U.S. patent application number 10/607712 was filed with the patent office on 2004-04-15 for human methionine synthase: cloning, and methods for evaluating risk of neural tube defects, cardiovascular disease, and cancer.
Invention is credited to Campeau, Eric, Goyette, Philippe, Gravel, Roy A., LeClerc, Daniel, Rozen, Rima.
Application Number | 20040073018 10/607712 |
Document ID | / |
Family ID | 31891943 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040073018 |
Kind Code |
A1 |
Gravel, Roy A. ; et
al. |
April 15, 2004 |
Human methionine synthase: cloning, and methods for evaluating risk
of neural tube defects, cardiovascular disease, and cancer
Abstract
The invention features a method for detecting an increased
likelihood of hyperhomocysteinemia and, in turn, an increased or
decreased likelihood of neural tube defects or cardiovascular
disease. The invention also features therapeutic methods for
reducing the risk of neural tube defects, colon cancers and related
cancers. Also provided are the sequences of the human methionine
synthase gene and protein and compounds and kits for performing the
methods of the invention.
Inventors: |
Gravel, Roy A.; (Westmont,
CA) ; Rozen, Rima; (Montreal West, CA) ;
LeClerc, Daniel; (Montreal, CA) ; Goyette,
Philippe; (Montreal, CA) ; Campeau, Eric;
(Montreal, CA) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
31891943 |
Appl. No.: |
10/607712 |
Filed: |
June 27, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10607712 |
Jun 27, 2003 |
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08980326 |
Nov 26, 1997 |
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60031964 |
Nov 27, 1996 |
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60050310 |
Jun 20, 1997 |
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Current U.S.
Class: |
536/23.2 ;
435/193; 435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C07H 21/02 20130101; C12Q 2600/156 20130101; C07H 21/04 20130101;
C12Q 1/6883 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
536/023.2 ;
435/069.1; 435/193; 435/320.1; 435/325 |
International
Class: |
C07H 021/04; C12N
009/10 |
Claims
What is claimed is:
1. A substantially pure human nucleic acid comprising at least 40
nucleotides that hybridizes under high stringency conditions to a
sequence found within the nucleic acid of SEQ ID NO:1.
2. The nucleic acid of claim 1, wherein said sequence has a
sequence complementary to at least 50% of at least 60 contiguous
nucleotides of the nucleic acid encoding the methionine synthase
polypeptide, said sequence sufficient to allow nucleic acid
hybridization under high stringency conditions.
3. The nucleic acid of claim 1, wherein said nucleic acid comprises
a mutation or a polymorphism, wherein said nucleic acid probe
detects a mutation or polymorphism selected from the group
consisting of D919G, H920D, and .DELTA.Ile881.
4. The nucleic acid of claim 3, wherein said sequence of said
nucleic acid comprises the cobalamin binding domain of the human
methionine synthase gene.
5. The nucleic acid of claim 2, wherein at least 18 contiguous
nucleotides of said sequence are complementary to at least 90% of
the corresponding nucleotides of the nucleic acid encoding the
methionine synthase polypeptide.
6. The nucleic acid of claim 1, wherein said high stringency
conditions comprise hybridization in 2.times.SSC at 40.degree.
C.
7. A substantially pure human nucleic acid, wherein the sequence of
said nucleic acid is at least 75% identical to the corresponding
region of at least 50 contiguous base pairs of the nucleic acid of
SEQ ID NO:1.
8. A substantially pure human nucleic acid, wherein the sequence of
said nucleic acid is at least 35% identical to the corresponding
region of at least 50 contiguous base pairs of the nucleic acid of
SEQ ID NO:1.
9. A kit for