a given species. Polymorphic variants also may encompass "single
nucleotide polymorphisms" (SNPs) in which the polynucleotide
sequence varies by one nucleotide base. The presence of SNPs may be
indicative of, for example, a certain population, a disease state,
or a propensity for a disease state.
[0135] A "variant" of a particular polypeptide sequence is defined
as a polypeptide sequence having at least 40% sequence identity to
the particular polypeptide sequence over a certain length of one of
the polypeptide sequences using blastp with the "BLAST 2 Sequences"
tool Version 2.0.9 (May 7, 1999) set at default parameters. Such a
pair of polypeptides may show, for example, at least 50%, at least
60%, at least 70%, at least 80%, at least 90%, at least 91%, at
least 92%, at least 93%, at least 94%, at least 95%, at least 96%,
at least 97%, at least 98%, or at least 99% or greater sequence
identity over a certain defined length of one of the
polypeptides.
The Invention
[0136] The invention is based on the discovery of new human lipid
metabolism enzymes (LME), the polynucleotides encoding LME, and the
use of these compositions for the diagnosis, treatment, or
prevention of cancer, neurological disorders,
autoimmune/inflammatory disorders, gastrointestinal disorders, and
cardiovascular disorders.
[0137] Table 1 summarizes the nomenclature for the full length
polynucleotide and polypeptide sequences of the invention. Each
polynucleotide and its corresponding polypeptide are correlated to
a single Incyte project identification number (Incyte Project ID).
Each polypeptide sequence is denoted by both a polypeptide sequence
identification number (Polypeptide SEQ ID NO:) and an Incyte
polypeptide sequence number (Incyte Polypeptide ID) as shown. Each
polynucleotide sequence is denoted by both a polynucleotide
sequence identification number (Polynucleotide SEQ ID NO:) and an
Incyte polynucleotide consensus sequence number (Incyte
Polynucleotide ID) as shown.
[0138] Table 2 shows sequences with homology to the polypeptides of
the invention as identified by BLAST analysis against the GenBank
protein (genpept) database. Columns 1 and 2 show the polypeptide
sequence identification number (Polypeptide SEQ ID NO:) and the
corresponding Incyte polypeptide sequence number (Incyte
Polypeptide ID) for polypeptides of the invention. Column 3 shows
the GenBank identification number (Genbank ID NO:) of the nearest
GenBank homolog. Column 4 shows the probability score for the match
between each polypeptide and its GenBank homolog. Column 5 shows
the annotation of the GenBank homolog along with relevant citations
where applicable, all of which are expressly incorporated by
reference herein.
[0139] Table 3 shows various structural features of the
polypeptides of the invention. Columns 1 and 2 show the polypeptide
sequence identification number (SEQ ID NO:) and the corresponding
Incyte polypeptide sequence number (Incyte Polypeptide ID) for each
polypeptide of the invention. Column 3 shows the number of amino
acid residues in each polypeptide. Column 4 shows potential
phosphorylation sites, and column 5 shows potential glycosylation
sites, as determined by the MOTIFS program of the GCG sequence
analysis software package (Genetics Computer Group, Madison Wis.).
Column 6 shows amino acid residues comprising signature sequences,
domains, and motifs. Column 7 shows analytical methods for protein
structure/function analysis and in some cases, searchable databases
to which the analytical methods were applied.
[0140] Together, Tables 2 and 3 summarize the properties of
polypeptides of the invention, and these properties establish that
the claimed polypeptides are lipid metabolism enzymes. For example,
SEQ ID NO: 1 is 50% identical, from residue N63 to residue H300, to
a C. elegans protein having similarity to human enoyl-CoA hydratase
(GenBank ID g3876901) as determined by the Basic Local Alignment
Search Tool (BLAST). (See Table 2.) The BLAST probability score is
2.50E-56, which indicates the probability of obtaining the observed
polypeptide sequence alignment by chance. SEQ ID NO: 1 also
contains an enoyl-CoA hydratase/isomerase family domain as
determined by searching for statistically significant matches in
the hidden Markov model (HMM)-based PFAM database of conserved
protein family domains. (See Table 3.) Data from BLIMPS and
PROFILESCAN analyses provide further corroborative evidence that
SEQ ID NO: 1 is an enoyl-CoA hydratase. SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 were analyzed and
annotated in a similar manner. The algorithms and parameters for
the analysis of SEQ ID NO:1-6 are described in Table 7.
[0141] As shown in Table 4, the full length polynucleotide
sequences of the present invention were assembled using cDNA
sequences or coding (exon) sequences derived from genomic DNA, or
any combination of these two types of sequences. Columns 1 and 2
list the polynucleotide sequence identification number
(Polynucleotide SEQ ID NO:) and the corresponding Incyte
polynucleotide consensus sequence number (Incyte Polynucleotide ID)
for each polynucleotide of the invention. Column 3 shows the length
of each polynucleotide sequence in basepairs. Column 4 lists
fragments of the polynucleotide sequences which are useful, for
example, in hybridization or amplification technologies that
identify SEQ ID NO: 7-12 or that distinguish between SEQ ID NO:
7-12 and related polynucleotide sequences. Column 5 shows
identification numbers corresponding to cDNA sequences, coding
sequences (exons) predicted from genomic DNA, and/or sequence
assemblages comprised of both cDNA and genomic DNA. These sequences
were used to assemble the full length polynucleotide sequences of
the invention. Columns 6 and 7 of Table 4 show the nucleotide start
(5') and stop (3') positions of the cDNA sequences in column 5
relative to their respective full length sequences.
[0142] The identification numbers in Column 5 of Table 4 may refer
specifically, for example, to Incyte cDNAs along with their
corresponding cDNA libraries. For example, 6827519J1 is the
identification number of an Incyte cDNA sequence, and SINTNOR01 is
the cDNA library from which it is derived. Incyte cDNAs for which
cDNA libraries are not indicated were derived from pooled cDNA
libraries (e.g., 71530085V1). Alternatively, the identification
numbers in column 5 may refer to GenBank cDNAs or ESTs which
contributed to the assembly of the full length polynucleotide
sequences: Alternatively, the identification numbers in column 5
may refer to coding regions predicted by Genscan analysis of
genomic DNA. The Genscan-predicted coding sequences may have been
edited prior to assembly. (See Example IV.) Alternatively, the
identification numbers in column 5 may refer to assemblages of both
cDNA and Genscan-predicted exons brought together by an "exon
stitching" algorithm. (See Example V.) Alternatively, the
identification numbers in column 5 may refer to assemblages of both
cDNA and Genscan-predicted exons brought together by an
"exon-stretching" algorithm. (See Example V.) In some cases, Incyte
cDNA coverage redundant with the sequence coverage shown in column
5 was obtained to confirm the final consensus polynucleotide
sequence, but the relevant Incyte cDNA identification numbers are
not shown.
[0143] Table 5 shows the representative cDNA libraries for those
full length polynucleotide sequences which were assembled using
Incyte cDNA sequences. The representative cDNA library is the
Incyte cDNA library which is most frequently represented by the
Incyte cDNA sequences which were used to assemble and confirm the
above polynucleotide sequences. The tissues and vectors which were
used to construct the cDNA libraries shown in Table 5 are described
in Table 6.
[0144] The invention also encompasses LME variants. A preferred LME
variant is one which has at least about 80%, or alternatively at
least about 90%, or even at least about 95% amino acid sequence
identity to the LME amino acid sequence, and which contains at
least one functional or structural characteristic of LME.
[0145] The invention also encompasses polynucleotides which encode
LME. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising a sequence selected from the
group consisting of SEQ ID NO: 7-12, which encodes LME. The
polynucleotide sequences of SEQ ID NO: 7-12, as presented in the
Sequence Listing, embrace the equivalent RNA sequences, wherein
occurrences of the nitrogenous base thymine are replaced with
uracil, and the sugar backbone is composed of ribose instead of
deoxyribose.
[0146] The invention also encompasses a variant of a polynucleotide
sequence encoding LME. In particular, such a variant polynucleotide
sequence will have at least about 70%, or alternatively at least
about 85%, or even at least about 95% polynucleotide sequence
identity to the polynucleotide sequence encoding LME. A particular
aspect of the invention encompasses a variant of a polynucleotide
sequence comprising a sequence selected from the group consisting
of SEQ ID NO: 7-12 which has at least about 70%, or alternatively
at least about 85%, or even at least about 95% polynucleotide
sequence identity to a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 7-12. Any one of the polynucleotide
variants described above can encode an amino acid sequence which
contains at least one functional or structural characteristic of
LME.
[0147] 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 LME, some bearing minimal
similarity 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 LME, and all such
variations are to be considered as being specifically
disclosed.
[0148] Although nucleotide sequences which encode LME and its
variants are generally capable of hybridizing to the nucleotide
sequence of the naturally occurring LME under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding LME or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. 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 LME 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.
[0149] The invention also encompasses production of DNA sequences
which encode LME and LME 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 well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding LME or any fragment thereof.
[0150] 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: 7-12 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) Hybridization conditions, including annealing and
wash conditions, are described in "Definitions."
[0151] Methods for DNA sequencing are well known in the art and may
be used to practice any of the embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE (US Biochemical, Cleveland Ohio), Taq
polymerase (Applied Biosystems), thermostable T7 polymerase
(Amersham Pharmacia Biotech, Piscataway N.J.), or combinations of
polymerases and proofreading exonucleases such as those found in
the ELONGASE amplification system (Life Technologies, Gaithersburg
Md.). Preferably, sequence preparation is automated with machines
such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno
Nev.), PTC200 thermal cycler (MJ Research, Watertown Mass.) and ABI
CATALYST 800 thermal cycler (Applied Biosystems). Sequencing is
then carried out using either the ABI 373 or 377 DNA sequencing
system (Applied Biosystems), the MEGABACE 1000 DNA sequencing
system (Molecular Dynamics, Sunnyvale Calif.), or other systems
known in the art. The resulting sequences are analyzed using a
variety of algorithms which are well known in the art. (See, e.g.,
Ausubel, F. M. (1997) Short Protocols in Molecular Biology, John
Wiley & Sons, New York N.Y., unit 7.7; Meyers, R. A. (1995)
Molecular Biology and Biotechnology, Wiley VCH, New York N.Y., pp.
856-853.)
[0152] The nucleic acid sequences encoding LME may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based 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 and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries (Clontech, Palo Alto Calif.) to walk genomic DNA. This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences, Plymouth
Minn.) or another appropriate program, to be about 22 to 30
nucleotides in length, to have a GC content of about 50% or more,
and to anneal to the template at temperatures of about 68.degree.
C. to 72.degree. C.
[0153] When screening for full length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are 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.
[0154] 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 nucleotide-specific, laser-stimulated
fluorescent dyes, 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, Applied Biosystems), and the entire process
from loading of samples to computer analysis and electronic data
display may be computer controlled. Capillary electrophoresis is
especially preferable for sequencing small DNA fragments which may
be present in limited amounts in a particular sample.
[0155] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode LME may be cloned in
recombinant DNA molecules that direct expression of LME, 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 used to express
LME.
[0156] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter LME-encoding sequences for a variety of purposes including,
but not limited to, modification of 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, oligonucleotide-mediated site-directed mutagenesis may
be used to introduce mutations that create new restriction sites,
alter glycosylation patterns, change codon preference, produce
splice variants, and so forth.
[0157] The nucleotides of the present invention may be subjected to
DNA shuffling techniques such as MOLECULAR BREEDING (Maxygen Inc.,
Santa Clara Calif.; described in U.S. Pat. No. 5,837,458; Chang,
C.-C. et al. (1999) Nat. Biotechnol. 17:793-797; Christians, F. C.
et al. (1999) Nat. Biotechnol. 17:259-264; and Crameri, A. et al.
(1996) Nat. Biotechnol. 14:315-319) to alter or improve the
biological properties of LME, such as its biological or enzymatic
activity or its ability to bind to other molecules or compounds.
DNA shuffling is a process by which a library of gene variants is
produced using PCR-mediated recombination of gene fragments. The
library is then subjected to selection or screening procedures that
identify those gene variants with the desired properties. These
preferred variants may then be pooled and further subjected to
recursive rounds of DNA shuffling and selection/screening. Thus,
genetic diversity is created through "artificial" breeding and
rapid molecular evolution. For example, fragments of a single gene
containing random point mutations may be recombined, screened, and
then reshuffled until the desired properties are optimized.
Alternatively, fragments of a given gene may be recombined with
fragments of homologous genes in the same gene family, either from
the same or different species, thereby maximizing the genetic
diversity of multiple naturally occurring genes in a directed and
controllable manner.
[0158] In another embodiment, sequences encoding LME 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) Nucleic
Acids Symp. Ser. 7:215-223; and Horn, T. et al. (1980) Nucleic
Acids Symp. Ser. 7:225-232.) Alternatively, LME itself or a
fragment thereof may be synthesized using chemical methods. For
example, peptide synthesis can be performed using various
solution-phase or solid-phase techniques. (See, e.g., Creighton, T.
(1984) Proteins, Structures and Molecular Properties, WH Freeman,
New York N.Y., pp. 55-60; and Roberge, J. Y. et al. (1995) Science
269:202-204.) Automated synthesis may be achieved using the ABI 431
A peptide synthesizer (Applied Biosystems). Additionally, the amino
acid sequence of LME, 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 or
a polypeptide having a sequence of a naturally occurring
polypeptide.
[0159] The peptide may be substantially purified by preparative
high performance. liquid chromatography. (See, e.g., Chiez, R. M.
and F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The
composition of the synthetic peptides may be confirmed by amino
acid analysis or by sequencing. (See, e.g., Creighton, supra, pp.
28-53.)
[0160] In order to express a biologically active LME, the
nucleotide sequences encoding LME or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding LME. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding LME. Such
signals include the ATG initiation codon and adjacent sequences,
e.g. the Kozak sequence. In cases where sequences encoding LME and
its initiation codon and upstream regulatory 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 an in-frame ATG initiation codon should be provided by
the vector. 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 host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0161] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding LME and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
N.Y., ch. 4, 8, and 16-17; Ausubel, F. M. et al. (1995) Current
Protocols in Molecular Biology, John Wiley & Sons, New York
N.Y., ch. 9, 13, and 16.)
[0162] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding LME. 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 viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral 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. (See, e.g., Sambrook,
supra; Ausubel, supra; Van Heeke, G. and S. M. Schuster (1989) J.
Biol. Chem. 264:5503-5509; Engelhard, E. K. et al. (1994) Proc.
Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum.
Gene Ther. 7:1937-1945; Takamatsu, N. (1987) EMBO J. 6:307-311; The
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill,
New York N.Y., pp. 191-196; Logan, J. and T. Shenk (1984) Proc.
Natl. Acad. Sci. USA 81:3655-3659; and Harrington, J. J. et al.
(1997) Nat. Genet. 15:345-355.) 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. (See,
e.g., Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5(6):350-356;
Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(13):6340-6344;
Buller, R. M. et al. (1985) Nature 317(6040):813-815; McGregor, D.
P. et al. (1994) Mol. Immunol. 31(3):219-226; and Verma, I. M. and
N. Somia (1997) Nature 389:239-242.) The invention is not limited
by the host cell employed.
[0163] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding LME. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding LME can be achieved using a multifunctional E. coli vector
such as PBLUESCRIPT (Stratagene, La Jolla Calif.) or PSPORT1
plasmid (Life Technologies). Ligation of sequences encoding LME
into the vector's multiple cloning site disrupts the lacZ gene,
allowing a colorimetric screening procedure for identification of
transformed bacteria containing recombinant molecules. In addition,
these vectors may be useful for in vitro transcription, dideoxy
sequencing, single strand rescue with helper phage, and creation of
nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S. M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) When large
quantities of LME are needed, e.g. for the production of
antibodies, vectors which direct high level expression of LME may
be used. For example, vectors containing the strong, inducible SP6
or T7 bacteriophage promoter may be used.
[0164] Yeast expression systems may be used for production of LME.
A number of vectors containing constitutive or inducible promoters,
such as alpha factor, alcohol oxidase, and PGH promoters, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastors. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, supra; Bitter, G. A. et al. (1987) Methods Enzymol.
153:516-544; and Scorer, C. A. et al. (1994) Bio/Technology
12:181-184.)
[0165] Plant systems may also be used for expression of LME.
Transcription of sequences encoding LME may be driven by viral
promoters, e.g., the 35S and 19S promoters of CaMV 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.
(See, e.g., 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. (See, e.g., The McGraw Hill
Yearbook of Science and Technology (1992) McGraw Hill, New York
N.Y., pp. 191-196.)
[0166] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding LME 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
infective virus which expresses LME in host cells. (See, e.g.,
Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA
81:3655-3659.) In addition, transcription enhancers, such as the
Rous sarcoma virus (RSV) enhancer, may be used to increase
expression in mammalian host cells. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0167] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from 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. (See, e.g., Harrington, J. J. et al. (1997)
Nat. Genet. 15:345-355.) For long term production of recombinant
proteins in mammalian systems, stable expression of LME in cell
lines is preferred. For example, sequences encoding LME can be
transformed into cell lines 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 a selective agent, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be propagated using tissue culture techniques
appropriate to the cell type.
[0168] 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 and adenine
phosphoribosyltransferase genes, for use in tk and apr cells,
respectively. (See, e.g., Wigler, M. et al. (1977) Cell 11:223-232;
Lowy, L. et al. (1980) Cell 22:817-823.) Also, antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for
selection. For example, dhfr confers resistance to methotrexate;
neo confers resistance to the aminoglycosides neomycin and G-418;
and als and pat confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively. (See, e.g.,
Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. USA 77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.)
Additional selectable genes have been described, e.g., trpB and
hisD, which alter cellular requirements for metabolites. (See,
e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.
Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green
fluorescent proteins (GFP; Clontech), .beta. glucuronidase and its
substrate .beta.-glucuronide, or luciferase and its substrate
luciferin may be used. These markers can be used not only to
identify transformants, but also to quantify the amount of
transient or stable protein expression attributable to a specific
vector system. (See, e.g., Rhodes, C. A. (1995) Methods Mol. Biol.
55:121-131.)
[0169] 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 LME is inserted within a marker gene
sequence, transformed cells containing sequences encoding LME can
be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding LME 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.
[0170] In general, host cells that contain the nucleic acid
sequence encoding LME and that express LME 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, PCR amplification, 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.
[0171] Immunological methods for detecting and measuring the
expression of LME using either specific polyclonal or monoclonal
antibodies 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
LME is preferred, but a competitive binding assay may be employed.
These and other assays are well known in the art. (See, e.g.,
Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual,
APS Press, St. Paul Minn., Sect. IV; Coligan, J. E. et al. (1997)
Current Protocols in Immunology, Greene Pub. Associates and
Wiley-Interscience, New York N.Y.; and Pound, J. D. (1998)
Immunochemical Protocols, Humana Press, Totowa N.J.)
[0172] 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 LME include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding LME, 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 Amersham Pharmacia Biotech, Promega (Madison Wis.), and
US Biochemical. Suitable reporter molecules or labels which may be
used for ease of detection include radionuclides, enzymes,
fluorescent, chemiluminescent, or cbromogenic agents, as well as
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0173] Host cells transformed with nucleotide sequences encoding
LME 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 retained 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 LME may be designed to
contain signal sequences which direct secretion of LME through a
prokaryotic or eukaryotic cell membrane.
[0174] 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" or "pro" form of the protein may also be used to
specify protein targeting, folding, and/or activity. Different host
cells which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and W138) are available from the American Type
Culture Collection (ATCC, Manassas Va.) and may be chosen to ensure
the correct modification and processing of the foreign protein.
[0175] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding LME may be ligated
to a heterologous sequence resulting in translation of a fusion
protein in any of the aforementioned host systems. For example, a
chimeric LME protein containing a heterologous moiety that can be
recognized by a commercially available antibody may facilitate the
screening of peptide libraries for inhibitors of LME activity.
Heterologous protein and peptide moieties may also facilitate
purification of fusion proteins using commercially available
affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG,
c-nryc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His
enable purification of their cognate fusion proteins on immobilized
glutathione, maltose, phenylarsine oxide, calmodulin, and
metal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin
(HA) enable immunoaffinity purification of fusion proteins using
commercially available monoclonal and polyclonal antibodies that
specifically recognize these epitope tags. A fusion protein may
also be engineered to contain a proteolytic cleavage site located
between the LME encoding sequence and the heterologous protein
sequence, so that LME may be cleaved away from the heterologous
moiety following purification. Methods for fusion protein
expression and purification are discussed in Ausubel (1995, supra,
ch. 10). A variety of commercially available kits may also be used
to facilitate expression and purification of fusion proteins.
[0176] In a further embodiment of the invention, synthesis of
radiolabeled LME may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract system (Promega). These
systems couple transcription and translation of protein-coding
sequences operably associated with the T7, T3, or SP6 promoters.
Translation takes place in the presence of a radiolabeled amino
acid precursor, for example, .sup.35S-methionine.
[0177] LME of the present invention or fragments thereof may be
used to screen for compounds that specifically bind to LME. At
least one and up to a plurality of test compounds may be screened
for specific binding to LME. Examples of test compounds include
antibodies, oligonucleotides, proteins (e.g., receptors), or small
molecules.
[0178] In one embodiment, the compound thus identified is closely
related to the natural ligand of LME, e.g., a ligand or fragment
thereof, a natural substrate, a structural or functional mimetic,
or a natural binding partner. (See, e.g., Coligan, J. E. et al.
(1991) Current Protocols in Immunology 1(2): Chapter 5.) Similarly,
the compound can be closely related to the natural receptor to
which LME binds, or to at least a fragment of the receptor, e.g.,
the ligand binding site. In either case, the compound can be
rationally designed using known techniques. In one embodiment,
screening for these compounds involves producing appropriate cells
which express LME, either as a secreted protein or on the cell
membrane. Preferred cells include cells from mammals, yeast,
Drosophila, or E. coli. Cells expressing LME or cell membrane
fractions which contain LME are then contacted with a test compound
and binding, stimulation, or inhibition of activity of either LME
or the compound is analyzed.
[0179] An assay may simply test binding of a test compound to the
polypeptide, wherein binding is detected by a fluorophore,
radioisotope, enzyme conjugate, or other detectable label. For
example, the assay may comprise the steps of combining at least one
test compound with LME, either in solution or affixed to a solid
support, and detecting the binding of LME to the compound.
Alternatively, the assay may detect or measure binding of a test
compound in the presence of a labeled competitor. Additionally, the
assay may be carried out using cell-free preparations, chemical
libraries, or natural product mixtures, and the test compound(s)
may be free in solution or affixed to a solid support.
[0180] LME of the present invention or fragments thereof may be
used to screen for compounds that modulate the activity of LME.
Such compounds may include agonists, antagonists, or partial or
inverse agonists. In one embodiment, an assay is performed under
conditions permissive for LME activity, wherein LME is combined
with at least one test compound, and the activity of LME in the
presence of a test compound is compared with the activity of LME in
the absence of the test compound. A change in the activity of LME
in the presence of the test compound is indicative of a compound
that modulates the activity of LME. Alternatively, a test compound
is combined with an in vitro or cell-free system comprising LME
under conditions suitable for LME activity, and the assay is
performed. In either of these assays, a test compound which
modulates the activity of LME may do so indirectly and need not
come in direct contact with the test compound. At least one and up
to a plurality of test compounds may be screened.
[0181] In another embodiment, polynucleotides encoding LME or their
mammalian homologs may be "knocked out" in an animal model system
using homologous recombination in embryonic stem (ES) cells. Such
techniques are well known in the art and are useful for the
generation of animal models of human disease. (See, e.g., U.S. Pat.
No. 5,175,383 and U.S. Pat. No. 5,767,337.) For example, mouse ES
cells, such as the mouse 129/SvJ cell line, are derived from the
early mouse embryo and grown in culture. The ES cells are
transformed with a vector containing the gene of interest disrupted
by a marker gene, e.g., the neomycin phosphotransferase gene (neo;
Capecchi, M. R. (1989) Science 244:1288-1292). The vector
integrates into the corresponding region of the host genome by
homologous recombination. Alternatively, homologous recombination
takes place using the Cre-loxP system to knockout a gene of
interest in a tissue- or developmental stage-specific manner
(Marth, J. D. (1996) Clin. Invest. 97:1999-2002; Wagner, K. U. et
al. (1997) Nucleic Acids Res. 25:4323-4330). Transformed ES cells
are identified and microinjected into mouse cell blastocysts such
as those from the C57BL/6 mouse strain. The blastocysts are
surgically transferred to pseudopregnant dams, and the resulting
chimeric progeny are genotyped and bred to produce heterozygous or
homozygous strains. Transgenic animals thus generated may be tested
with potential therapeutic or toxic agents.
[0182] Polynucleotides encoding LME may also be manipulated in
vitro in ES cells derived from human blastocysts. Human ES cells
have the potential to differentiate into at least eight separate
cell lineages including endoderm, mesoderm, and ectodermal cell
types. These cell lineages differentiate into, for example, neural
cells, hematopoietic lineages, and cardiomyocytes (Thomson, J. A.
et al. (1998) Science 282:1145-1147).
[0183] Polynucleotides encoding LME can also be used to create
"knockin" humanized animals (pigs) or transgenic animals (mice or
rats) to model human disease. With knockin technology, a region of
a polynucleotide encoding LME is injected into animal ES cells, and
the injected sequence integrates into the animal cell genome.
Transformed cells are injected into blastulae, and the blastulae
are implanted as described above. Transgenic progeny or inbred
lines are studied and treated with potential pharmaceutical agents
to obtain information on treatment of a human disease.
Alternatively, a mammal inbred to overexpress LME, e.g., by
secreting LME in its milk, may also serve as a convenient source of
that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev.
4:55-74).
Therapeutics
[0184] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between regions of LME and lipid
metabolism enzymes. In addition, the expression of LME is closely
associated with reproductive tissues, reproductive disorders,
cancer, and the representative libraries listed in Table 6.
Therefore, LME appears to play a role in cancer, neurological
disorders, autoimmune/inflammatory disorders, gastrointestinal
disorders, and cardiovascular disorders. In the treatment of
disorders associated with increased LME expression or activity, it
is desirable to decrease the expression or activity of LME. In the
treatment of disorders associated with decreased LME expression or
activity, it is desirable to increase the expression or activity of
LME.
[0185] Therefore, in one embodiment, LME or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of LME. Examples of such disorders include, but are not limited to,
a cancer, such as adenocarcinoma, leukemia, lymphoma, melanoma,
myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of
the adrenal gland, bladder, bone, bone marrow, brain, breast,
cervix, gall bladder, ganglia, gastrointestinal tract, heart,
kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus, thyroid,
and uterus; a neurological disorder such as epilepsy, ischemic
cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's
disease, Pick's disease, Huntington's disease, dementia,
Parkinson's disease and other extrapyramidal disorders, amyotrophic
lateral sclerosis and other motor neuron disorders, progressive
neural muscular atrophy, retinitis pigmentosa, hereditary ataxias,
multiple sclerosis and other demyelinating diseases, bacterial and
viral meningitis, brain abscess, subdural empyema, epidural
abscess, suppurative intracranial thrombophlebitis, myelitis and
radiculitis, viral central nervous system disease, prion diseases
including kuru, Creutzfeldt-Jakob disease, and
Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia,
nutritional and metabolic diseases of the nervous system,
neurofibromatosis, tuberous sclerosis, cerebelloretinal
hemangioblastomatosis, encephalotrigeminal syndrome, mental
retardation and other developmental disorders of the central
nervous system including Down syndrome, cerebral palsy,
neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis and polymyositis, inherited, metabolic,
endocrine, and toxic myopathies, myasthenia gravis, periodic
paralysis, mental disorders including mood, anxiety, and
schizophrenic disorders, seasonal affective disorder (SAD),
akathesia, amnesia, catatonia, diabetic neuropathy, tardive
dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia,
Tourette's disorder, progressive supranuclear palsy, corticobasal
degeneration, and familial frontotemporal dementia; an
autoimmune/inflammatory disorder such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis,
Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus, emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; a gastrointestinal disorder such
as dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis,
gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral
or pyloric edema, abdominal angina, pyrosis, gastroenteritis,
intestinal obstruction, infections of the intestinal tract, peptic
ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis,
pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis, passive congestion of the liver,
hepatoma, infectious colitis, ulcerative colitis, ulcerative
proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss
syndrome, colonic carcinoma, colonic obstruction, irritable bowel
syndrome, short bowel syndrome, diarrhea, constipation,
gastrointestinal hemorrhage, acquired immunodeficiency syndrome
(AIDS) enteropathy, jaundice, hepatic encephalopathy, hepatorenal
syndrome, hepatic steatosis, hemochromatosis, Wilson's disease,
alphal-antitrypsin deficiency, Reye's syndrome, primary sclerosing
cholangitis, liver infarction, portal vein obstruction and
thrombosis, 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 a cardiovascular disorder such as arteriovenous
fistula, atherosclerosis, hypertension, vasculitis, Raynaud's
disease, aneurysms, arterial dissections, varicose veins,
thrombophlebitis and phlebothrombosis, vascular tumors, and
complications of thrombolysis, balloon angioplasty, vascular
replacement, and coronary artery bypass graft surgery, congestive
heart failure, ischemic heart disease, angina pectoris, myocardial
infarction, hypertensive heart disease, degenerative valvular heart
disease, calcific aortic valve stenosis, congenitally bicuspid
aortic valve, mitral annular calcification, mitral valve prolapse,
rheumatic fever and rheumatic heart disease, infective
endocarditis, nonbacterial thrombotic endocarditis, endocarditis of
systemic lupus erythematosus, carcinoid heart disease,
cardiomyopathy, myocarditis, pericarditis, neoplastic heart
disease, congenital heart disease, and complications of cardiac
transplantation, congenital lung anomalies, atelectasis, pulmonary
congestion and edema, pulmonary embolism, pulmonary hemorrhage,
pulmonary infarction, pulmonary hypertension, vascular sclerosis,
obstructive pulmonary disease, restrictive pulmonary disease,
chronic obstructive pulmonary disease, emphysema, chronic
bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia,
viral and mycoplasmal pneumonia, lung abscess, pulmonary
tuberculosis, diffuse interstitial diseases, pneumoconioses,
sarcoidosis, idiopathic pulmonary fibrosis, desquamative
interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary
eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse
pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic
pulmonary hemosiderosis, pulmonary involvement in collagen-vascular
disorders, pulmonary alveolar proteinosis, lung tumors,
inflammatory and noninflammatory pleural effusions, pneumothorax,
pleural tumors, drug-induced lung disease, radiation-induced lung
disease, and complications of lung transplantation. In another
embodiment, a vector capable of expressing LME or a fragment or
derivative thereof may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of LME including, but not limited to, those described above.
[0186] In a further embodiment, a composition comprising a
substantially purified LME in conjunction with a suitable
pharmaceutical carrier may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of LME including, but not limited to, those provided above.
[0187] In still another embodiment, an agonist which modulates the
activity of LME may be administered to a subject to treat or
prevent a disorder associated with decreased expression or activity
of LME including, but not limited to, those listed above.
[0188] In a further embodiment, an antagonist of LME may be
administered to a subject to treat or prevent a disorder associated
with increased expression or activity of LME. Examples of such
disorders include, but are not limited to, those cancers,
neurological disorders, autoimmune/inflammatory disorders,
gastrointestinal disorders, and cardiovascular disorders described
above. In one aspect, an antibody which specifically binds LME may
be used directly as an antagonist or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissues which express LME.
[0189] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding LME may be administered
to a subject to treat or prevent a disorder associated with
increased expression or activity of LME including, but not limited
to, those described above.
[0190] 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.
[0191] An antagonist of LME may be produced using methods which are
generally known in the art. In particular, purified LME may be used
to produce antibodies or to screen libraries of pharmaceutical
agents to identify those which specifically bind LME. Antibodies to
LME 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 generally preferred for therapeutic use. For the production of
antibodies, various hosts including goats, rabbits, rats, mice,
humans, and others may be immunized by injection with LME 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.
[0192] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to LME have an amino acid
sequence consisting of at least about 5 amino acids, and generally
will consist 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. Short stretches of LME amino acids may be fused with those
of another protein, such as KLH, and antibodies to the chimeric
molecule may be produced.
[0193] Monoclonal antibodies to LME may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G.
et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl.
Acad. Sci. USA 80:2026-2030; and Cole, S. P. et al. (1984) Mol.
Cell. Biol. 62:109-120.)
[0194] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci. USA
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
LME-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton, D. R. (1991) Proc.
Natl. Acad. Sci. USA 88:10134-10137.)
[0195] 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. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et
al. (1991) Nature 349:293-299.)
[0196] Antibody fragments which contain specific binding sites for
LME may also be generated. For example, such fragments include, but
are not limited to, F (ab').sub.2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F (ab').sub.2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0197] 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 LME and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering LME epitopes
is generally used, but a competitive binding assay may also be
employed (Pound, supra).
[0198] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for LME. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
LME-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple LME epitopes,
represents the average affinity, or avidity, of the antibodies for
LME. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular LME epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
LME-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of LME, preferably in active form, from the antibody
(Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL
Press, Washington D.C.; Liddell, J. E. and A. Cryer (1991) A
Practical Guide to Monoclonal Antibodies, John Wiley & Sons,
New York N.Y.).
[0199] The titer and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is generally employed in procedures requiring precipitation of
LME-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0200] In another embodiment of the invention, the polynucleotides
encoding LME, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, modifications of gene
expression can be achieved by designing complementary sequences or
antisense molecules (DNA, RNA, PNA, or modified oligonucleotides)
to the coding or regulatory regions of the gene encoding LME. Such
technology is well known in the art, and antisense oligonucleotides
or larger fragments can be designed from various locations along
the coding or control regions of sequences encoding LME. (See,
e.g., Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press
Inc., Totawa N.J.)
[0201] In therapeutic use, any gene delivery system suitable for
introduction of the antisense sequences into appropriate target
cells can be used. Antisense sequences can be delivered
intracellularly in the form of an expression plasmid which, upon
transcription, produces a sequence complementary to at least a
portion of the cellular sequence encoding the target protein. (See,
e.g., Slater, J. E. et al. (1998) J. Allergy Cli. Immunol.
102(3):469-475; and Scanlon, K. J. et al. (1995) 9(13):1288-1296.)
Antisense sequences can also be introduced intracellularly through
the use of viral vectors, such as retrovirus and adeno-associated
virus vectors. (See, e.g., Miller, A. D. (1990) Blood 76:271;
Ausubel, supra; Uckert, W. and W. Walther (1994) Pharmacol. Ther.
63 (3):323-347.) Other gene delivery mechanisms include
liposome-derived systems, artificial viral envelopes, and other
systems known in the art. (See, e.g., Rossi, J. J. (1995) Br. Med.
Bull. 51(1):217-225; Boado, R. J. et al. (1998) J. Pharm. Sci.
87(11):1308-1315; and Morris, M. C. et al. (1997) Nucleic Acids
Res. 25(14):2730-2736.)
[0202] In another embodiment of the invention, polynucleotides
encoding LME may be used for somatic or germline gene therapy. Gene
therapy may be performed to (i) correct a genetic deficiency (e.g.,
in the cases of severe combined immunodeficiency (SCID)-X1 disease
characterized by X-linked inheritance (Cavazzana-Calvo, M. et al.
(2000) Science 288:669-672), severe combined immunodeficiency
syndrome associated with an inherited adenosine deaminase (ADA)
deficiency (Blaese, R. M. et al. (1995) Science 270:475-480;
Bordignon, C. et al. (1995) Science 270:470-475), cystic fibrosis
(Zabner, J. et al. (1993) Cell 75:207-216; Crystal, R. G. et al.
(1995) Hum. Gene Therapy 6:643-666; Crystal, R. G. et al. (1995)
Hum. Gene Therapy 6:667-703), thalassamias, familial
hypercholesterolemia, and hemophilia resulting from Factor VIII or
Factor IX deficiencies (Crystal, R. G. (1995) Science 270:404-410;
Verma, I. M. and N. Somia (1997) Nature 389:239-242)), (ii) express
a conditionally lethal gene product (e.g., in the case of cancers
which result from unregulated cell proliferation), or (iii) express
a protein which affords protection against intracellular parasites
(e.g., against human retroviruses, such as human immunodeficiency
virus (HIV) (Baltimore, D. (1988) Nature 335:395-396; Poeschla, E.
et al. (1996) Proc. Natl. Acad. Sci. USA. 93:11395-11399),
hepatitis B or C virus (HBV, HCV); fungal parasites, such as
Candida albicans and Paracoccidioides brasiliensis; and protozoan
parasites such as Plasmodium falciparum and Trypanosoma cruzi). In
the case where a genetic deficiency in LME expression or regulation
causes disease, the expression of LME from an appropriate
population of transduced cells may alleviate the clinical
manifestations caused by the genetic deficiency.
[0203] In a further embodiment of the invention, diseases or
disorders caused by deficiencies in LME are treated by constructing
mammalian expression vectors encoding LME and introducing these
vectors by mechanical means into LME-deficient cells. Mechanical
transfer technologies for use with cells in vivo or ex vitro
include (i) direct DNA microinjection into individual cells, (ii)
ballistic gold particle delivery, (iii) liposome-mediated
transfection, (iv) receptor-mediated gene transfer, and (v) the use
of DNA transposons (Morgan, R. A. and W. F. Anderson (1993) Annu.
Rev. Biochem. 62:191-217; Ivics, Z. (1997) Cell 91:501-510; Boulay,
J-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
[0204] Expression vectors that may be effective for the expression
of LME include, but are not limited to, the PCDNA 3.1, EPITAG,
PRCCMV2, PREP, PVAX vectors (Invitrogen, Carlsbad Calif.),
PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La Jolla Calif.),
and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo
Alto Calif.). LME may be expressed using (i) a constitutively
active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma
virus (RSV), SV40 virus, thymidine kinase (TK), or .beta.-actin
genes), (ii) an inducible promoter (e.g., the
tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992)
Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995)
Science 268:1766-1769; Rossi, F. M. V. and H. M. Blau (1998) Curr.
Opin. Biotechnol. 9:451-456), commercially available in the T-REX
plasmid (Invitrogen)); the ecdysone-inducible promoter (available
in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapamycin
inducible promoter; or the RU486/mifepristone inducible promoter
(Rossi, F. M. V. and Blau, H. M. supra)), or (iii) a
tissue-specific promoter or the native promoter of the endogenous
gene encoding LME from a normal individual. Commercially available
liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION
KIT, available from Invitrogen) allow one with ordinary skill in
the art to deliver polynucleotides to target cells in culture and
require minimal effort to optimize experimental parameters. In the
alternative, transformation is performed using the calcium
phosphate method (Graham, F. L. and A. J. Eb (1973) Virology
52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO
J. 1:841-845).
[0205] The introduction of DNA to primary cells requires
modification of these standardized mammalian transfection
protocols.
[0206] In another embodiment of the invention, diseases or
disorders caused by genetic defects with respect to LME expression
are treated by constructing a retrovirus vector consisting of (i)
the polynucleotide encoding LME under the control of an independent
promoter or the retrovirus long terminal repeat (LTR) promoter,
(ii) appropriate RNA packaging signals, and (iii) a Rev-responsive
element (RRE) along with additional retrovirus cis-acting RNA
sequences and coding sequences required for efficient vector
propagation. Retrovirus vectors (e.g., PFB and PFBNEO) are
commercially available (Stratagene) and are based on published data
(Riviere, I. et al. (1995) Proc. Natl. Acad. Sci. USA
92:6733-6737), incorporated by reference herein.
[0207] The vector is propagated in an appropriate vector producing
cell line (VPCL) that expresses an envelope gene with a tropism for
receptors on the target cells or a promiscuous envelope protein
such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650;
Bender, M. A. et al. (1987) J. Virol. 61:1639-1646; Adam, M. A. and
A. D. Miller (1988) J. Virol. 62:3802-3806; Dull, T. et al. (1998)
J. Virol. 72:8463-8471; Zufferey, R. et al. (1998) J. Virol.
72:9873-9880). U.S. Pat. No. 5,910,434 to Rigg ("Method for
obtaining retrovirus packaging cell lines producing high
transducing efficiency retroviral supernatant") discloses a method
for obtaining retrovirus packaging cell lines and is hereby
incorporated by reference. Propagation of retrovirus vectors,
transduction of a population of cells (e.g., CD4.sup.+ T-cells),
and the return of transduced cells to a patient are procedures well
known to persons skilled in the art of gene therapy and have been
well documented (Ranga, U. et al. (1997) J. Virol. 71:7020-7029;
Bauer, G. et al. (1997) Blood 89:2259-2267; Bonyhadi, M. L. (1997)
J. Virol. 71:4707-4716; Ranga, U. et al. (1998) Proc. Natl. Acad.
Sci. USA 95:1201-1206; Su, L. (1997) Blood 89:2283-2290).
[0208] In the alternative, an adenovirus-based gene therapy
delivery system is used to deliver polynucleotides encoding LME to
cells which have one or more genetic abnormalities with respect to
the expression of LME. The construction and packaging of
adenovirus-based vectors are well known to those with ordinary
skill in the art. Replication defective adenovirus vectors have
proven to be versatile for importing genes encoding
immunoregulatory proteins into intact islets in the pancreas
(Csete, M. E. et al. (1995) Transplantation 27:263-268).
Potentially useful adenoviral vectors are described in U.S. Pat.
No. 5,707,618 to Armentano ("Adenovirus vectors for gene therapy"),
hereby incorporated by reference. For adenoviral vectors, see also
Antinozzi, P. A. et al. (1999) Annu. Rev. Nutr. 19:511-544 and
Verma, I. M. and N. Somia (1997) Nature 18:389:239-242, both
incorporated by reference herein. In another alternative, a
herpes-based, gene therapy delivery system is used to deliver
polynucleotides encoding LME to target cells which have one or more
genetic abnormalities with respect to the expression of LME. The
use of herpes simplex virus (HSV)-based vectors may be especially
valuable for introducing LME to cells of the central nervous
system, for which HSV has a tropism. The construction and packaging
of herpes-based vectors are well known to those with ordinary skill
in the art. A replication-competent herpes simplex virus (HSV) type
1-based vector has been used to deliver a reporter gene to the eyes
of primates (Liu, X. et al. (1999) Exp. Eye Res. 169:385-395). The
construction of a HSV-1 virus vector has also been disclosed in
detail in U.S. Pat. No. 5,804,413 to DeLuca ("Herpes simplex virus
strains for gene transfer"), which is hereby incorporated by
reference. U.S. Pat. No. 5,804,413 teaches the use of recombinant
HSV d92 which consists of a genome containing at least one
exogenous gene to be transferred to a cell under the control of the
appropriate promoter for purposes including human gene therapy.
Also taught by this patent are the construction and use of
recombinant HSV strains deleted for ICP4, ICP27 and ICP22. For HSV
vectors, see also Goins, W. F. et al. (1999) J. Virol. 73:519-532
and Xu, H. et al. (1994) Dev. Biol. 163:152-161, hereby
incorporated by reference. The manipulation of cloned herpesvirus
sequences, the generation of recombinant virus following the
transfection of multiple plasmids containing different segments of
the large herpesvirus genomes, the growth and propagation of
herpesvirus, and the infection of cells with herpesvirus are
techniques well known to those of ordinary skill in the art.
[0209] In another alternative, an alphavirus (positive,
single-stranded RNA virus) vector is used to deliver
polynucleotides encoding LME to target cells. The biology of the
prototypic alphavirus, Semliki Forest Virus (SFV), has been studied
extensively and gene transfer vectors have been based on the SFV
genome (Garoff, H. and K.-J. Li (1998) Curr. Opin. Biotechnol.
9:464-469). During alphavirus RNA replication, a subgenomic RNA is
generated that normally encodes the viral capsid proteins. This
subgenomic RNA replicates to higher levels than the full length
genomic RNA, resulting in the overproduction of capsid proteins
relative to the viral proteins with enzymatic activity (e.g.,
protease and polymerase). Similarly, inserting the coding sequence
for LME into the alphavirus genome in place of the capsid-coding
region results in the production of a large number of LME-coding
RNAs and the synthesis of high levels of LME in vector transduced
cells. While alphavirus infection is typically associated with cell
lysis within a few days, the ability to establish a persistent
infection in hamster normal kidney cells (BHK-21) with a variant of
Sindbis virus (SIN) indicates that the lytic replication of
alphaviruses can be altered to suit the needs of the gene therapy
application (Dryga, S. A. et al. (1997) Virology 228:74-83). The
wide host range of alphaviruses will allow the introduction of LME
into a variety of cell types. The specific transduction of a subset
of cells in a population may require the sorting of cells prior to
transduction. The methods of manipulating infectious cDNA clones of
alphaviruses, performing alphavirus cDNA and RNA transfections, and
performing alphavirus infections, are well known to those with
ordinary skill in the art.
[0210] Oligonucleotides derived from the transcription initiation
site, e.g., between about positions -10 and +10 from the start
site, may also be employed to inhibit gene expression. Similarly,
inhibition can be achieved using triple helix base-pairing
methodology. Triple helix pairing is useful because it causes
inhibition of the ability of the double helix to open sufficiently
for the binding of polymerases, transcription factors, or
regulatory molecules. Recent therapeutic advances using triplex DNA
have been described in the literature. (See, e.g., Gee, J. E. et
al. (1994) in Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing, Mt. Kisco N.Y., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0211] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding LME.
[0212] 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.
[0213] 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 LME. 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.
[0214] 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.
[0215] An additional embodiment of the invention encompasses a
method for screening for a compound which is effective in altering
expression of a polynucleotide encoding LME. Compounds which may be
effective in altering expression of a specific polynucleotide may
include, but are not limited to, oligonucleotides, antisense
oligonucleotides, triple helix-forming oligonucleotides,
transcription factors and other polypeptide transcriptional
regulators, and non-macromolecular chemical entities which are
capable of interacting with specific polynucleotide sequences.
Effective compounds may alter polynucleotide expression by acting
as either inhibitors or promoters of polynucleotide expression.
Thus, in the treatment of disorders associated with increased LME
expression or activity, a compound which specifically inhibits
expression of the polynucleotide encoding LME may be
therapeutically useful, and in the treatment of disorders
associated with decreased LME expression or activity, a compound
which specifically promotes expression of the polynucleotide
encoding LME may be therapeutically useful.
[0216] At least one, and up to a plurality, of test compounds may
be screened for effectiveness in altering expression of a specific
polynucleotide. A test compound may be obtained by any method
commonly known in the art, including chemical modification of a
compound known to be effective in altering polynucleotide
expression; selection from an existing, commercially-available or
proprietary library of naturally-occurring or non-natural chemical
compounds; rational design of a compound based on chemical and/or
structural properties of the target polynucleotide; and selection
from a library of chemical compounds created combinatorially or
randomly. A sample comprising a polynucleotide encoding LME is
exposed to at least one test compound thus obtained. The sample may
comprise, for example, an intact or permeabilized cell, or an in
vitro cell-free or reconstituted biochemical system. Alterations in
the expression of a polynucleotide encoding LME are assayed by any
method commonly known in the art. Typically, the expression of a
specific nucleotide is detected by hybridization with a probe
having a nucleotide sequence complementary to the sequence of the
polynucleotide encoding LME. The amount of hybridization may be
quantified, thus forming the basis for a comparison of the
expression of the polynucleotide both with and without exposure to
one or more test compounds. Detection of a change in the expression
of a polynucleotide exposed to a test compound indicates that the
test compound is effective in altering the expression of the
polynucleotide. A screen for a compound effective in altering
expression of a specific polynucleotide can be carried out, for
example, using a Schizosaccharomyces pombe gene expression system
(Atkins, D. et al. (1999) U.S. Pat. No. 5,932,435; Arndt, G. M. et
al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as
HeLa cell (Clarke, M. L. et al. (2000) Biochem. Biophys. Res.
Commun. 268:8-13). A particular embodiment of the present invention
involves screening a combinatorial library of oligonucleotides
(such as deoxyribonucleotides, ribonucleotides, peptide nucleic
acids, and modified oligonucleotides) for antisense activity
against a specific polynucleotide sequence (Bruice, T. W. et al.
(1997) U.S. Pat. No. 5,686,242; Bruice, T. W. et al. (2000) U.S.
Pat. No. 6,022,691).
[0217] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nat. Biotechnol. 15:462-466.)
[0218] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as humans, dogs, cats, cows, horses, rabbits,
and monkeys.
[0219] An additional embodiment of the invention relates to the
administration of a composition which generally comprises an active
ingredient formulated with a pharmaceutically acceptable excipient.
Excipients may include, for example, sugars, starches, celluloses,
gums, and proteins. Various formulations are commonly known and are
thoroughly discussed in the latest edition of Remington's
Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such
compositions may consist of LME, antibodies to LME, and mimetics,
agonists, antagonists, or inhibitors of LME.
[0220] The 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, pulmonary, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0221] Compositions for pulmonary administration may be prepared in
liquid or dry powder form. These compositions are generally
aerosolized immediately prior to inhalation by the patient. In the
case of small molecules (e.g. traditional low molecular weight
organic drugs), aerosol delivery of fast-acting formulations is
well-known in the art. In the case of macromolecules (e.g. larger
peptides and proteins), recent developments in the field of
pulmonary delivery via the alveolar region of the lung have enabled
the practical delivery of drugs such as insulin to blood
circulation (see, e.g., Patton, J. S. et al., U.S. Pat. No.
5,997,848). Pulmonary delivery has the advantage of administration
without needle injection, and obviates the need for potentially
toxic penetration enhancers.
[0222] 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.
[0223] Specialized forms of compositions may be prepared for direct
intracellular delivery of macromolecules comprising LME or
fragments thereof. For example, liposome preparations containing a
cell-impermeable macromolecule may promote cell fusion and
intracellular delivery of the macromolecule. Alternatively, LME or
a fragment thereof may be joined to a short cationic N-terminal
portion from the HIV Tat-1 protein. Fusion proteins thus generated
have been found to transduce into the cells of all tissues,
including the brain, in a mouse model system (Schwarze, S. R. et
al. (1999) Science 285:1569-1572).
[0224] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models such as mice, rats, rabbits,
dogs, monkeys, 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.
[0225] A therapeutically effective dose refers to that amount of
active ingredient, for example LME or fragments thereof, antibodies
of LME, and agonists, antagonists or inhibitors of LME, 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 ED.sub.50 (the dose therapeutically effective in 50% of the
population) or LD.sub.50 (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. Compositions which exhibit large
therapeutic indices are preferred. The data obtained from cell
culture assays and animal studies are used to formulate a range of
dosage for human use. The dosage contained in such compositions is
preferably within a range of circulating concentrations that
includes the ED.sub.50 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.
[0226] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring treatment. Dosage
and administration are adjusted to provide sufficient levels of the
active moiety or to maintain the desired effect. Factors which may
be taken into account include the severity of the disease state,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to
therapy.
[0227] Long-acting compositions may be administered every 3 to 4
days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0228] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, depending upon
the route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
Diagnostics
[0229] In another embodiment, antibodies which specifically bind
LME may be used for the diagnosis of disorders characterized by
expression of LME, or in assays to monitor patients being treated
with LME or agonists, antagonists, or inhibitors of LME. Antibodies
useful for diagnostic purposes may be prepared in the same manner
as described above for therapeutics. Diagnostic assays for LME
include methods which utilize the antibody and a label to detect
LME in human body fluids or in extracts of cells or tissues. The
antibodies may be used with or without modification, and may be
labeled by covalent or non-covalent attachment of a reporter
molecule. A wide variety of reporter molecules, several of which
are described above, are known in the art and may be used.
[0230] A variety of protocols for measuring LME, including ELISAs,
RIAs, and FACS, are known in the art and provide a basis for
diagnosing altered or abnormal levels of LME expression. Normal or
standard values for LME expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
for example, human subjects, with antibodies to LME under
conditions suitable for complex formation. The amount of standard
complex formation may be quantitated by various methods, such as
photometric means. Quantities of LME 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.
[0231] In another embodiment of the invention, the polynucleotides
encoding LME 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 quantify gene expression
in biopsied tissues in which expression of LME may be correlated
with disease. The diagnostic assay may be used to determine
absence, presence, and excess expression of LME, and to monitor
regulation of LME levels during therapeutic intervention.
[0232] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding LME or closely related molecules may be used to
identify nucleic acid sequences which encode LME. The specificity
of the probe, whether it is made from a highly specific region,
e.g., the 5' regulatory region, or from a less specific region,
e.g., a conserved motif, and the stringency of the hybridization or
amplification will determine whether the probe identifies only
naturally occurring sequences encoding LME, allelic variants, or
related sequences.
[0233] Probes may also be used for the detection of related
sequences, and may have at least 50% sequence identity to any of
the LME encoding sequences. The hybridization probes of the subject
invention may be DNA or RNA and may be derived from the sequence of
SEQ ID NO: 7-12 or from genomic sequences including promoters,
enhancers, and introns of the LME gene.
[0234] Means for producing specific hybridization probes for DNAs
encoding LME include the cloning of polynucleotide sequences
encoding LME or LME derivatives into vectors for the production of
mRNA probes. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerases and the
appropriate labeled nucleotides. Hybridization probes may be
labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0235] Polynucleotide sequences encoding LME may be used for the
diagnosis of disorders associated with expression of LME. Examples
of such disorders include, but are not limited to, cancer, such as
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in particular, cancers of the adrenal gland,
bladder, bone, bone marrow, brain, breast, cervix, gall bladder,
ganglia, gastrointestinal tract, heart, kidney, liver, lung,
muscle, ovary, pancreas, parathyroid, penis, prostate, salivary
glands, skin, spleen, testis, thymus, thyroid, and uterus; a
neurological disorder such as epilepsy, ischemic cerebrovascular
disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's
disease, Huntington's disease, dementia, Parkinson's disease and
other extrapyramidal disorders, amyotrophic lateral sclerosis and
other motor neuron disorders, progressive neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system disease, prion diseases including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome, fatal familial insomnia, nutritional and metabolic
diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other
developmental disorders of the central nervous system including
Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic
nervous system disorders, cranial nerve disorders, spinal cord
diseases, muscular dystrophy and other neuromuscular disorders,
peripheral nervous system disorders, dermatomyositis and
polymyositis, inherited, metabolic, endocrine, and toxic
myopathies, myasthenia gravis, periodic paralysis, mental disorders
including mood, anxiety, and schizophrenic disorders, seasonal
affective disorder (SAD), akathesia, amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia, Tourette's disorder, progressive
supranuclear palsy, corticobasal degeneration, and familial
frontotemporal dementia; an autoimmune/inflammatory disorder such
as acquired immunodeficiency syndrome (AIDS), Addison's disease,
adult respiratory distress syndrome, allergies, ankylosing
spondylitis, amyloidosis, anemia, asthma, atherosclerosis,
autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune
polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED),
bronchitis, cholecystitis, contact dermatitis, Crohn's disease,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis
fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis,
Goodpasture's syndrome, gout, Graves' disease, Hashimoto's
thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis, myasthenia gravis, myocardial or pericardial
inflammation, osteoarthritis, osteoporosis, pancreatitis,
polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,
scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic
lupus erythematosus, systemic sclerosis, thrombocytopenic purpura,
ulcerative colitis, uveitis, Werner syndrome, complications of
cancer, hemodialysis, and extracorporeal circulation, viral,
bacterial, fungal, parasitic, protozoal, and helminthic infections,
and trauma; a gastrointestinal disorder such as dysphagia, peptic
esophagitis, esophageal spasm, esophageal stricture, esophageal
carcinoma, dyspepsia, indigestion, gastritis, gastric carcinoma,
anorexia, nausea, emesis, gastroparesis, antral or pyloric edema,
abdominal angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma,
biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis,
ulcerative colitis, ulcerative proctitis, Crohn's disease,
Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage,
acquired immunodeficiency syndrome (AIDS) enteropathy, jaundice,
hepatic encephalopathy, hepatorenal syndrome, hepatic steatosis,
hemochromatosis, Wilson's disease, alpha1-antitrypsin deficiency,
Reye's syndrome, primary sclerosing cholangitis, liver infarction,
portal vein obstruction and thrombosis, 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 a
cardiovascular disorder such as arteriovenous fistula,
atherosclerosis, hypertension, vasculitis, Raynaud's disease,
aneurysms, arterial dissections, varicose veins, thrombophlebitis
and phlebothrombosis, vascular tumors, and complications of
thrombolysis, balloon angioplasty, vascular replacement, and
coronary artery bypass graft surgery, congestive heart failure,
ischemic heart disease, angina pectoris, myocardial infarction,
hypertensive heart disease, degenerative valvular heart disease,
calcific aortic valve stenosis, congenitally bicuspid aortic valve,
mitral annular calcification, mitral valve prolapse, rheumatic
fever and rheumatic heart disease, infective endocarditis,
nonbacterial thrombotic endocarditis, endocarditis of systemic
lupus erythematosus, carcinoid heart disease, cardiomyopathy,
myocarditis, pericarditis, neoplastic heart disease, congenital
heart disease, and complications of cardiac transplantation,
congenital lung anomalies, atelectasis, pulmonary congestion and
edema, pulmonary embolism, pulmonary hemorrhage, pulmonary
infarction, pulmonary hypertension, vascular sclerosis, obstructive
pulmonary disease, restrictive pulmonary disease, chronic
obstructive pulmonary disease, emphysema, chronic bronchitis,
bronchial asthma, bronchiectasis, bacterial pneumonia, viral and
mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis,
diffuse interstitial diseases, pneumoconioses, sarcoidosis,
idiopathic pulmonary fibrosis, desquamative interstitial
pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia
bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary
hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary
hemosiderosis, pulmonary involvement in collagen-vascular
disorders, pulmonary alveolar proteinosis, lung tumors,
inflammatory and noninflammatory pleural effusions, pneumothorax,
pleural tumors, drug-induced lung disease, radiation-induced lung
disease, and complications of lung transplantation. The
polynucleotide sequences encoding LME may be used in Southern or
northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; in dipstick, pin, and multiformat ELISA-like
assays; and in microarrays utilizing fluids or tissues from
patients to detect altered LME expression. Such qualitative or
quantitative methods are well known in the art.
[0236] In a particular aspect, the nucleotide sequences encoding
LME may be useful in assays that detect the presence of associated
disorders, particularly those mentioned above. The nucleotide
sequences encoding LME 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
quantified and compared with a standard value. If the amount of
signal in the patient sample is significantly altered in comparison
to a control sample then the presence of altered levels of
nucleotide sequences encoding LME in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0237] In order to provide a basis for the diagnosis of a disorder
associated with expression of LME, 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
LME, under conditions suitable for hybridization or amplification.
Standard hybridization may be quantified by comparing the values
obtained from normal subjects with values from an experiment in
which a known amount of a substantially purified polynucleotide is
used. Standard values obtained in this manner may be compared with
values obtained from samples from patients who are symptomatic for
a disorder. Deviation from standard values is used to establish the
presence of a disorder.
[0238] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0239] With respect to cancer, the presence of an abnormal amount
of transcript (either under- or overexpressed) 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.
[0240] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding LME 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 LME, or a fragment of a polynucleotide
complementary to the polynucleotide encoding LME, 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 quantification of
closely related DNA or RNA sequences.
[0241] In a particular aspect, oligonucleotide primers derived from
the polynucleotide sequences encoding LME may be used to detect
single nucleotide polymorphisms (SNPs). SNPs are substitutions,
insertions and deletions that are a frequent cause of inherited or
acquired genetic disease in humans. Methods of SNP detection
include, but are not limited to, single-stranded conformation
polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods. In SSCP,
oligonucleotide primers derived from the polynucleotide sequences
encoding LME are used to amplify DNA using the polymerase chain
reaction (PCR). The DNA may be derived, for example, from diseased
or normal tissue, biopsy samples, bodily fluids, and the like. SNPs
in the DNA cause differences in the secondary and tertiary
structures of PCR products in single-stranded form, and these
differences are detectable using gel electrophoresis in
non-denaturing gels. In fSCCP, the oligonucleotide primers are
fluorescently labeled, which allows detection of the amplimers in
high-throughput equipment such as DNA sequencing machines.
Additionally, sequence database analysis methods, termed in silico
SNP (is SNP), are capable of identifying polymorphisms by comparing
the sequence of individual overlapping DNA fragments which assemble
into a common consensus sequence. These computer-based methods
filter out sequence variations due to laboratory preparation of DNA
and sequencing errors using statistical models and automated
analyses of DNA sequence chromatograms. In the alternative, SNPs
may be detected and characterized by mass spectrometry using, for
example, the high throughput MASSARRAY system (Sequenom, Inc., San
Diego Calif.).
[0242] Methods which may also be used to quantify the expression of
LME include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993)
Anal. Biochem. 212:229-236.) The speed of quantitation of multiple
samples may be accelerated by running the assay in a
high-throughput format where the oligomer or polynucleotide of
interest is presented in various dilutions and a spectrophotometric
or colorimetric response gives rapid quantitation.
[0243] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as elements on a microarray. The microarray can be used
in transcript imaging techniques which monitor the relative
expression levels of large numbers of genes simultaneously as
described below. The microarray may also be used to identify
genetic variants, mutations, and polymorphisms. This information
may be used to determine gene function, to understand the genetic
basis of a disorder, to diagnose a disorder, to monitor
progression/regression of disease as a function of gene expression,
and to develop and monitor the activities of therapeutic agents in
the treatment of disease. In particular, this information may be
used to develop a pharmacogenomic profile of a patient in order to
select the most appropriate and effective treatment regimen for
that patient. For example, therapeutic agents which are highly
effective and display the fewest side effects may be selected for a
patient based on his/her pharmacogenomic profile.
[0244] In another embodiment, LME, fragments of LME, or antibodies
specific for LME may be used as elements on a microarray. The
microarray may be used to monitor or measure protein-protein
interactions, drug-target interactions, and gene expression
profiles, as described above.
[0245] A particular embodiment relates to the use of the
polynucleotides of the present invention to generate a transcript
image of a tissue or cell type. A transcript image represents the
global pattern of gene expression by a particular tissue or cell
type. Global gene expression patterns are analyzed by quantifying
the number of expressed genes and their relative abundance under
given conditions and at a given time. (See Seilhamer et al.,
"Comparative Gene Transcript Analysis," U.S. Pat. No. 5,840,484,
expressly incorporated by reference herein.) Thus a transcript
image may be generated by hybridizing the polynucleotides of the
present invention or their complements to the totality of
transcripts or reverse transcripts of a particular tissue or cell
type. In one embodiment, the hybridization takes place in
high-throughput format, wherein the polynucleotides of the present
invention or their complements comprise a subset of a plurality of
elements on a microarray. The resultant transcript image would
provide a profile of gene activity.
[0246] Transcript images may be generated using transcripts
isolated from tissues, cell lines, biopsies, or other biological
samples. The transcript image may thus reflect gene expression in
vivo, as in the case of a tissue or biopsy sample, or in vitro, as
in the case of a cell line.
[0247] Transcript images which profile the expression of the
polynucleotides of the present invention may also be used in
conjunction with in vitro model systems and preclinical evaluation
of pharmaceuticals, as well as toxicological testing of industrial
and naturally-occurring environmental compounds. All compounds
induce characteristic gene expression patterns, frequently termed
molecular fingerprints or toxicant signatures, which are indicative
of mechanisms of action and toxicity (Nuwaysir, E. F. et al. (1999)
Mol. Carcinog. 24:153-159; Steiner, S, and N. L. Anderson (2000)
Toxicol. Lett. 112-113:467-471, expressly incorporated by reference
herein). If a test compound has a signature similar to that of a
compound with known toxicity, it is likely to share those toxic
properties. These fingerprints or signatures are most useful and
refined when they contain expression information from a large
number of genes and gene families. Ideally, a genome-wide
measurement of expression provides the highest quality signature.
Even genes whose expression is not altered by any tested compounds
are important as well, as the levels of expression of these genes
are used to normalize the rest of the expression data. The
normalization procedure is useful for comparison of expression data
after treatment with different compounds. While the assignment of
gene function to elements of a toxicant signature aids in
interpretation of toxicity mechanisms, knowledge of gene function
is not necessary for the statistical matching of signatures which
leads to prediction of toxicity. (See, for example, Press Release
00-02 from the National Institute of Environmental Health Sciences,
released Feb. 29, 2000, available from the internet.) Therefore, it
is important and desirable in toxicological screening using
toxicant signatures to include all expressed gene sequences.
[0248] In one embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing nucleic acids
with the test compound. Nucleic acids that are expressed in the
treated biological sample are hybridized with one or more probes
specific to the polynucleotides of the present invention, so that
transcript levels corresponding to the polynucleotides of the
present invention may be quantified. The transcript levels in the
treated biological sample are compared with levels in an untreated
biological sample. Differences in the transcript levels between the
two samples are indicative of a toxic response caused by the test
compound in the treated sample.
[0249] Another particular embodiment relates to the use of the
polypeptide sequences of the present invention to analyze the
proteome of a tissue or cell type. The term proteome refers to the
global pattern of protein expression in a particular tissue or cell
type. Each protein component of a proteome can be subjected
individually to further analysis. Proteome expression patterns, or
profiles, are analyzed by quantifying the number of expressed
proteins and their relative abundance under given conditions and at
a given time. A profile of a cell's proteome may thus be generated
by separating and analyzing the polypeptides of a particular tissue
or cell type. In one embodiment, the separation is achieved using
two-dimensional gel electrophoresis, in which proteins from a
sample are separated by isoelectric focusing in the first
dimension, and then according to molecular weight by sodium dodecyl
sulfate slab gel electrophoresis in the second dimension (Steiner
and Anderson, supra). The proteins are visualized in the gel as
discrete and uniquely positioned spots, typically by staining the
gel with an agent such as Coomassie Blue or silver or fluorescent
stains. The optical density of each protein spot is generally
proportional to the level of the protein in the sample. The optical
densities of equivalently positioned protein spots from different
samples, for example, from biological samples either treated or
untreated with a test compound or therapeutic agent, are compared
to identify any changes in protein spot density related to the
treatment. The proteins in the spots are partially sequenced using,
for example, standard methods employing chemical or enzymatic
cleavage followed by mass spectrometry. The identity of the protein
in a spot may be determined by comparing its partial sequence,
preferably of at least 5 contiguous amino acid residues, to the
polypeptide sequences of the present invention. In some cases,
further sequence data may be obtained for definitive protein
identification.
[0250] A proteomic profile may also be generated using antibodies
specific for LME to quantify the levels of LME expression. In one
embodiment, the antibodies are used as elements on a microarray,
and protein expression levels are quantified by exposing the
microarray to the sample and detecting the levels of protein bound
to each array element (Lueking, A. et al. (1999) Anal. Biochem.
270:103-111; Mendoze, L. G. et al. (1999) Biotechniques
27:778-788). Detection may be performed by a variety of methods
known in the art, for example, by reacting the proteins in the
sample with a thiol- or amino-reactive fluorescent compound and
detecting the amount of fluorescence bound at each array
element.
[0251] Toxicant signatures at the proteome level are also useful
for toxicological screening, and should be analyzed in parallel
with toxicant signatures at the transcript level. There is a poor
correlation between transcript and protein abundances for some
proteins in some tissues (Anderson, N. L. and J. Seilhamer (1997)
Electrophoresis 18:533-537), so proteome toxicant signatures may be
useful in the analysis of compounds which do not significantly
affect the transcript image, but which alter the proteomic profile.
In addition, the analysis of transcripts in body fluids is
difficult, due to rapid degradation of mRNA, so proteomic profiling
may be more reliable and informative in such cases.
[0252] In another embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing proteins with
the test compound. Proteins that are expressed in the treated
biological sample are separated so that the amount of each protein
can be quantified. The amount of each protein is compared to the
amount of the corresponding protein in an untreated biological
sample. A difference in the amount of protein between the two
samples is indicative of a toxic response to the test compound in
the treated sample. Individual proteins are identified by
sequencing the amino acid residues of the individual proteins and
comparing these partial sequences to the polypeptides of the
present invention.
[0253] In another embodiment, the toxicity of a test compound is
assessed by treating a biological sample containing proteins with
the test compound. Proteins from the biological sample are
incubated with antibodies specific to the polypeptides of the
present invention. The amount of protein recognized by the
antibodies is quantified. The amount of protein in the treated
biological sample is compared with the amount in an untreated
biological sample. A difference in the amount of protein between
the two samples is indicative of a toxic response to the test
compound in the treated sample.
[0254] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. USA 93:10614-10619; Baldeschweiler et al. (1995) PCT
application WO95/251116; Shalon, D. et al. (1995) PCT application
WO95/35505; Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. USA
94:2150-2155; and Heller, M. J. et al. (1997) U.S. Pat. No.
5,605,662.) Various types of microarrays are well known and
thoroughly described in DNA Microarrays: A Practical Approach, M.
Schena, ed. (1999) Oxford University Press, London, hereby
expressly incorporated by reference.
[0255] In another embodiment of the invention, nucleic acid
sequences encoding LME may be used to generate hybridization probes
useful in mapping the naturally occurring genomic sequence. Either
coding or noncoding sequences may be used, and in some instances,
noncoding sequences may be preferable over coding sequences. For
example, conservation of a coding sequence among members of a
multi-gene family may potentially cause undesired cross
hybridization during chromosomal mapping. The sequences may be
mapped to a particular chromosome, to a specific region of a
chromosome, or to artificial chromosome constructions, e.g., human
artificial chromosomes (HACs), yeast artificial chromosomes (YACs),
bacterial artificial chromosomes (BACs), bacterial PI
constructions, or single chromosome cDNA libraries. (See, e.g.,
Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355; Price, C.
M. (1993) Blood Rev. 7:127-134; and Trask, B. J. (1991) Trends
Genet. 7:149-154.) Once mapped, the nucleic acid sequences of the
invention may be used to develop genetic linkage maps, for example,
which correlate the inheritance of a disease state with the
inheritance of a particular chromosome region or restriction
fragment length polymorphism (RFLP). (See, for example, Lander, E.
S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA
83:7353-7357.)
[0256] Fluorescent in situ hybridization (FISH) may be correlated
with other physical and genetic map data. (See, e.g., Heinz-Ulrich,
et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic
map data can be found in various scientific journals or at the
Online Mendelian Inheritance in Man (OMIM) World Wide Web site.
Correlation between the location of the gene encoding LME on a
physical map and a specific disorder, or a predisposition to a
specific disorder, may help define the region of DNA associated
with that disorder and thus may further positional cloning efforts.
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
exact chromosomal locus is not known. This information is valuable
to investigators searching for disease genes using positional
cloning or other gene discovery techniques. Once the gene or genes
responsible for a disease or syndrome have been crudely localized
by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to 11q22-23, any sequences mapping to that
area may represent associated or regulatory genes for further
investigation. (See, e.g., Gatti, R. A. et al. (1988) Nature
336:577-580.) The nucleotide sequence of the instant invention may
also be used to detect differences in the chromosomal location due
to translocation, inversion, etc., among normal, carrier, or
affected individuals.
[0257] In another embodiment of the invention, LME, 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 LME and the agent being tested may be
measured.
[0258] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate. The test
compounds are reacted with LME, or fragments thereof, and washed.
Bound LME is then detected by methods well known in the art.
Purified LME 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.
[0259] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding LME specifically compete with a test compound for binding
LME. In this manner, antibodies can be used to detect the presence
of any peptide which shares one or more antigenic determinants with
LME. In additional embodiments, the nucleotide sequences which
encode LME 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.
[0260] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever.
[0261] The disclosures of all patents, applications and
publications, mentioned above and below, including U.S. Ser. No.
60/186,480, U.S. Ser. No. 60/190,415, and U.S. Ser. No. 60/198,437,
are expressly incorporated by reference herein.
EXAMPLES
I. Construction of cDNA Libraries
[0262] Incyte cDNAs were derived from cDNA libraries described in
the LIFESEQ GOLD database (Incyte Genomics, Palo Alto Calif.) and
shown in Table 4, column 5. Some tissues were homogenized and lysed
in guanidinium isothiocyanate, while others were homogenized and
lysed in phenol or in a suitable mixture of denaturants, such as
TRIZOL (Life Technologies), a monophasic solution of phenol and
guanidine isothiocyanate. The resulting lysates were centrifuged
over C.sub.8Cl cushions or extracted with chloroform. RNA was
precipitated from the lysates with either isopropanol or sodium
acetate and ethanol, or by other routine methods.
[0263] Phenol extraction and precipitation of RNA were repeated as
necessary to increase RNA purity. In some cases, RNA was treated
with DNase. For most libraries, poly(A)+ RNA was isolated using
oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex
particles (QIAGEN, Chatsworth Calif.), or an OLIGOTEX mRNA
purification kit (QIAGEN). Alternatively, RNA was isolated directly
from tissue lysates using other RNA isolation kits, e.g., the
POLY(A)PURE mRNA purification kit (Ambion, Austin Tex.).
[0264] In some cases, Stratagene was provided with RNA and
constructed the corresponding cDNA libraries. Otherwise, cDNA was
synthesized and cDNA libraries were constructed with the UNIZAP
vector system (Stratagene) or SUPERSCRIPT plasmid system (Life
Technologies), using the recommended procedures or similar methods
known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.)
Reverse transcription was initiated using oligo d(T) or random
primers. Synthetic oligonucleotide adapters were ligated to double
stranded cDNA, and the cDNA was digested with the appropriate
restriction enzyme or enzymes. For most libraries, the cDNA was
size-selected (300-1000 bp) using SEPHACRYL S1000, SEPHAROSE CL2B,
or SEPHAROSE CL4B column chromatography (Amersham Pharmacia
Biotech) or preparative agarose gel electrophoresis. cDNAs were
ligated into compatible restriction enzyme sites of the polylinker
of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene),
PSPORT1 plasmid (Life Technologies), PCDNA2.1 plasmid (Invitrogen,
Carlsbad Calif.), PBK-CMV plasmid (Stratagene), or pINCY (Incyte
Genomics, Palo Alto Calif.), or derivatives thereof. Recombinant
plasmids were transformed into competent E. coli cells including
XLl-Blue, XL1-BlueMRF, or SOLR from Stratagene or DH5.alpha.,
DH10B, or ElectroMAX DH10B from Life Technologies.
II. Isolation of cDNA Clones
[0265] Plasmids obtained as described in Example I were recovered
from host cells by in vivo excision using the UNIZAP vector system
(Stratagene) or by cell lysis. Plasmids were purified using at
least one of the following: a Magic or WIZARD Minipreps DNA
purification system (Promega); an AGTC Miniprep purification kit
(Edge Biosystems, Gaithersburg Md.); and QIAWELL 8 Plasmid, QIAWELL
8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the
R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. Following
precipitation, plasmids were resuspended in 0.1 ml of distilled
water and stored, with or without lyophilization, at 4.degree.
C.
[0266] Alternatively, plasmid DNA was amplified from host cell
lysates using direct link PCR in a high-throughput format (Rao, V.
B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal
cycling steps were carried out in a single reaction mixture.
Samples were processed and stored in 384-well plates, and the
concentration of amplified plasmid DNA was quantified
fluorometrically using PICOGREEN dye (Molecular Probes, Eugene
Oreg.) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy,
Helsinki, Finland).
III. Sequencing and Analysis
[0267] Incyte cDNA recovered in plasmids as described in Example II
were sequenced as follows. Sequencing reactions were processed
using standard methods or high-throughput instrumentation such as
the ABI CATALYST 800 (Applied Biosystems) thermal cycler or the
PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA
microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton)
liquid transfer system. cDNA sequencing reactions were prepared
using reagents provided by Amersham Pharmacia Biotech or supplied
in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Applied Biosystems).
Electrophoretic separation of cDNA sequencing reactions and
detection of labeled polynucleotides were carried out using the
MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI
PRISM 373 or 377 sequencing system (Applied Biosystems) in
conjunction with standard ABI protocols and base calling software;
or other sequence analysis systems known in the art. Reading frames
within the cDNA sequences were identified using standard methods
(reviewed in Ausubel, 1997, supra, unit 7.7). Some of the cDNA
sequences were selected for extension using the techniques
disclosed in Example VIII.
[0268] The polynucleotide sequences derived from Incyte cDNAs were
validated by removing vector, linker, and poly(A) sequences and by
masking ambiguous bases, using algorithms and programs based on
BLAST, dynamic programming, and dinucleotide nearest neighbor
analysis. The Incyte cDNA sequences or translations thereof were
then queried against a selection of public databases such as the
GenBank primate, rodent, mammalian, vertebrate, and eukaryote
databases, and BLOCKS, PRINTS, DOMO, PRODOM, and hidden Markov
model (HMM)-based protein family databases such as PFAM. (HMM is a
probabilistic approach which analyzes consensus primary structures
of gene families. See, for example, Eddy, S. R. (1996) Curr. Opin.
Struct. Biol. 6:361-365.) The queries were performed using programs
based on BLAST, FASTA, BLIMPS, and HMMER. The Incyte cDNA sequences
were assembled to produce full length polynucleotide sequences.
Alternatively, GenBank cDNAs, GenBank ESTs, stitched sequences,
stretched sequences, or Genscan-predicted coding sequences (see
Examples IV and V) were used to extend Incyte cDNA assemblages to
full length. Assembly was performed using programs based on Phred,
Phrap, and Consed, and cDNA assemblages were screened for open
reading frames using programs based on GeneMark, BLAST, and FASTA.
The full length polynucleotide sequences were translated to derive
the corresponding full length polypeptide sequences. Alternatively,
a polypeptide of the invention may begin at any of the methionin
residues of the full length translated polypeptide. Full length
polypeptide sequences were subsequently analyzed by querying
against databases such as the GenBank protein databases (genpept),
SwissProt, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, and hidden Markov
model (HMM)-based protein family databases such as PFAM. Full
length polynucleotide sequences are also analyzed using MAcDNASIS
PRO software (Hitachi Software Engineering, South San Francisco
Calif.) and LASERGENE software (DNASTAR). Polynucleotide and
polypeptide sequence alignments are generated using default
parameters specified by the CLUSTAL algorithm as incorporated into
the MEGALIGN multisequence alignment program (DNASTAR), which also
calculates the percent identity between aligned sequences.
[0269] Table 7 summarizes the tools, programs, and algorithms used
for the analysis and assembly of Incyte cDNA and full length
sequences and provides applicable descriptions, references, and
threshold parameters. The first column of Table 7 shows the tools,
programs, and algorithms used, the second column provides brief
descriptions thereof, the third column presents appropriate
references, all of which are incorporated by reference herein in
their entirety, and the fourth column presents, where applicable,
the scores, probability values, and other parameters used to
evaluate the strength of a match between two sequences (the higher
the score or the lower the probability value, the greater the
identity between two sequences).
[0270] The programs described above for the assembly and analysis
of full length polynucleotide and polypeptide sequences were also
used to identify polynucleotide sequence fragments from SEQ ID NO:
7-12. Fragments from about 20 to about 4000 nucleotides which are
useful in hybridization and amplification technologies are
described in Table 4, column 4.
IV. Identification and Editing of Coding Sequences from Genomic
DNA
[0271] Putative lipid metabolism enzymes were initially identified
by running the Genscan gene identification program against public
genomic sequence databases (e.g., gbpri and gbhtg). Genscan is a
general-purpose gene identification program which analyzes genomic
DNA sequences from a variety of organisms (See Burge, C. and S.
Karlin (1997) J. Mol. Biol. 268:78-94, and Burge, C. and S. Karlin
(1998) Curr. Opin. Struct. Biol. 8:346-354). The program
concatenates predicted exons to form an assembled cDNA sequence
extending from a methionin to a stop codon. The output of Genscan
is a FASTA database of polynucleotide and polypeptide sequences.
The maximum range of sequence for Genscan to analyze at once was
set to 30 kb. To determine which of these Genscan predicted cDNA
sequences encode lipid metabolism enzymes, the encoded polypeptides
were analyzed by querying against PFAM models for lipid metabolism
enzymes. Potential lipid metabolism enzymes were also identified by
homology to Incyte cDNA sequences that had been annotated as lipid
metabolism enzymes. These selected Genscan-predicted sequences were
then compared by BLAST analysis to the genpept and gbpri public
databases. Where necessary, the Genscan-predicted sequences were
then edited by comparison to the top BLAST hit from genpept to
correct errors in the sequence predicted by Genscan, such as extra
or omitted exons. BLAST analysis was also used to find any Incyte
cDNA or public cDNA coverage of the Genscan-predicted sequences,
thus providing evidence for transcription. When Incyte cDNA
coverage was available, this information was used to correct or
confirm the Genscan predicted sequence. Full length polynucleotide
sequences were obtained by assembling Genscan-predicted coding
sequences with Incyte cDNA sequences and/or public cDNA sequences
using the assembly process described in Example III. Alternatively,
full length polynucleotide sequences were derived entirely from
edited or unedited Genscan-predicted coding sequences.
V. Assembly of Genomic Sequence Data with cDNA Sequence Data
"Stitched" Sequences
[0272] Partial cDNA sequences were extended with exons predicted by
the Genscan gene identification program described in Example IV.
Partial cDNAs assembled as described in Example III were mapped to
genomic DNA and parsed into clusters containing related cDNAs and
Genscan exon predictions from one or more genomic sequences. Each
cluster was analyzed using an algorithm based on graph theory and
dynamic programming to integrate cDNA and genomic information,
generating possible splice variants that were subsequently
confirmed, edited, or extended to create a full length sequence.
Sequence intervals in which the entire length of the interval was
present on more than one sequence in the cluster were identified,
and intervals thus identified were considered to be equivalent by
transitivity. For example, if an interval was present on a cDNA and
two genomic sequences, then all three intervals were considered to
be equivalent. This process allows unrelated but consecutive
genomic sequences to be brought together, bridged by cDNA sequence.
Intervals thus identified were then "stitched" together by the
stitching algorithm in the order that they appear along their
parent sequences to generate the longest possible sequence, as well
as sequence variants. Linkages between intervals which proceed
along one type of parent sequence (cDNA to cDNA or genomic sequence
to genomic sequence) were given preference over linkages which
change parent type (cDNA to genomic sequence). The resultant
stitched sequences were translated and compared by BLAST analysis
to the genpept and gbpri public databases. Incorrect exons
predicted by Genscan were corrected by comparison to the top BLAST
hit from genpept. Sequences were further extended with additional
cDNA sequences, or by inspection of genomic DNA, when
necessary.
"Stretched" Sequences
[0273] Partial DNA sequences were extended to full length with an
algorithm based on BLAST analysis. First, partial cDNAs assembled
as described in Example III were queried against public databases
such as the GenBank primate, rodent, mammalian, vertebrate, and
eukaryote databases using the BLAST program. The nearest GenBank
protein homolog was then compared by BLAST analysis to either
Incyte cDNA sequences or GenScan exon predicted sequences described
in Example IV. A chimeric protein was generated by using the
resultant high-scoring segment pairs (HSPs) to map the translated
sequences onto the GenB ank protein homolog. Insertions or
deletions may occur in the chimeric protein with respect to the
original GenBank protein homolog. The GenBank protein homolog, the
chimeric protein, or both were used as probes to search for
homologous genomic sequences from the public human genome
databases. Partial DNA sequences were therefore "stretched" or
extended by the addition of homologous genomic sequences. The
resultant stretched sequences were examined to determine whether it
contained a complete gene.
VI. Chromosomal Mapping of LME Encoding Polynucleotides
[0274] The sequences which were used to assemble SEQ ID NO: 7-12
were compared with sequences from the Incyte LIFESEQ database and
public domain databases using BLAST and other implementations of
the Smith-Waterman algorithm. Sequences from these databases that
matched SEQ ID NO: 7-12 were assembled into clusters of contiguous
and overlapping sequences using assembly algorithms such as Phrap
(Table 7). Radiation hybrid and genetic mapping data available from
public resources such as the Stanford Human Genome Center (SHGC),
Whitehead Institute for Genome Research (WIGR), and Genethon were
used to determine if any of the clustered sequences had been
previously mapped. Inclusion of a mapped sequence in a cluster
resulted in the assignment of all sequences of that cluster,
including its particular SEQ ID NO:, to that map location.
[0275] Map locations are represented by ranges, or intervals, or
human chromosomes. The map position of an interval, in
centiMorgans, is measured relative to the terminus of the
chromosome's p-arm. (The centiMorgan (cM) is a unit of measurement
based on recombination frequencies between chromosomal markers. On
average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in
humans, although this can vary widely due to hot and cold spots of
recombination.) The cM distances are based on genetic markers
mapped by Genethon which provide boundaries for radiation hybrid
markers whose sequences were included in each of the clusters.
Human genome maps and other resources available to the public, such
as the NCBI "GeneMap'99" on the internet, can be employed to
determine if previously identified disease genes map within or in
proximity to the intervals indicated above.
VII. Analysis of Polynucleotide Expression
[0276] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; Ausubel (1995) supra, ch. 4 and
16.) Analogous computer techniques applying BLAST were used to
search for identical or related molecules in cDNA databases such as
GenBank or LIFESEQ (Incyte Genomics). 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 similar.
The basis of the search is the product score, which is defined
as:
BLAST Score .times. Percent Identity 5 .times. minimum { length (
Seq . 1 ) , length ( Seq . 2 ) } ##EQU00001##
[0277] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. The product score is a normalized value between 0 and 100,
and is calculated as follows: the BLAST score is multiplied by the
percent nucleotide identity and the product is divided by (5 times
the length of the shorter of the two sequences). The BLAST score is
calculated by assigning a score of +5 for every base that matches
in a high-scoring segment pair (HSP), and -4 for every mismatch.
Two sequences may share more than one HSP (separated by gaps). If
there is more than one HSP, then the pair with the highest BLAST
score is used to calculate the product score. The product score
represents a balance between fractional overlap and quality in a
BLAST alignment. For example, a product score of 100 is produced
only for 100% identity over the entire length of the shorter of the
two sequences being compared. A product score of 70 is produced
either by 100% identity and 70% overlap at one end, or by 88%
identity and 100% overlap at the other. A product score of 50 is
produced either by 100% identity and 50% overlap at one end, or 79%
identity and 100% overlap.
[0278] Alternatively, polynucleotide sequences encoding LME are
analyzed with respect to the tissue sources from which they were
derived. For example, some full length sequences are assembled, at
least in part, with overlapping Incyte cDNA sequences (see Example
III). Each cDNA sequence is derived from a cDNA library constructed
from a human tissue. Each human tissue is classified into one of
the following organ/tissue categories: cardiovascular system;
connective tissue; digestive system; embryonic structures;
endocrine system; exocrine glands; genitalia, female; genitalia,
male; germ cells; hemic and immune system; liver; musculoskeletal
system; nervous system; pancreas; respiratory system; sense organs;
skin; stomatognathic system; unclassified/mixed; or urinary tract.
The number of libraries in each category is counted and divided by
the total number of libraries across all categories. Similarly,
each human tissue is classified into one of the following
disease/condition categories: cancer, cell line, developmental,
inflammation, neurological, trauma, cardiovascular, pooled, and
other, and the number of libraries in each category is counted and
divided by the total number of libraries across all categories. The
resulting percentages reflect the tissue- and disease-specific
expression of cDNA encoding LME. cDNA sequences and cDNA
library/tissue information are found in the LIFESEQ GOLD database
(Incyte Genomics, Palo Alto Calif.).
VIII. Extension of LME Encoding Polynucleotides
[0279] Full length polynucleotide sequences were also produced by
extension of an appropriate fragment of the full length molecule
using oligonucleotide primers designed from this fragment. One
primer was synthesized to initiate 5' extension of the known
fragment, and the other primer was synthesized to initiate 3'
extension of the known fragment. The initial primers were designed
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.
[0280] Selected human cDNA libraries were used to extend the
sequence. If more than one extension was necessary or desired,
additional or nested sets of primers were designed.
[0281] High fidelity amplification was obtained by PCR using
methods well known in the art. PCR was performed in 96-well plates
using the PTC-200 thermal cycler (MJ Research, Inc.). The reaction
mix contained DNA template, 200 nmol of each primer, reaction
buffer containing Mg.sup.2+, (NH.sub.4).sub.2SO.sub.4, and
2-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase
(Stratagene), with the following parameters for primer pair PCI A
and PCI B: Step 1: 94.degree. C., 3 min; Step 2: 94.degree. C., 15
sec; Step 3: 60.degree. C., 1 min; Step 4: 68.degree. C., 2 min;
Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68.degree. C.,
5 min; Step 7: storage at 4.degree. C. In the alternative, the
parameters for primer pairT7 and SK+ were as follows: Step 1:
94.degree. C., 3 min; Step 2: 94.degree. C., 15 sec; Step 3:
57.degree. C., 1 min; Step 4: 68.degree. C., 2 min; Step 5: Steps
2, 3, and 4 repeated 20 times; Step 6: 68.degree. C., 5 min; Step
7: storage at 4.degree. C.
[0282] The concentration of DNA in each well was determined by
dispensing 1001 PICOGREEN quantitation reagent (0.25% (v/v)
PICOGREEN; Molecular Probes, Eugene Oreg.) dissolved in 1.times.TE
and 0.5 .mu.l of undiluted PCR product into each well of an opaque
fluorimeter plate (Corning Costar, Acton Mass.), allowing the DNA
to bind to the reagent. The plate was scanned in a Fluoroskan II
(Labsystems Oy, Helsinki, Finland) to measure the fluorescence of
the sample and to quantify the concentration of DNA. A 5 .mu.l to
10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a 1% agarose gel to determine which reactions
were successful in extending the sequence.
[0283] The extended nucleotides were desalted and concentrated,
transferred to 384-well plates, digested with CviJI cholera virus
endonuclease (Molecular Biology Research, Madison Wis.), and
sonicated or sheared prior to religation into pUC 18 vector
(Amersham Pharmacia Biotech). For shotgun sequencing, the digested
nucleotides were separated on low concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar
ACE (Promega). Extended clones were religated using T4 ligase (New
England Biolabs, Beverly Mass.) into pUC 18 vector (Amersham
Pharmacia Biotech), treated with Pfu DNA polymerase (Stratagene) to
fill-in restriction site overhangs, and transfected into competent
E. coli cells. Transformed cells were selected on
antibiotic-containing media, and individual colonies were picked
and cultured overnight at 37.degree. C. in 384-well plates in LB/2x
carb liquid media.
[0284] The cells were lysed, and DNA was amplified by PCR using Taq
DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase
(Stratagene) with the following parameters: Step 1: 94.degree. C.,
3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min;
Step 4: 72.degree. C., 2 min; Step 5: steps 2, 3, and 4 repeated 29
times; Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree.
C. DNA was quantified by PICOGREEN reagent (Molecular Probes) as
described above. Samples with low DNA recoveries were reamplified
using the same conditions as described above. Samples were diluted
with 20% dimethysulfoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit
(Amersham Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator
cycle sequencing ready reaction kit (Applied Biosystems).
[0285] In like manner, full length polynucleotide sequences are
verified using the above procedure or are used to obtain 5'
regulatory sequences using the above procedure along with
oligonucleotides designed for such extension, and an appropriate
genomic library.
IX. Labeling and Use of Individual Hybridization Probes
[0286] Hybridization probes derived from SEQ ID NO: 7-12 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, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham
Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN,
Boston Mass.). The labeled oligonucleotides are substantially
purified using a SEPHADEX G-25 superfine size exclusion dextran
bead column (Amersham Pharmacia Biotech). An aliquot containing
10.sup.7 counts per minute of the labeled probe is used in a
typical membrane-based hybridization analysis of human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
[0287] The DNA from each digest is fractionated on a 0.7% 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 conditions of up to,
for example, 0.1.times. saline sodium citrate and 0.5% sodium
dodecyl sulfate. Hybridization patterns are visualized using
autoradiography or an alternative imaging means and compared.
X. Microarrays
[0288] The linkage or synthesis of array elements upon a microarray
can be achieved utilizing photolithography, piezoelectric printing
(inkjet printing, See, e.g., Baldeschweiler, supra), mechanical
microspotting technologies, and derivatives thereof. The substrate
in each of the aforementioned technologies should be uniform and
solid with a non-porous surface (Schena (1999), supra). Suggested
substrates include silicon, silica, glass slides, glass chips, and
silicon wafers. Alternatively, a procedure analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced using available
methods and machines well known to those of ordinary skill in the
art and may contain any appropriate number of elements. (See, e.g.,
Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al.
(1996) Genome Res. 6:639-645; Marshall, A. and J. Hodgson (1998)
Nat. Biotechnol. 16:27-31.)
[0289] Full length cDNAs, Expressed Sequence Tags (ESTs), or
fragments or oligomers thereof may comprise the elements of the
microarray. Fragments or oligomers suitable for hybridization can
be selected using software well known in the art such as LASERGENE
software (DNASTAR). The array elements are hybridized with
polynucleotides in a biological sample. The polynucleotides in the
biological sample are conjugated to a fluorescent label or other
molecular tag for ease of detection. After hybridization,
nonhybridized nucleotides from the biological sample are removed,
and a fluorescence scanner is used to detect hybridization at each
array element. Alternatively, laser desorbtion and mass
spectrometry may be used for detection of hybridization. The degree
of complementarity and the relative abundance of each
polynucleotide which hybridizes to an element on the microarray may
be assessed. In one embodiment, microarray preparation and usage is
described in detail below.
Tissue or Cell Sample Preparation
[0290] Total RNA is isolated from tissue samples using the
guanidinium thiocyanate method and poly(A).sup.+ RNA is purified
using the oligo-(dT) cellulose method. Each poly(A).sup.+ RNA
sample is reverse transcribed using MMLV reverse-transcriptase,
0.05 pg/.mu.l oligo-(dT) primer (21mer), 1.times. first strand
buffer, 0.03 units/.mu.l RNase inhibitor, 500 .mu.M dATP, 500 .mu.M
dGTP, 500 .mu.M dTTP, 40 .mu.M dCTP, 40 .mu.M dCTP-Cy3 (BDS) or
dCTP-Cy5 (Amersham Pharmacia Biotech). The reverse transcription
reaction is performed in a 25 ml volume containing 200 ng
poly(A).sup.+ RNA with GEMBRIGHT kits (Incyte). Specific control
poly(A).sup.+ RNAs are synthesized by in vitro transcription from
non-coding yeast genomic DNA. After incubation at 37.degree. C. for
2 hr, each reaction sample (one with Cy3 and another with Cy5
labeling) is treated with 2.5 ml of 0.5M sodium hydroxide and
incubated for 20 minutes at 85.degree. C. to the stop the reaction
and degrade the RNA. Samples are purified using two successive
CHROMA SPIN 30 gel filtration spin columns (CLONTECH Laboratories,
Inc. (CLONTECH), Palo Alto Calif.) and after combining, both
reaction samples are ethanol precipitated using 1 ml of glycogen (1
mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The
sample is then dried to completion using a SpeedVAC (Savant
Instruments Inc., Holbrook N.Y.) and resuspended in 14 .mu.l
5.times.SSC/0.2% SDS.
Microarray Preparation
[0291] Sequences of the present invention are used to generate
array elements. Each array element is amplified from bacterial
cells containing vectors with cloned cDNA inserts. PCR
amplification uses primers complementary to the vector sequences
flanking the cDNA insert. Array elements are amplified in thirty
cycles of PCR from an initial quantity of 1-2 ng to a final
quantity greater than 5 .mu.g. Amplified array elements are then
purified using SEPHACRYL-400 (Amersham Pharmacia Biotech). Purified
array elements are immobilized on polymer-coated glass slides.
Glass microscope slides (Corning) are cleaned by ultrasound in 0.1%
SDS and acetone, with extensive distilled water washes between and
after treatments. Glass slides are etched in 4% hydrofluoric acid
(VWR Scientific Products Corporation (VWR), West Chester Pa.),
washed extensively in distilled water, and coated with 0.05%
aminopropyl silane (Sigma) in 95% ethanol. Coated slides are cured
in a 110.degree. C. oven.
[0292] Array elements are applied to the coated glass substrate
using a procedure described in U.S. Pat. No. 5,807,522,
incorporated herein by reference. 1 .mu.l of the array element DNA,
at an average concentration of 100 ng/.mu.l, is loaded into the
open capillary printing element by a high-speed robotic apparatus.
The apparatus then deposits about 5 nl of array element sample per
slide.
[0293] Microarrays are UV-crosslinked using a STRATALINKER
UV-crosslinker (Stratagene). Microarrays are washed at room
temperature once in 0.2% SDS and three times in distilled water.
Non-specific binding sites are blocked by incubation of microarrays
in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc.,
Bedford Mass.) for 30 minutes at 60.degree. C. followed by washes
in 0.2% SDS and distilled water as before.
Hybridization
[0294] Hybridization reactions contain 9 .mu.l of sample mixture
consisting of 0.2 .mu.g each of Cy3 and d Cy5 labeled cDNA
synthesis products in 5.times.SSC, 0.2% SDS hybridization buffer.
The sample mixture is heated to 65.degree. C. for 5 minutes and is
aliquoted onto the microarray surface and covered with an 1.8 cm
coverslip. The arrays are transferred to a waterproof chamber
having a cavity just slightly larger than a microscope slide. The
chamber is kept at 100% humidity internally by the addition of 140
.mu.l of 5.times.SSC in a corner of the chamber. The chamber
containing the arrays is incubated for about 6.5 hours at
60.degree. C. The arrays are washed for 10 min at 45.degree. C. in
a first wash buffer (1.times.SSC, 0.1% SDS), three times for 10
minutes each at 45.degree. C. in a second wash buffer
(0.1.times.SSC), and dried.
Detection
[0295] Reporter-labeled hybridization complexes are detected with a
microscope equipped with an Innova 70 mixed gas 10 W laser
(Coherent, Inc., Santa Clara Calif.) capable of generating spectral
lines at 488 nm for excitation of Cy3 and at 632 nm for excitation
of Cy5. The excitation laser light is focused on the array using a
20.times. microscope objective (Nikon, Inc., Melville N.Y.). The
slide containing the array is placed on a computer-controlled X-Y
stage on the microscope and raster-scanned past the objective. The
1.8 cm.times.1.8 cm array used in the present example is scanned
with a resolution of 20 micrometers.
[0296] In two separate scans, a mixed gas multiline laser excites
the two fluorophores sequentially. Emitted light is split, based on
wavelength, into two photomultiplier tube detectors (PMT R1477,
Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the
two fluorophores. Appropriate filters positioned between the array
and the photomultiplier tubes are used to filter the signals. The
emission maxima of the fluorophores used are 565 mu for Cy3 and 650
nm for Cy5. Each array is typically scanned twice, one scan per
fluorophore using the appropriate filters at the laser source,
although the apparatus is capable of recording the spectra from
both fluorophores simultaneously.
[0297] The sensitivity of the scans is typically calibrated using
the signal intensity generated by a cDNA control species added to
the sample mixture at a known concentration. A specific location on
the array contains a complementary DNA sequence, allowing the
intensity of the signal at that location to be correlated with a
weight ratio of hybridizing species of 1:100,000. When two samples
from different sources (e.g., representing test and control cells),
each labeled with a different fluorophore, are hybridized to a
single array for the purpose of identifying genes that are
differentially expressed, the calibration is done by labeling
samples of the calibrating cDNA with the two fluorophores and
adding identical amounts of each to the hybridization mixture.
[0298] The output of the photomultiplier tube is digitized using a
12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog
Devices, Inc., Norwood Mass.) installed in an IBM-compatible PC
computer. The digitized data are displayed as an image where the
signal intensity is mapped using a linear 20-color transformation
to a pseudocolor scale ranging from blue (low signal) to red
(high-signal). The data is also analyzed quantitatively. Where two
different fluorophores are excited and measured simultaneously, the
data are first corrected for optical crosstalk (due to overlapping
emission spectra) between the fluorophores using each fluorophore's
emission spectrum.
[0299] A grid is superimposed over the fluorescence signal image
such that the signal from each spot is centered in each element of
the grid. The fluorescence signal within each element is then
integrated to obtain a numerical value corresponding to the average
intensity of the signal. The software used for signal analysis is
the GEMTOOLS gene expression analysis program (Incyte).
XI. Complementary Polynucleotides
[0300] Sequences complementary to the LME-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring LME. 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 (National Biosciences) and the
coding sequence of LME. 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 LME-encoding transcript.
XII. Expression of LME
[0301] Expression and purification of LME is achieved using
bacterial or virus-based expression systems. For expression of LME
in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express LME upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of LME in
eukaryotic cells is achieved by infecting insect or mammalian cell
lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding LME by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.) In most expression systems, LME is
synthesized as a fusion protein with, e.g., glutathione
S-transferase (GST) or a peptide epitope tag, such as FLAG or
6-His, permitting rapid, single-step, affinity-based purification
of recombinant fusion protein from crude cell lysates. GST, a
26-kilodalton enzyme from Schistosoma iaponicum, enables the
purification of fusion proteins on immobilized glutathione under
conditions that maintain protein activity and antigenicity
(Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from LME at specifically
engineered sites. FLAG, an 8-amino acid peptide, enables
immunoaffinity purification using commercially available monoclonal
and polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a
stretch of six consecutive histidine residues, enables purification
on metal-chelate resins (QIAGEN). Methods for protein expression
and purification are discussed in Ausubel (1995, supra. ch. 10 and
16). Purified LME obtained by these methods can be used directly in
the assays shown in Examples XVI and XVII, where applicable.
XIII. Functional Assays
[0302] LME function is assessed by expressing the sequences
encoding LME at physiologically elevated levels in mammalian cell
culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life
Technologies) and PCR3.1 (Invitrogen, Carlsbad Calif.), both of
which contain the cytomegalovirus promoter. 5-10 .mu.g of
recombinant vector are transiently transfected into a human cell
line, for example, an endothelial or hematopoietic cell line, using
either liposome formulations or electroporation. 1-2 .mu.g of an
additional plasmid containing sequences encoding a marker protein
are co-transfected. Expression of a marker protein provides a means
to distinguish transfected cells from nontransfected cells and is a
reliable predictor of cDNA expression from the recombinant vector.
Marker proteins of choice include, e.g., Green Fluorescent Protein
(GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry
(FCM), an automated, laser optics-based technique, is used to
identify transfected cells expressing GFP or CD64-GFP and to
evaluate the apoptotic state of the cells and other cellular
properties. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose events preceding or coincident with cell
death. These events include changes in nuclear DNA content as
measured by staining of DNA with propidium iodide; changes in cell
size and granularity as measured by forward light scatter and 90
degree side light scatter; down-regulation of DNA synthesis as
measured by decrease in bromodeoxyuridine uptake; alterations in
expression of cell surface and intracellular proteins as measured
by reactivity with specific antibodies; and alterations in plasma
membrane composition as measured by the binding of
fluorescein-conjugated Annexin V protein to the cell surface.
Methods in flow cytometry are discussed in Ormerod, M. G. (1994)
Flow Cytometry, Oxford, New York N.Y.
[0303] The influence of LME on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding LME and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding LME and other genes of interest can be
analyzed by northern analysis or microarray techniques.
XIV. Production of LME Specific Antibodies
[0304] LME substantially purified using polyacrylamide gel
electrophoresis (PAGE; see, e.g., 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.
[0305] Alternatively, the LME amino acid sequence is analyzed using
LASERGENE software (DNASTAR) to determine regions of high
immunogenicity, and a corresponding oligopeptide is synthesized and
used to raise antibodies by means known to those of skill in the
art. Methods for selection of appropriate epitopes, such as those
near the C-terminus or in hydrophilic regions are well described in
the art. (See, e.g., Ausubel, 1995, supra, ch. 11.)
[0306] Typically, oligopeptides of about 15 residues in length are
synthesized using an ABI 431A peptide synthesizer (Applied
Biosystems) using FMOC chemistry and coupled to KLH (Sigma-Aldrich,
St. Louis Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase
immunogenicity. (See, e.g., Ausubel, 1995, supra.) Rabbits are
immunized with the oligopeptide-KLH complex in complete Freund's
adjuvant. Resulting antisera are tested for antipeptide and
anti-LME activity by, for example, binding the peptide or LME to a
substrate, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio-iodinated goat anti-rabbit
IgG.
XV. Purification of Naturally Occurring LME Using Specific
Antibodies
[0307] Naturally occurring or recombinant LME is substantially
purified by immunoaffinity chromatography using antibodies specific
for LME. An immunoaffinity column is constructed by covalently
coupling anti-LME antibody to an activated chromatographic resin,
such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0308] Media containing LME are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of LME (e.g., high ionic strength buffers
in the presence of detergent). The column is eluted under
conditions that disrupt antibody/LME 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 LME is collected.
XVI. Identification of Molecules Which Interact with LME
[0309] LME, or biologically active fragments thereof, are labeled
with 1251 Bolton-Hunter reagent. (See, e.g., Bolton A. E. and W. M.
Hunter (1973) Biochem. J. 133:529-539.) Candidate molecules
previously arrayed in the wells of a multi-well plate are incubated
with the labeled LME, washed, and any wells with labeled LME
complex are assayed. Data obtained using different concentrations
of LME are used to calculate values for the-number, affinity, and
association of LME with the candidate molecules.
[0310] Alternatively, molecules interacting with LME are analyzed
using the yeast two-hybrid system as described in Fields, S, and O,
Song (1989) Nature 340:245-246, or using commercially available
kits based on the two-hybrid system, such as the MATCHMAKER system
(Clontech).
[0311] LME may also be used in the PATHCALLING process (CuraGen
Corp., New Haven Conn.) which employs the yeast two-hybrid system
in a high-throughput manner to determine all interactions between
the proteins encoded by two large libraries of genes (Nandabalan,
K. et al. (2000) U.S. Pat. No. 6,057,101).
XVII. Demonstration of LME Activity
[0312] LME activity can be demonstrated by an in vitro hydrolysis
assay with vesicles containing 1-palmitoyl-2-[1-.sup.14C]oleoyl
phosphatidylcholine (Sigma-Aldrich). LME triglyceride lipase
activity and phospholipase A.sub.2 activity are demonstrated by
analysis of the cleavage products isolated from the hydrolysis
reaction mixture. Vesicles containing
l-palmitoyl-2-[1-.sup.14C]oleoyl phosphatidylcholine (Amersham
Pharmacia Biotech.) are prepared by mixing 2.0 .mu.Ci of the
radiolabeled phospholipid with 12.5 mg of unlabeled
1-palmitoyl-2-oleoyl phosphatidylcholine and drying the mixture
under N.sub.2. 2.5 ml of 150 mM Tris-HCl, pH 7.5, is added, and the
mixture is sonicated and centrifuged. The supernatant may be stored
at 4.degree. C. The final reaction mixtures contain 0.25 ml of
Hanks buffered salt solution supplemented with 2.0 mM
taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mM
CaCl.sub.2, pH 7.4, 150 .mu.g of 1-palmitoyl-2-[1-.sup.14C]oleoyl
phosphatidylcholine vesicles, and various amounts of LME diluted in
PBS. After incubation for 30 min at 37.degree. C., 20 .mu.g each of
lyso-phosphatidylcholine and oleic acid are added as carriers and
each sample is extracted for total lipids. The lipids are separated
by thin layer chromatography using a two solvent system of
chloroform:methanol:acetic acid:water (65:35:8:4) until the solvent
front is halfway up the plate. The process is then continued with
hexane:ether:acetic:acid (86:16:1) until the solvent front is at
the top of the plate. The lipid-containing areas are visualized
with 12 vapor; the spots are scraped, and their radioactivity is
determined by scintillation counting. The amount of radioactivity
released as fatty acids will increase as a greater amount of LME is
added to the assay mixture while the amount of radioactivity
released as lyso-phosphatidylcholine will remain low. This
demonstrates that LME cleaves at the sn-2 and not the sn-1
position, as is characteristic of phospholipase A.sub.2
activity.
[0313] Alternatively, LME phospholipase activity is measured by the
hydrolysis of a fatty acyl residue at the sn-1 position of
phosphatidylserine. LME is combined with the Tritium [.sup.3H]
labeled substrate phosphatidylserine at stoichiometric quantities
in a suitable buffer. Following an appropriate incubation time, the
hydrolyzed reaction products are separated from the substrates by
chromatographic methods. The amount of acylglycerophosphoserine
produced is measured by counting tritiated product with the help of
a scintillation counter. Various control groups are set up to
account for background noise and unincorporated substrate. The
final counts represent the tritiated enzyme product
[.sup.3H]-acylglycerophosphoserine, which is directly proportional
to the activity of LME in biological samples.
[0314] LME lipoxygenase activity can be measured by chromatographic
methods. Extracted LME lipoxygenase protein is incubated with 100
.mu.M [1-.sup.14C] arachidonic acid or other unlabeled fatty acids
at 37.degree. C. for 30 min. After the incubation, stop solution
(acetonitrile:methanol:water, 350:150:1) is added. The samples are
extracted and analyzed by reverse-phase HPLC by using a solvent
system of methanol/water/acetic acid, 85:15:0.01 (vol/vol) at a
flow rate of 1 ml/min. The effluent is monitored at 235 nm and
analyzed for the presence of the major arachidonic metabolite such
as 12-PETE (catalyzed by 12-LOX). The fractions are also subjected
to liquid scintillation counting. The final counts represent the
products, which is directly proportional to the activity of LME in
biological samples. For stereochemical analysis, the metabolites of
aracbidonic acid are analyzed further by chiral phase-HPLC and by
mass spectrometry (Sun, D. et al. (1998) J. Biol. Chem.
273:33540-33547).
[0315] 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 certain 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.
TABLE-US-00002 TABLE 1 Incyte Incyte Incyte Polypeptide Polypeptide
Polynucleotide Polynucleotide Project ID SEQ ID NO: ID SEQ ID NO:
ID 2372651 1 2372651CD1 7 2372651CB1 2470792 2 2470792CD1 8
2470792CB1 1506182 3 1506182CD1 9 1506182CB1 2690842 4 2690842CD1
10 2690842CB1 5027764 5 5027764CD1 11 5027764CB1 2488174 6
2488174CD1 12 2488174CB1
TABLE-US-00003 TABLE 2 Polypeptide Incyte GenBank Probability
GenBank SEQ ID NO: Polypeptide ID ID NO: Score Homolog 1 2372651CD1
g3876901 2.50E-56 Similarity to Human enoyl-CoA hydratase (SW:
ECHM_HUMAN) [Caenorhabditis elegans] ((1998) Science 282:
2012-2018) 2 2470792CD1 g7024433 0 Male sterility protein 2-like
protein [torpedo marmorata] 3 1506182CD1 g11245478 1.00e-162
Nicotinamide mononucleotide adenylyl transferase [Homo sapiens] 4
2690842CD1 g6503307 3.20E-18 Phospholipid biosynthetic
acyltransferase family member [Arabidopsis thaliana] (Neuwald, A.
F. (1997) Curr. Biol. 7: R465-466) 5 5027764CD1 g4090960 1.40E-64
Phosphatidylserine-specific phospholipase A1 [Homo sapiens] (Nagai,
Y. et al. (1999) J. Biol. Chem. 274: 11-53-11059) 6 2488174CD1
g7274380 1.00E-151 Group III secreted phospholipase A2 [Homo
sapiens] (Valentin, E. et al. (2000) J. Biol. Chem. 275:
7492-7496)
TABLE-US-00004 TABLE 3 SEQ Incyte Amino Potential Potential
Analytical ID Polypeptide Acid Phosphorylation Glycosylation
Signature Sequences, Methods and NO: ID Residues Sites Sites
Domains and Motifs Databases 1 2372651CD1 303 S49 S167 T233
Enoyl-CoA hydratase/isomerase ProfileScan S43 T69 S105 signature:
F12-L181 S210 Enoyl-CoA hydratase/isomerase family: HMMER-PFAM
R57-P225 Enoyl-CoA hydratase/isomerase: BLIMPS- BL00166A: G55-K66
BLOCKS BL00166B: V93-E114 BL00166C: V140-A166 BL00166D: L190-P225
BL00166E: A289-R294 Enoyl CoA hydratase isomerase: BLAST- PD000432:
I59-P225 PRODOM Enoyl-CoA hydratase/isomerase: BLAST-DOMO
DM00366|P30084|34-285: I59-K295 2 2470792CD1 515 T33 S94 T235 N128
N341 ATP/GTP-binding site motif A (P-loop); MOTIFS S389 S414 T114
N396 A5-K11 T120 T193 T257 Male sterility protein 2: BLAST- S356
T371 T512 PD018334; W200-L445 PRODOM Y168 Y404 3 1506182CD1 279
T130 S109 S256 N36 Signal cleavage: M1-M22 SPScan S4 T38 T95 S136
Lipopolysaccharide core BLIMPS- S223 S235 biosynthesis protein
signature: PRINTS PR01020A: V9-L27 PR01020C: H65-Q89 Protein
F26H9.4: BLAST- PD023338: V9-L106 PRODOM Membrane protein YLR328W:
BLAST-DOMO DM07979|P53204|1-394: M1-L106 4 2690842CD1 432 S35 S74
S76 T84 N111 Phospholipid biosynthetic acyltransferase: HMMER-PFAM
S176 S178 S203 R18-S203 T208 S287 S333 T353 T370 T394 S398 S80 T110
T117 S254 Y253 5 5027764CD1 451 T72 S14 S16 S97 N50 N58 N66 Vespid
venom allergen PLA1 signature BLIMPS- S144 T175 S206 N357 PR00825A:
PRINTS S258 S272 S287 P188-H205, L211-P231 T320 T47 T68 Lipase
precursor, signal, hydrolase, lipid BLAST- S121 T124 T348
degradation, glycoprotein PD001492: PRODOM N64-I335 Triacylglycerol
lipase: BLAST-DOMO DM00344/A49488/25-326: L39-V333 Signal petide:
M1-A18 HMMER Signal cleavage: M1-A18 SPScan Lipase: H31-D319
HMMER-PFAM Lipases, serine proteins BL00120: BLIMPS- N102-I116,
D146-S160, Y223-C233 BLOCKS Triacylglycerol lipase family signature
BLIMPS- PR00821: PRINTS S59-F78, V104-H119, D147-E165, C246-E261,
P325-K340 6 2488174CD1 312 S125 S57 S190 N15 N83 N128 Phospholipase
A2 histidine active site MOTIFS S212 S87 S96 N199 N242 (PDOC00109):
S121 S138 S166 C28-C35 T201 S253 Phosphollipase A2 isozymes: BLAST-
PD033132: M1-R66 PRODOM A2, phospholipase, histidine: BLAST-DOMO
DM05541|P80003|1-141: M1-R66 DM05541|B56338|1-136: P2-R66
TABLE-US-00005 TABLE 4 Incyte Polynucleotide Polynucleotide
Sequence Selected Sequence 5' 3' SEQ ID NO: ID Length Framents
Fragments Position Position 7 2372651CB1 1667 1-177 6827519J1 235
827 (SINTNOR01) 7001351H1 531 1136 (HEALDIR01) 5601906H1 1339 1652
(UTRENON03) 6515361H1 1116 1650 (THYMDIT01) 5602106H1 1340 1653
(UTRENON03) 3574443H1 1 297 (BRONNOT01) 8 2470792CB1 2124 1-863,
70774783V1 1678 2066 2075-2124 70780150V1 682 1195 70067901V1 1548
2065 70067625V1 1209 1685 6830177J1 1 688 (SINTNOR01) 70781158V1
633 1157 70778451V1 946 1600 622417H1 1871 2124 (PGANNOT01) 9
1506182CB1 2955 1-52, 1433938R1 1718 2183 550-587, (BEPINON01) 10
2690842CB1 1579 1-313 2690842H1 (LUNGNOT23) 969 1210 7362670H1
(LUNLTUE02) 1 615 7261254H1 (UTRETMC01) 1010 1579 6913602H1
(PITUDIR01) 587 1043 11 5027764CB1 3170 2450-2737, 841054T6
(PROSTUT05) 2557 3170 507-1843 3224086R6 (COLNNON03) 2477 3021
70705531V1 1621 2291 4438670H1 (SINTNOT22) 1 272 6933434H1
(SINTTMR02) 14 652 70635824V1 2273 2892 70750908V1 1723 2330
70743692V1 999 1595 71051604V1 470 1054 71246013V1 1078 1692 12
2488174CB1 1900 1-1772 70906023V1 1368 1889 71530085V1 497 1187
71531355V1 566 1284 70905361V1 1263 1888 2488174F6 (LUNGNOT22) 1
532 71527244V1 1426 1899
TABLE-US-00006 TABLE 5 Polynucleotide Incyte Representative SEQ ID
NO: Project ID Library 7 2372651CB1 PROSNOT18 8 2470792CB1
EOSIHET02 9 1506182CB1 BARITUT02 10 2690842CB1 UTRETMC01 11
5027764CB1 PROSTUT05 12 2488174CB1 LUNGNOT22
TABLE-US-00007 TABLE 6 Library Vector Library Description BRAITUT02
PSRORT1 Library was constructed using RNA isolated from brain tumor
tissue removed from the frontal lobe of a 58-year-old Caucasian
male during excision of a cerebral meningeal lesion. Pathology
indicated a grade 2 metastatic hypernephroma. Patient history
included a grade 2 renal cell carcinoma, insomnia, and chronic
airway obstruction. Family history included a malignant neoplasm of
the kindney. EOSIHET02 PBLUESCRIPT Library was constructed using
RNA isolated from peripheral blood cells apheresed from a 48-
year-old Caucasian male. Patient history included
hypereosinophilia. The cell population was determined to be greater
than 77% eosinophils by Wright's staining. LUNGNOT22 pINCY Library
was constructed using RNA isolated from lung tissue removed from a
58-year-old Caucasian female. The tissue sample used to construct
this library was found to have tumor contaminant upon microscopic
examination. Pathology for the assocfiated tumor tissue indicated a
caseating granuloma. Family history included congestive heart
failure, breast cancer, secondary bone cancer, acute myocardial
infarction and athereosclerotic coronary artery disease. PROSNOT18
pINCY Library was constructed using RNA isolated from diseased
prostate tissue removed from a 58- year-old Caucasian male during a
radical cystectomy, radical prostatectomy, and gastrostomy.
Pathology indicated adenofibromatous hyperplasia; this tissue was
associated with a grade 3 transitional cell carcinoma. patient
history included angina and emphysema. Family history included
acute myocardial infarction, atherosclerotic coronary artery
disease, and type II diabetes. PROSTUT05 PSPORT1 Library was
constructed using RNA isolated from diseased prostate tissue
removed from a 69- year-old Caucasian male during a radical
prostatectomy. Pathology indicated adenocarcinoma (Gleason grade 3
+ 4). Adenofibromatous hyperplasia was also present. Family history
included congestive heart failure, multiple myeloma,
hyperlipidemia, and rheumatoid arthritis. UTRETMC01 pINCY This
large size-fractionated library was constructed using pooled cDNA
from two different donors. cDNA was generated using mRNA isolated
from endometrial tissue removed from a 32- year-old Caucasian
female (donor A) during total abdominal hysterectomy, bilateral
salpingo- oophorectomy, and cystocele repair; and from endometrial
tissue removed from a 48-year-old Caucasian female (donor B) during
a vaginal hysterectomy, rectocele repair, and bilateral
salpingo-oophorectomy. Pathology for donor A indicated the
endometrium was in the Proliferative phase. The right ovary showed
a corpus luteal cyst. For donor B, pathology indicated chronic
cervicitis and the endometrium was weakly proliferative. The right
ovary and specimen from the peritoneum indicated endometriosis
focally involving the surface of the right ovary and the
peritoneum. Pathology for the matched tumor tissue indicated a
single submucosal leiomyoma, which exhibited extensive hyalin
change with hyalin-type necrosis. The left ovary contained a corpus
luteum cyst. Donor A presented with abdominal pain, stress
incontinence, and dysmenorrhea. Patient history included
hemorrhagic ovarian cysts, uterine endometriosis, normal delivery,
and casearean deliveries. Donor B presented with metrorrhagia,
extrinsic asthma, depressive disorder, and anxiety state, Patient
history included alcohol abuse, hyperlipidemia, a normal delivery,
tobacco abuse in remission, and meningitis. Patient medications (B)
included Prozac, Trazodone, Clorazepate, and Medrol.
TABLE-US-00008 TABLE 7 Program Description Reference Parameter
Threshold ABIFACTURA A program that removes vector Applied
Biosystems, Foster City, CA. sequences and masks ambiguous bases in
nucleic acid sequences. ABI/PARACEL FDF A Fast Data Finder useful
in Applied Biosystems, Foster City, CA; Mismatch <50% comparing
and annotating amino acid Paracel Inc., Pasadena, CA. or nucleic
acid sequences. ABI AutoAssembler A program that assembles nucleic
Applied Biosystems, Foster City, CA. acid sequences. BLAST A Basic
Local Alignment Search Tool Altschul, S. F. et al. (1990) J. Mol.
Biol. EsTs: Probability useful in sequence similarity search 215:
403-410; Altschul, S. F. et al. (1997) value = 1.0E-8 or less for
amino acid and nucleic acie Nucleic Acids Res. 25: 3389-3402. Full
Length sequences: sequences. BLAST includes five Probability value
= 1.0E-10 functions: blastp, blastn, blastx, or less tblastn, and
tblastx. FASTA A Pearson and Lipman algorithm that Pearson, W. R.
and D. J. Lipman (1988) ESTs: fasta E searches for similarity
between a Proc. Natl. Acad Sci. USA 85: 2444-2448; value = 1.06E-6
query sequence and a group of Pearson, W. R. (1990) Methods
Enzymol. Assembled ESTs: fasta sequences of the same type. FASTA
183: 63-98: and Smith, T. F. and M. S. Waterman Identity = 95% or
greater comprises as least five functions: (1981) Adv. Appl. Math.
2: 482-489. and Match length = 200 fasta, tfasta, fastx, tfastx,
and search. bases or greater; fastx E value = 1:0E-8 or less Full
Length sequences: fastx score = 100 or greater BLIMPS A BLocks
IMProved Searcher that Henikoff, S. and J. G. Henikoff (1991)
Probability value = 1.0E-3 matches a sequence against those in
Nucleic Acids Res. 19: 6565-6572; or less BLOCKS, PRINTS, DOMO,
PRODOM, Henikoff, J. G. and S. Henikoff (1996) and PFAM databases
to search for gene Methods Enzymol. 266: 88-105; and families,
sequence homology, and Attwood, T. K. et al. (1997) J. Chem.
structural fingerprint regions. Inf. Comput. Sci. 37: 471-424.
HMMER An algorithm for searching a query Krogh, A. et al. (1994) J.
Mol. Biol. PFAM hits: Probability sequence against hidden Markov
model 235: 1501-1531; Sonnhammer, E. L. L. value = 1.0E-3 or less
(HMM)-based databases of protein family et al. (1988) Nucleic Acids
Res. Signal peptide hits: consensus sequences, such as PFAM. 26:
320-322; Durbin, R. et al. (1998) Score = 0 or greater Our World
View, in a Nutshell, Cambridge Univ. Press, pp. 1-350. ProfileScan
An algorithm that searches for structureal Gribskov, M. et al.
(1988) CABIOS Normalized quality and sequence motifs in protein
sequences 4: 61-66; Gribskov, M. et al. (1989) score
.gtoreq.GCG-specified that match sequence patterns defined in
Methods Enzymol. 183: 146-159; "HIGH" value for that Prosite.
Bairoch, A. et al. (1997) Nucleic Acids particular Prosite motif.
Res. 25: 217-221. Generally, score = 1.4-2.1. Phred A base-calling
algoritm that examines Ewing, B. et al. (1998) Genome Res.
automated sequencer traces with high 8: 175-185; Ewing, B. and P.
Green sensitivity and probability. (1998) Genome Res. 8: 186-194.
Phrap A Phils Revised Assembly Program Smith, T. F. and M. S.
Waterman (1981) Score = 120 or greater; including SWAT and
CrossMatch, Adv. Appl. Math. 2: 482-489; Smith, T. F. Match length
= 56 or programs based on efficient and M. S. Waterman (1981) J.
Mol. Biol. greater implementation of the Smith- 147: 195-197; and
Green, P., University of Waterman algorithm, useful in Washington,
Seattle, WA. searching sequence homology and assembling DNA
sequences. Consed A graphical tool for viewing and Gordon, D. et
al. (1998) Genome Res. editing Phraph assemblies. 8: 195-202.
SPScan A weight matrix analysis program Nielson, H. et al. (1997)
Protein Score = 3.5 or greater that scans protein sequences for the
Engineering 10: 1-6; Claverie, J. M. and S. Audic presence of
secretory signal peptides. (1997) CABIOS 12: 431-439. TMAP A
program that uses weight matrices Persson, B. and P. Argos (1994)
J. Mol. to delineate transmembrane Biol. 237: 182-192; Persson, B.
and P. Argos segments on protein sequences and (1996) Protein Sci.
5: 363-371. determine orientation. TMHMMER A program that uses a
hidden Sonnhammer, E. L. et al. (1998) Proc. Sixth Markov model
(HMM) to delineate Intl. Conf, on Intelligent Systems for Mol.
transmembrane segments on protein Biol., Glasgow et al., eds., The
Am. Assoc. sequences and determine orientation. for Artificial
Intelligence Press, Menlo Park, CA, pp. 175-182. Motifs A program
that searches amino acid Bairoch, A. et al. (1997) Nucleic Acids
Res. sequences for patterns that matched 25: 217-221; Wisconsin
Package Program those defined in Prosite. Manual, version 9, page
M51-59, Genetics Computer Group, Madison, WI.
Sequence CWU 1
1
121303PRTHomo sapiensmisc_featureIncyte ID No 2372651CD1 1Met Ala
Ala Val Ala Val Leu Arg Ala Phe Gly Ala Ser Gly Pro 1 5 10 15Met
Cys Leu Arg Arg Gly Pro Trp Ala Gln Leu Pro Ala Arg Phe 20 25 30Cys
Ser Arg Asp Pro Ala Gly Ala Gly Arg Arg Glu Ser Glu Pro 35 40 45Arg
Pro Thr Ser Ala Arg Gln Leu Asp Gly Ile Arg Asn Ile Val 50 55 60Leu
Ser Asn Pro Lys Lys Arg Asn Thr Leu Ser Leu Ala Met Leu 65 70 75Lys
Ser Leu Gln Ser Asp Ile Leu His Asp Ala Asp Ser Asn Asp 80 85 90Leu
Lys Val Ile Ile Ile Ser Ala Glu Gly Pro Val Phe Ser Ser 95 100
105Gly His Asp Leu Lys Glu Leu Thr Glu Glu Gln Gly Arg Asp Tyr 110
115 120His Ala Glu Val Phe Gln Thr Cys Ser Lys Val Met Met His Ile
125 130 135Arg Asn His Pro Val Pro Val Ile Ala Met Val Asn Gly Leu
Ala 140 145 150Thr Ala Ala Gly Cys Gln Leu Val Ala Ser Cys Asp Ile
Ala Val 155 160 165Ala Ser Asp Lys Ser Ser Phe Ala Thr Pro Gly Val
Asn Val Gly 170 175 180Leu Phe Cys Ser Thr Pro Gly Val Ala Leu Ala
Arg Ala Val Pro 185 190 195Arg Lys Val Ala Leu Glu Met Leu Phe Thr
Gly Glu Pro Ile Ser 200 205 210Ala Gln Glu Ala Leu Leu His Gly Leu
Leu Ser Lys Val Val Pro 215 220 225Glu Ala Glu Leu Gln Glu Glu Thr
Met Arg Ile Ala Arg Lys Ile 230 235 240Ala Ser Leu Ser Arg Pro Val
Val Ser Leu Gly Lys Ala Thr Phe 245 250 255Tyr Lys Gln Leu Pro Gln
Asp Leu Gly Thr Ala Tyr Tyr Leu Thr 260 265 270Ser Gln Ala Met Val
Asp Asn Leu Ala Leu Arg Asp Gly Gln Glu 275 280 285Gly Ile Thr Ala
Phe Leu Gln Lys Arg Lys Pro Val Trp Ser His 290 295 300Glu Pro
Val2515PRTHomo sapiensmisc_featureIncyte ID No 2470792CD1 2Met Ser
Thr Ile Ala Ala Phe Tyr Gly Gly Lys Ser Ile Leu Ile 1 5 10 15Thr
Gly Ala Thr Gly Phe Leu Gly Lys Val Leu Met Glu Lys Leu 20 25 30Phe
Arg Thr Ser Pro Asp Leu Lys Val Ile Tyr Ile Leu Val Arg 35 40 45Pro
Lys Ala Gly Gln Thr Leu Gln Gln Arg Val Phe Gln Ile Leu 50 55 60Asp
Ser Lys Leu Phe Glu Lys Val Lys Glu Val Cys Pro Asn Val 65 70 75His
Glu Lys Ile Arg Ala Ile Tyr Ala Asp Leu Asn Gln Asn Asp 80 85 90Phe
Ala Ile Ser Lys Glu Asp Met Gln Glu Leu Leu Ser Cys Thr 95 100
105Asn Ile Ile Phe His Cys Ala Ala Thr Val Arg Phe Asp Asp Thr 110
115 120Leu Arg His Ala Val Gln Leu Asn Val Thr Ala Thr Arg Gln Leu
125 130 135Leu Leu Met Ala Ser Gln Met Pro Lys Leu Glu Ala Phe Ile
His 140 145 150Ile Ser Thr Ala Tyr Ser Asn Cys Asn Leu Lys His Ile
Asp Glu 155 160 165Val Ile Tyr Pro Cys Pro Val Glu Pro Lys Lys Ile
Ile Asp Ser 170 175 180Leu Glu Trp Leu Asp Asp Ala Ile Ile Asp Glu
Ile Thr Pro Lys 185 190 195Leu Ile Arg Asp Trp Pro Asn Ile Tyr Thr
Tyr Thr Lys Ala Leu 200 205 210Gly Glu Met Val Val Gln Gln Glu Ser
Arg Asn Leu Asn Ile Ala 215 220 225Ile Ile Arg Pro Ser Ile Val Gly
Ala Thr Trp Gln Glu Pro Phe 230 235 240Pro Gly Trp Val Asp Asn Ile
Asn Gly Pro Asn Gly Ile Ile Ile 245 250 255Ala Thr Gly Lys Gly Phe
Leu Arg Ala Ile Lys Ala Thr Pro Met 260 265 270Ala Val Ala Asp Val
Ile Pro Val Asp Thr Val Val Asn Leu Met 275 280 285Leu Ala Val Gly
Trp Tyr Thr Ala Val His Arg Pro Lys Ser Thr 290 295 300Leu Val Tyr
His Ile Thr Ser Gly Asn Met Asn Pro Cys Asn Trp 305 310 315His Lys
Met Gly Val Gln Val Leu Ala Thr Phe Glu Lys Ile Pro 320 325 330Phe
Glu Arg Pro Phe Arg Arg Pro Asn Ala Asn Phe Thr Ser Asn 335 340
345Ser Phe Thr Ser Gln Tyr Trp Asn Ala Val Ser His Arg Ala Pro 350
355 360Ala Ile Ile Tyr Asp Cys Tyr Leu Arg Leu Thr Gly Arg Lys Pro
365 370 375Arg Met Thr Lys Leu Met Asn Arg Leu Leu Arg Thr Val Ser
Met 380 385 390Leu Glu Tyr Phe Ile Asn Arg Ser Trp Glu Trp Ser Thr
Tyr Asn 395 400 405Thr Glu Met Leu Met Ser Glu Leu Ser Pro Glu Asp
Gln Arg Val 410 415 420Phe Asn Phe Asp Val Arg Gln Leu Asn Trp Leu
Glu Tyr Ile Glu 425 430 435Asn Tyr Val Leu Gly Val Lys Lys Tyr Leu
Leu Lys Glu Asp Met 440 445 450Ala Gly Ile Pro Lys Ala Lys Gln Arg
Leu Lys Arg Leu Arg Asn 455 460 465Ile His Tyr Leu Phe Asn Thr Ala
Leu Phe Leu Ile Ala Trp Arg 470 475 480Leu Leu Ile Ala Arg Ser Gln
Met Ala Arg Asn Val Trp Phe Phe 485 490 495Ile Val Ser Phe Cys Tyr
Lys Phe Leu Ser Tyr Phe Arg Ala Ser 500 505 510Ser Thr Leu Lys Val
5153279PRTHomo sapiensmisc_featureIncyte ID No 1506182CD1 3Met Glu
Asn Ser Glu Lys Thr Glu Val Val Leu Leu Ala Cys Gly 1 5 10 15Ser
Phe Asn Pro Ile Thr Asn Met His Leu Arg Leu Phe Glu Leu 20 25 30Ala
Lys Asp Tyr Met Asn Gly Thr Gly Arg Tyr Thr Val Val Lys 35 40 45Gly
Ile Ile Ser Pro Val Gly Asp Ala Tyr Lys Lys Lys Gly Leu 50 55 60Ile
Pro Ala Tyr His Arg Val Ile Met Ala Glu Leu Ala Thr Lys 65 70 75Asn
Ser Lys Trp Val Glu Val Asp Thr Trp Glu Ser Leu Gln Lys 80 85 90Glu
Trp Lys Glu Thr Leu Lys Val Leu Arg His His Gln Glu Lys 95 100
105Leu Glu Ala Ser Asp Cys Asp His Gln Gln Asn Ser Pro Thr Leu 110
115 120Glu Arg Pro Gly Arg Lys Arg Lys Trp Thr Glu Thr Gln Asp Ser
125 130 135Ser Gln Lys Lys Ser Leu Glu Pro Lys Thr Lys Ala Val Pro
Lys 140 145 150Val Lys Leu Leu Cys Gly Ala Asp Leu Leu Glu Ser Phe
Ala Val 155 160 165Pro Asn Leu Trp Lys Ser Glu Asp Ile Thr Gln Ile
Val Ala Asn 170 175 180Tyr Gly Leu Ile Cys Val Thr Arg Ala Gly Asn
Asp Ala Gln Lys 185 190 195Phe Ile Tyr Glu Ser Asp Val Leu Trp Lys
His Arg Ser Asn Ile 200 205 210His Val Val Asn Glu Trp Ile Ala Asn
Asp Ile Ser Ser Thr Lys 215 220 225Ile Arg Arg Ala Leu Arg Arg Gly
Gln Ser Ile Arg Tyr Leu Val 230 235 240Pro Asp Leu Val Gln Glu Tyr
Ile Glu Lys His Asn Leu Tyr Ser 245 250 255Ser Glu Ser Glu Asp Arg
Asn Ala Gly Val Ile Leu Ala Pro Leu 260 265 270Gln Arg Asn Thr Ala
Glu Ala Lys Thr 2754432PRTHomo sapiensmisc_featureIncyte ID No
2690842CD1 4Met Arg Thr Met Trp Phe Ala Gly Gly Phe His Arg Val Ala
Val 1 5 10 15Lys Gly Arg Gln Ala Leu Pro Thr Glu Ala Ala Ile Leu
Thr Leu 20 25 30Ala Pro His Ser Ser Tyr Phe Asp Ala Ile Pro Val Thr
Met Thr 35 40 45Met Ser Ser Ile Val Met Lys Ala Glu Ser Arg Asp Ile
Pro Ile 50 55 60Trp Gly Thr Leu Ile Gln Tyr Ile Arg Pro Val Phe Val
Ser Arg 65 70 75Ser Asp Gln Asp Ser Arg Arg Lys Thr Val Glu Glu Ile
Lys Arg 80 85 90Arg Ala Gln Ser Asn Gly Lys Trp Pro Gln Ile Met Ile
Phe Pro 95 100 105Glu Gly Thr Cys Thr Asn Arg Thr Cys Leu Ile Thr
Phe Lys Pro 110 115 120Gly Ala Phe Ile Pro Gly Ala Pro Val Gln Pro
Val Val Leu Arg 125 130 135Tyr Pro Asn Lys Leu Asp Thr Ile Thr Trp
Thr Trp Gln Gly Pro 140 145 150Gly Ala Leu Glu Ile Leu Trp Leu Thr
Leu Cys Gln Phe His Asn 155 160 165Gln Val Glu Ile Glu Phe Leu Pro
Val Tyr Ser Pro Ser Glu Glu 170 175 180Glu Lys Arg Asn Pro Ala Leu
Tyr Ala Ser Asn Val Arg Arg Val 185 190 195Met Ala Glu Ala Leu Gly
Val Ser Val Thr Asp Tyr Thr Phe Glu 200 205 210Asp Cys Gln Leu Ala
Leu Ala Glu Gly Gln Leu Arg Leu Pro Ala 215 220 225Asp Thr Cys Leu
Leu Glu Phe Ala Arg Leu Val Arg Gly Leu Gly 230 235 240Leu Lys Pro
Glu Lys Leu Glu Lys Asp Leu Asp Arg Tyr Ser Glu 245 250 255Arg Ala
Arg Met Lys Gly Gly Glu Lys Ile Gly Ile Ala Glu Phe 260 265 270Ala
Ala Ser Leu Glu Val Pro Val Ser Asp Leu Leu Glu Asp Met 275 280
285Phe Ser Leu Phe Asp Glu Ser Gly Ser Gly Glu Val Asp Leu Arg 290
295 300Glu Cys Val Val Ala Leu Ser Val Val Cys Arg Pro Ala Arg Thr
305 310 315Leu Asp Thr Ile Gln Leu Ala Phe Lys Thr Tyr Gly Ala Gln
Glu 320 325 330Asp Gly Ser Val Gly Glu Gly Asp Leu Ser Cys Ile Leu
Lys Thr 335 340 345Ala Leu Gly Val Ala Glu Leu Thr Val Thr Asp Leu
Phe Arg Ala 350 355 360Ile Asp Gln Glu Glu Lys Gly Lys Ile Thr Phe
Ala Asp Phe His 365 370 375Arg Phe Ala Glu Met Tyr Pro Ala Phe Ala
Glu Glu Tyr Leu Tyr 380 385 390Pro Asp Gln Thr His Phe Glu Ser Cys
Ala Glu Thr Ser Pro Ala 395 400 405Pro Ile Pro Asn Gly Phe Cys Ala
Asp Phe Ser Pro Glu Asn Ser 410 415 420Asp Ala Gly Arg Lys Pro Val
Arg Lys Lys Leu Asp 425 4305451PRTHomo sapiensmisc_featureIncyte ID
No 5027764CD1 5Met Leu Arg Phe Tyr Leu Phe Ile Ser Leu Leu Cys Leu
Ser Arg 1 5 10 15Ser Asp Ala Glu Glu Thr Cys Pro Ser Phe Thr Arg
Leu Ser Phe 20 25 30His Ser Ala Val Val Gly Thr Gly Leu Asn Val Arg
Leu Met Leu 35 40 45Tyr Thr Arg Lys Asn Leu Thr Cys Ala Gln Thr Ile
Asn Ser Ser 50 55 60Ala Phe Gly Asn Leu Asn Val Thr Lys Lys Thr Thr
Phe Ile Val 65 70 75His Gly Phe Arg Pro Thr Gly Ser Pro Pro Val Trp
Met Asp Asp 80 85 90Leu Val Lys Gly Leu Leu Ser Val Glu Asp Met Asn
Val Val Val 95 100 105Val Asp Trp Asn Arg Gly Ala Thr Thr Leu Ile
Tyr Thr His Ala 110 115 120Ser Ser Lys Thr Arg Lys Val Ala Met Val
Leu Lys Glu Phe Ile 125 130 135Asp Gln Met Leu Ala Glu Gly Ala Ser
Leu Asp Asp Ile Tyr Met 140 145 150Ile Gly Val Ser Leu Gly Ala His
Ile Ser Gly Phe Val Gly Glu 155 160 165Met Tyr Asp Gly Trp Leu Gly
Arg Ile Thr Gly Leu Asp Pro Ala 170 175 180Gly Pro Leu Phe Asn Gly
Lys Pro His Gln Asp Arg Leu Asp Pro 185 190 195Ser Asp Ala Gln Phe
Val Asp Val Ile His Ser Asp Thr Asp Ala 200 205 210Leu Gly Tyr Lys
Glu Pro Leu Gly Asn Ile Asp Phe Tyr Pro Asn 215 220 225Gly Gly Leu
Asp Gln Pro Gly Cys Pro Lys Thr Ile Leu Gly Gly 230 235 240Phe Gln
Tyr Phe Lys Cys Asp His Gln Arg Ser Val Tyr Leu Tyr 245 250 255Leu
Ser Ser Leu Arg Glu Ser Cys Thr Ile Thr Ala Tyr Pro Cys 260 265
270Asp Ser Tyr Gln Asp Tyr Arg Asn Gly Lys Cys Val Ser Cys Gly 275
280 285Thr Ser Gln Lys Glu Ser Cys Pro Leu Leu Gly Tyr Tyr Ala Asp
290 295 300Asn Trp Lys Asp His Leu Arg Gly Lys Asp Pro Pro Met Thr
Lys 305 310 315Ala Phe Phe Asp Thr Ala Glu Glu Ser Pro Phe Cys Met
Tyr His 320 325 330Tyr Phe Val Asp Ile Ile Thr Trp Asn Lys Asn Val
Arg Arg Gly 335 340 345Asp Ile Thr Ile Lys Leu Arg Asp Lys Ala Gly
Asn Thr Thr Glu 350 355 360Ser Lys Ile Asn His Glu Pro Thr Thr Phe
Gln Lys Tyr His Gln 365 370 375Val Ser Leu Leu Ala Arg Phe Asn Gln
Asp Leu Asp Lys Val Ala 380 385 390Ala Ile Ser Leu Met Phe Ser Thr
Gly Ser Leu Ile Gly Pro Arg 395 400 405Tyr Lys Leu Arg Ile Leu Arg
Met Lys Leu Arg Ser Leu Ala His 410 415 420Pro Glu Arg Pro Gln Leu
Cys Arg Tyr Asp Leu Val Leu Met Glu 425 430 435Asn Val Glu Thr Val
Phe Gln Pro Ile Leu Cys Pro Glu Leu Gln 440 445 450Leu6312PRTHomo
sapiensmisc_featureIncyte ID No 2488174CD1 6Met Pro Gly Thr Leu Trp
Cys Gly Val Gly Asp Ser Ala Gly Asn 1 5 10 15Ser Ser Glu Leu Gly
Val Phe Gln Gly Pro Asp Leu Cys Cys Arg 20 25 30Glu His Asp Arg Cys
Pro Gln Asn Ile Ser Pro Leu Gln Tyr Asn 35 40 45Tyr Gly Ile Arg Asn
Tyr Arg Phe His Thr Ile Ser His Cys Asp 50 55 60Cys Asp Thr Arg Cys
Arg Met Tyr Gly Thr Val Pro Leu Ala Arg 65 70 75Leu Gln Pro Arg Thr
Phe Tyr Asn Ala Ser Trp Ser Ser Arg Ala 80 85 90Thr Ser Pro Thr Pro
Ser Ser Arg Ser Pro Ala Pro Pro Lys Pro 95 100 105Arg Gln Lys Gln
His Leu Arg Lys Gly Pro Pro His Gln Lys Gly 110 115 120Ser Lys Arg
Pro Ser Lys Ala Asn Thr Thr Ala Leu Gln Asp Pro 125 130 135Met Val
Ser Pro Arg Leu Asp Val Ala Pro Thr Gly Leu Gln Gly 140 145 150Pro
Gln Gly Gly Leu Lys Pro Gln Gly Ala Arg Trp Val Cys Arg 155 160
165Ser Phe Arg Arg His Leu Asp Gln Cys Glu His Gln Ile Gly Pro 170
175 180Arg Glu Ile Glu Phe Gln Leu Leu Asn Ser Ala Gln Glu Pro Leu
185 190 195Phe His Cys Asn Cys Thr Arg Arg Leu Ala Arg Phe Leu Arg
Leu 200 205 210His Ser Pro Pro Glu Val Thr Asn Met Leu Trp Glu Leu
Leu Gly 215 220 225Thr Thr Cys Phe Lys Leu Ala Pro Pro Leu Asp Cys
Val Glu Gly 230 235 240Lys Asn Cys Ser Arg Asp Pro Arg Ala Ile Arg
Val Ser Ala Arg 245 250 255His Leu Arg Arg Leu Gln Gln Arg Arg His
Gln Leu Gln Asp Lys 260 265 270Gly Thr Asp Glu Arg Gln Pro Trp Pro
Ser Glu Pro Leu Arg Gly 275 280 285Pro Met Ser Phe Tyr Asn Gln Cys
Leu Gln Leu Thr Gln Ala Ala 290 295 300Arg Arg Pro Asp Arg Gln Gln
Lys Ser Trp
Ser Gln 305 31071667DNAHomo sapiensmisc_featureIncyte ID No
2372651CB1 7cgcctggcct ggggcgtctc cgcgaacctg ggcctgtcag gcggttccgt
ccgggtctcg 60gccaccgtcg agttccgtcg agttccgtcc cggccctgct cacagcagcg
ccctcggagc 120gcccagcacc tgcggccggc caggcagcgc gatcctgcgg
cgtctggcca tcccgaatgc 180tatggccgcc gtcgccgtct tgcgggcctt
cggggcaagt gggcccatgt gtctccggcg 240cggcccctgg gcccagctcc
ccgcccgctt ctgcagccgg gacccggccg gggcggggcg 300gcgggagtcg
gagccgcggc ccaccagcgc gcggcagctg gacggcataa ggaacatcgt
360cttgagcaat cccaagaaga ggaacacgtt gtcacttgca atgctgaaat
ctctccaaag 420tgacattctt catgacgctg acagcaacga tctgaaagtc
attatcatct cggctgaggg 480gcctgtgttt tcttctgggc atgacttaaa
ggagctgaca gaggagcaag gccgtgatta 540ccatgccgaa gtatttcaga
cctgttccaa ggtcatgatg cacatccgga accaccccgt 600ccccgtcatt
gccatggtca atggcctggc cacggctgcc ggctgtcaac tggttgccag
660ctgcgacatt gccgtggcga gcgacaagtc ctcttttgcc actcctgggg
tgaacgtcgg 720gctcttctgt tctacccctg gggttgcctt ggcaagagca
gtgcctagaa aggtggcctt 780ggagatgctc tttactggtg agcccatttc
tgcccaggag gccctgctcc acgggctgct 840tagcaaggtg gtgccagagg
cggagctgca ggaggagacc atgcggatcg ctaggaagat 900cgcatcgctg
agccgtccgg tggtgtccct gggcaaagcc accttctaca agcagctgcc
960ccaggacctg gggacggctt actacctcac ctcccaggcc atggtggaca
acctggccct 1020gcgggacggg caggagggca tcacggcctt cctccagaag
agaaaacctg tctggtcaca 1080cgagccagtg tgagtggagg cagaggagtg
aggcccacgg gcagcgccca ggagcccacc 1140ttcccctctg gcccagccac
cactgcctct cagcttcaac aggtgacagg ctgctttcgt 1200gacttgatat
tggtgtcata gcatttggcc tacattaaaa gccacaattt catggggaaa
1260ggacaaaatg gagagtgact gaggtgctga cctcagtgca aggctggtga
accctgcagc 1320gggccagcta tggtgggaag cctggcattt ggggtgctcc
ttgcaacgtc ttaagcaagc 1380gacccccctg acatagcaaa aggtggcaac
ccatggaggc agaaagaagg acgccagcct 1440gacccttatc tgaaacgtcc
taagcagagt taatcctggc tgctcaggag aggcgacaca 1500tttcaaatct
ccacgagata ttctccacac agaaaatctt cttgattcta tagagactta
1560atcatgccta tggctttgaa taatcttatg tgatttaaat aaattaaatc
tttatagaga 1620aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa
166782124DNAHomo sapiensmisc_featureIncyte ID No 2470792CB1
8cggagccggt gaaggtcgga gggagggtgg tttcctcccg ccccacaccc agtctccgag
60ccggatatat agagtgtcac gtttgggagc cgaaagactg gagccgtttc cttgtggctg
120gagcgcttcc cgtagcctcg gggaaggagc aggatttaga ggaccactag
ttggacccca 180tcctcgtgct ggaggaacag gaacctcttt caggagctat
aaaagaaagg gaggaatcat 240gtccacaatt gcagctttct atggcggcaa
gtccattctc atcacggggg ccacaggctt 300tctgggcaaa gtgctgatgg
agaagctgtt tcgcaccagc ccagacctga aagtcattta 360catccttgtg
aggcccaagg ctggccagac actgcagcag agggttttcc agatcctaga
420cagtaagcta tttgagaaag tcaaagaagt ttgtccaaat gtgcatgaga
agatcagagc 480tatttatgca gatctcaatc agaatgactt tgccatcagc
aaagaggaca tgcaggagct 540tctctcctgt acaaacataa tatttcactg
tgcagccact gtacgctttg acgacactct 600cagacatgct gtgcaactta
acgtcactgc cacccggcag ctcttgctta tggctagtca 660gatgccaaag
ctggaagcct ttatacatat ctctactgcc tattcaaatt gtaacctgaa
720gcacatcgat gaagttatct atccgtgccc tgtggagcca aaaaaaatca
ttgattccct 780tgagtggtta gacgatgcta ttattgacga gattacaccc
aagctgatca gagattggcc 840caatatttat acctacacca aggccttggg
agaaatggtg gtgcagcaag agagcaggaa 900cctgaacatt gccatcataa
ggccctccat tgtgggagca acttggcagg agcctttccc 960aggttgggtt
gataatataa atggacctaa tggaatcatt attgcgactg ggaaagggtt
1020tcttcgggcc ataaaagcta ctccaatggc tgtggcagac gtaattccag
ttgatacagt 1080cgtcaatctc atgctagctg taggatggta tactgcagtt
cacagaccta agtcaacatt 1140agtctaccac attacatctg gtaacatgaa
tccctgcaat tggcacaaaa tgggagtcca 1200agtcttggca acctttgaaa
aaatcccatt tgagagacct ttcaggaggc caaatgctaa 1260ttttaccagc
aacagcttca catcacagta ctggaatgcg gtcagccacc gggcccctgc
1320cattatctat gactgctatc tgcggctcac tggaaggaag cccaggatga
caaagctcat 1380gaatcggctt ttaagaactg tttccatgtt ggagtatttc
atcaaccgga gttgggaatg 1440gagcacgtac aatacagaaa tgctgatgtc
tgagctgagt cctgaagacc agagagtatt 1500caactttgac gtgcgccagt
tgaactggtt ggaatacatt gaaaattatg ttttgggagt 1560taaaaaatac
ttattgaaag aggatatggc tgggatccca aaagcaaagc aacgcttaaa
1620aaggctccga aatattcact acctctttaa tactgccctc ttccttatcg
cctggcgcct 1680tctcattgca agatctcaga tggctcggaa tgtctggttc
ttcattgtaa gcttctgtta 1740taaattcctc tcctacttta gagcatccag
cacgctcaaa gtttaagagc atttagccat 1800cgccttttat ctggaacctc
tcagatacct ctaaaacagc aaactgtgat tctcaagatt 1860agaaagtaac
aaggaatatg cccaaactgt caaatgtcac ctgttatgta ttcgtcccta
1920ttccttaact atgtattttt atttcagtga gagaaggaaa gttgtaaact
agcccatagt 1980cacctatatt ttagggaaaa aaatccaaat tgtttcctaa
cattctattt tatgcccttg 2040cgtattaaac gtgaaagtac tcccactttt
ctatatttag tttttctttt ctctctgaga 2100tgattcattt aaactcagta aata
212492955DNAHomo sapiensmisc_featureIncyte ID No 1506182CB1
9ccgggccgct ggtgatctcc ggtagcactc gggccggcgg acagtgaggg cgcgacaaca
60agggaggtgt cacagttttc catttagatc aacaacttca agttcttacc atggaaaatt
120ccgagaagac tgaagtggtt ctccttgctt gtggttcatt caatcccatc
accaacatgc 180acctcaggtt gtttgagctg gccaaggact acatgaatgg
aacaggaagg tacacagttg 240tcaaaggcat catctctcct gttggtgatg
cctacaagaa gaaaggactc attcctgcct 300atcaccgggt catcatggca
gaacttgcta ccaagaattc taaatgggtg gaagttgata 360catgggaaag
tcttcagaag gagtggaaag agactctgaa ggtgctaaga caccatcaag
420agaaattgga ggctagtgac tgtgatcacc agcagaactc acctactcta
gaaaggcctg 480gaaggaagag gaagtggact gaaacacaag attctagtca
aaagaaatcc ctagagccaa 540aaacaaaagc tgtgccaaag gtcaagctgc
tgtgtggggc agatttattg gagtcctttg 600ctgttcccaa tttgtggaag
agtgaagaca tcacccaaat cgtggccaac tatgggctca 660tatgtgttac
tcgggctgga aatgatgctc agaagtttat ctatgaatcg gatgtgctgt
720ggaaacaccg gagcaacatt cacgtggtga atgaatggat cgctaatgac
atctcatcca 780caaaaatccg gagagccctc agaaggggcc agagcattcg
ctacttggta ccagatcttg 840tccaagaata cattgaaaag cataatttgt
acagctctga gagtgaagac aggaatgctg 900gggtcatcct ggcccctttg
cagagaaaca ctgcagaagc taagacatag gaattctaca 960gcatgatatt
tcagacttcc catttgggga tctgaaacaa tctgggagtt aataactggg
1020gaaagaagtt gtgatctgtt gcctaaacta aagcttaaaa gtttagtaaa
aatcgtctgg 1080gcacagtggc tcacgcctgt aatcccagca ctttgggagg
ctgaggcagg tggatcacgg 1140ggtcaggaga tcgagaccat cctggccaat
atggtgagac cccatctcta ctaaaaatac 1200aaaaattagc tgtgtgtggt
ggcacgtgcc tgtggtctca gcatgctgag tggctgggat 1260tacaggcacc
cactaccatg tccggctaat tctgtatttt tagtagagat ggggtttcgc
1320catgttagac aggctggtct tgaactcctg acctcaggtg atctgcccac
ctcggccttc 1380caaagtgctg ggattacagg catgagccac tgcacccagc
ctgatcctat tgttgcacta 1440tttatggagc aacaactttg tacaaagaac
aagctttgta cagagaacaa gcttggcttt 1500ttctcccaac gccgaggatg
ctgttgatgc tgccacgtaa tagcataatt ttgggtgtcc 1560tcaaggacag
aacttccact ttgaataatg gaagttagaa caatgaattt cacaggggaa
1620taaatattaa tgactgacgt gaagaaaata tgccattgtt tattccctcc
tgcatcattt 1680ccataatttg cttttgtact gtcaatttag aggaaatgtg
tgatgctggt gttttgtttg 1740gcctgtttgt ttgatgctgg gggttttatg
tgttgtaccc tttacccctt acattgtgta 1800atttgaaagt ggcaaacaaa
cctgcagtaa aagtccttga ttggcatctt cattcggatg 1860atggagagcc
tttgtggtag tgtttgctta tgtgaacagc aggcctttca gataagagaa
1920gtggcttttc cttggtgatg aaggggtaga gattgagcca tggggatggt
ttaggttaaa 1980gaatgctttt tttttggcca tcatgaggat ctaacaacag
agtagaagga aggatgccct 2040aggtcagcac gcagggtggt gggagggctt
tcatcttcct tacccaagcc tctcttttca 2100cttttctaga agttcggaag
ttgttatatg atgaaatagc ctcctttaac gtttatttct 2160gggtgccaag
ggaggcccat tcctctaaca ttctgataat tcttctcaaa ggcctatgat
2220ctaaacattt caccatggca tccacttagc tgtggggctg catacacagt
ctccacctct 2280gaaatctgaa cttcatttac cagtggtgct gtttgaactt
cataatgcca gcacttcctg 2340aacacttact gtgtgcctgg cttgtgttcc
tgagtgcctt atatcacaag gaaacggcaa 2400aatcagggga ctggtataag
tggtgaagct gggcttgaat ctaagctttg tcttcagagc 2460cagtacccct
aacctctctt tctgtaaaac attacttttc aaagaatgaa gttgtagcca
2520aatcttgaaa tttttcattt accctaagtg agaacaaata aagtttcagc
aaaataataa 2580taataataat aatccactat agcttttggt tctctaggcc
aaagaaagct ttcacaatca 2640ttttttctgt tctttggtct cctggaaagc
tttcagtgga agcgatgttt gggacctgga 2700gtatgacata gtgggataaa
ttcaagttaa acttgaatct gaagcccaac ttgcctcagt 2760ttcctcacca
ataaattaag ggtcataaga gtatgtgcct catgaaaccg ttgggaaatc
2820taaacttgac catctacaaa gtggctggca cagaaacaag tgctcaacac
atagacatta 2880cagtgatcca ggccacatcc aaccaatgca gagaccaaca
gagcctcttc aggatcggga 2940acatccagtt aaaat 2955101579DNAHomo
sapiensmisc_featureIncyte ID No 2690842CB1 10tttcaagcca agaaagcttt
ccttccccaa agaaagaaat gggtccagta gtgctgacac 60actcaagaac ccgcagaaac
ccagctaagt tcccagttga gataaaccag tggccctcat 120gacactgacg
ctcttcccgg tccggctcct ggttgccgct gccatgatgc tgctggcctg
180gcccctcgca cttgtcgcat cctgggctct gcggagaagg aacccgagca
gcccccggcc 240ctgtggagga aggttgtgga cttcctgctg aaggccatca
tgcgcaccat gtggttcgcc 300ggcggcttcc accgggtggc cgtgaagggg
cggcaggcgc tgcccaccga ggcggccatc 360ctcacgctcg cgcctcactc
gtcctacttc gacgccatcc ctgtgaccat gacgatgtcc 420tccatcgtga
tgaaggcaga gagcagagac atcccgatct ggggaactct gatccagtat
480atacggcctg tgttcgtgtc ccggtcagac caggattctc gcaggaaaac
agtagaagaa 540atcaagagac gggcgcagtc caacggaaag tggccacaga
taatgatttt tccagaagga 600acttgtacaa acaggacctg cctaattacc
ttcaaacctg gtgcattcat ccctggagcg 660cccgtccagc ctgtggtttt
acgatatcca aataaactgg acaccatcac atggacgtgg 720caaggacctg
gagcgctgga aatcctgtgg ctcacgctgt gtcagtttca caaccaagtg
780gaaatcgagt tccttcctgt gtacagccct tctgaggagg agaagaggaa
ccccgcgctg 840tatgccagca acgtgcggcg agtcatggcc gaggccttgg
gtgtctccgt gactgactac 900acgttcgagg actgccagct ggccctggcg
gaaggacagc tccgtctccc cgctgacact 960tgccttttag aatttgccag
gctcgtgcgg ggcctcgggc taaaaccaga aaagcttgaa 1020aaagatctgg
acagatactc agaaagagcc aggatgaagg gaggagagaa gataggtatt
1080gcggagtttg ccgcctccct ggaagtcccc gtttctgact tgctggaaga
catgttttca 1140ctgttcgacg agagcggcag cggcgaggtg gacctgcgag
agtgtgtggt tgccctgtct 1200gtcgtctgcc ggccggcccg gaccctggac
accatccagc tggctttcaa gacgtacgga 1260gcgcaagagg acggcagcgt
cggcgaaggt gacctgtcct gcatcctcaa gacggccctg 1320ggggtggcag
agctcaccgt gaccgaccta ttccgagcca ttgaccaaga ggagaagggg
1380aagatcacat tcgctgactt ccacaggttt gcagaaatgt accctgcctt
cgcagaggaa 1440tacctgtacc cggatcagac acatttcgaa agctgtgcag
agacctcacc tgcgccaatc 1500ccaaacggct tctgtgccga tttcagcccg
gaaaactcag acgctgggcg gaagcctgtt 1560cgcaagaagc tggattagg
1579113170DNAHomo sapiensmisc_featureIncyte ID No 5027764CB1
11aaaatcccac agtggaaact cttaagcctc tgcgaagtaa atcattcttg tgaatgtgac
60acacgatctc tccagtttcc atatgttgag attctactta ttcatcagtt tgttgtgctt
120gtcaagatca gacgcagaag aaacatgtcc ttcattcacc aggctgagct
ttcacagtgc 180agtggttggt acgggactaa atgtgaggct gatgctctac
acaaggaaaa acctgacctg 240cgcacaaacc atcaactcct cagcttttgg
gaacttgaat gtgaccaaga aaaccacctt 300cattgtccat ggattcaggc
caacaggctc ccctcctgtt tggatggatg acttagtaaa 360gggtttgctc
tctgttgaag acatgaacgt agttgttgtt gattggaatc gaggagctac
420aactttaata tatacccatg cctctagtaa gaccagaaaa gtagccatgg
tcttgaagga 480atttattgac cagatgttgg cagaaggagc ttctcttgat
gacatttaca tgatcggagt 540aagtctagga gcccacatat ctgggtttgt
tggagagatg tacgatggat ggctggggag 600aattacaggc ctcgaccctg
caggcccttt attcaacggg aaacctcacc aagacagatt 660agatcccagt
gatgcgcagt ttgttgatgt catccattcc gacactgatg cactgggcta
720caaggagcca ttaggaaaca tagacttcta cccaaatgga ggattggatc
aacctggctg 780ccccaaaaca atattgggag gatttcagta ttttaaatgt
gaccaccaga ggtctgtata 840cctgtacctg tcttccctga gagagagctg
caccatcact gcgtatccct gtgactccta 900ccaggattat aggaatggca
agtgtgtcag ctgcggcacg tcacaaaaag agtcctgtcc 960ccttctgggc
tattatgctg ataattggaa agaccatcta agggggaaag atcctccaat
1020gacgaaggca ttctttgaca cagctgagga gagcccattc tgcatgtatc
attactttgt 1080ggatattata acatggaaca agaatgtaag aagaggggac
attaccatca aattgagaga 1140caaagctgga aacaccacag aatccaaaat
caatcatgaa cccaccacat ttcagaagta 1200tcaccaagtg agtctacttg
caagatttaa tcaagatctg gataaagtgg ctgcaatttc 1260cttgatgttc
tctacaggat ctctaatagg cccaaggtac aagctcagga ttctccgaat
1320gaagttaagg tcccttgccc atccggagag gcctcagctg tgtcggtatg
atcttgtcct 1380gatggaaaac gttgaaacag tcttccaacc tattctttgc
ccagagttgc agttgtaact 1440gttgccagga cacatggcca taaataatag
aaagaaagct acaaccacag gctgtttgaa 1500agcttcacct cacctttctg
caaggcagaa aaagtatgaa aaaaaccaag gcttttttca 1560gtagcgtcct
atggatgtca cattgtacat caaacaacct tgtgattata aaacgatccc
1620gggaaggagc ccctaactag ggcaagtcag aaatagccag gctcgcagca
gcgcagcgct 1680gtgtctgctg tgtcctgggg cctcccttgt tccgacctgt
caattctgct gcctgtcacg 1740cgggtggttc tgcccatcgc ggctgcgggt
caagcatctt caagggaagg acggactgga 1800ggcctcaccg tggactcaac
tctgcattct ccgtgccaca ttcctccagt tcccacacgt 1860agaagggaac
gaaactgacg tctacctcat ggggctgctg tgtgggtttg ggaggcaaaa
1920atctatgaag ggttttttga aatcccatag gtgccacatc tatgagatgt
ttgataaatg 1980tgaatatgct tttacatttg ggcttatcta atttgcaata
agagagcctc tctctatcaa 2040caccagcttc tctctcgggc tgtttgctca
gggaaggcaa gaaagccacg tgctggccct 2100ctgccttctc taaagtgctg
ttggagcatg gaggagctgg aggagatggg gatggactga 2160cagctaagag
ggcggctgct gggactagat agtggatgaa gaaagaagga cgaggaagcc
2220gtggggcagc ctcttcacat ggggacaggg gatggagcat gaggcaaggg
aaggaaaagc 2280agagcttatt tttcacctaa ggtggagaag gatcacttta
caggcaacgc tcattttaag 2340caacccttaa gaaatgttta tgtttcttta
ttaccaatgt aatctatgat tattgaagga 2400aatttagaaa atgcgtagat
acaaaattaa aaaaaaatac tgtccacgat cctattagag 2460gtaattaatg
ttagcctttt ggaacaaggc tgtcacctat tttgccaaca cgtgaattca
2520aaacatgaac cggtttgctt ttggagaatc tgaagactcc agtttgagga
atcctttgct 2580tccctggagg tagatgctgt ctgcaaatct agaatgacag
caggagtcca gtcaagaggt 2640cctgtcaggc caaggccaga aagaagggag
gacaatccct ggggccagat gcccagtgtg 2700aggggaggca tgatctgtcc
catggctgtg gccactgcag gaaggtctgt gaaaaggagg 2760tgacaggccc
agtcacctcc tcttcaccca agtgattgct ccttcaactg ctatctgtga
2820aaatagcctt tgttatgaag aaattgactc tctctctttt tttttttttg
gagttgccta 2880ggctggagtg caatggtacg atctcagctc actgcaacct
ccacctccca ggttcaattg 2940attctcctgc ctcagcctcc tgagtagctg
ggattacagg catgtgccac cacacccggc 3000taatttttgt atttttatta
gagacagggt ttcaccacgt tagccaggct cgtctcgaac 3060tcctgtcctc
aggtgactac ccgtctcggc ctcccaaagt gctgggatta caggcatgag
3120ccaccacacc cggccaaaaa tggattctct atgtcataaa ttaaaggagt
3170121900DNAHomo sapiensmisc_featureIncyte ID No 2488174CB1
12gcgagagaag agaggatgga ccatgcctgg cacactgtgg tgtggagttg gagattctgc
60tgggaactcc tcggagctgg gggtcttcca gggacctgat ctctgttgcc gggaacatga
120ccgctgccca cagaacatct cacccttgca gtacaactat ggcatccgaa
actaccgatt 180ccacaccatc tcccactgtg actgtgacac caggtgtagg
atgtacggca cagtgcccct 240cgctcgcctg cagcccagga ccttctacaa
tgcctcctgg agctcccggg ccacctcccc 300aactcccagc tcccggagcc
cagcccctcc caagcctcga cagaagcagc accttcggaa 360ggggccacca
catcagaaag ggtccaagcg ccccagcaaa gccaacacca cagccctcca
420ggaccctatg gtctctccca ggcttgatgt ggcccccaca ggcctccagg
gcccacaggg 480tggcctaaaa cctcagggtg cccgctgggt ctgccgcagc
ttccgccgcc acctggacca 540gtgtgagcac cagattgggc cccgggaaat
cgagttccag ctgctcaaca gcgcccaaga 600gcccctcttc cactgcaact
gcacgcgccg tctggcacgc ttcctgaggc tccacagccc 660acccgaggtt
accaacatgc tttgggagct gctgggcaca acctgcttca agctggcccc
720tccactggac tgtgtggaag gcaaaaactg ttccagagac cctagggcca
tcagggtgtc 780agcccggcac ttgcggaggc ttcagcagag gcgacaccag
ctccaggata aaggcacaga 840tgagaggcag ccatggcctt cagagcccct
gagaggcccc atgtcattct acaaccagtg 900cctgcagcta acccaggcag
ccaggagacc cgacaggcag cagaagtcct ggagccagtg 960acctcagttt
cagctttcct gggcaccagc ctggaccttg cccatggcta tgccaagcct
1020tgggaatctc agcctcccct ccgtaggtta gactgaagca tggcagaggc
tgttgtggac 1080aatcaagagg atgaatgggg ggatctcaag gcccaaatgc
tggaccacat ctcctgctgt 1140tctgggtaac cttgagctat gtatgacaca
actcttctat gcctggatgt ggtgttcagg 1200aagctcattc tgatgccctg
ggctttggcc ttgccaggga acttcacata cagatgagaa 1260tggggaaagg
gtaacttatt gcagcagccc caggcagtac caggaggagg tacatgtatg
1320tccgtgttgc aaaaataata catgcctcaa aaacctgcct aggggagccc
tagtgcctgg 1380gtgctgtggc ctgaggtagc aggtgggaag ttagggatgt
cacagaaatg tctgtgtctg 1440aatccaggat tggggtgggt gttggagagg
gctttcagct cccctcctcc caggggggcc 1500tcttttttta acggctgcca
tgcccttcct ggcccagccc taaacctaaa ttcaaatctc 1560ctccatgcct
ttgcgcaaag gacctccctc ttgcactcta agccttagtt tcctcctcta
1620aaaaaagggg gtctctaaac aggagctacc tcatagggtt gttgaggatt
aagtgaacca 1680atacatatac agtgcttagc acttaataag tactcccccc
tgcgacacct agctgaacta 1740tggtttggtg tctgatcttg agaggttgat
gtaacctttt aaaggcctca gttcgctcac 1800ctgtgaaatg ggtctaagaa
tagcactgat ctcacagggt tgtgatgcag attaaaggag 1860atggcatgtg
taatgtaaaa aaaaaaaaaa aaaaaaaaaa 1900
* * * * *
References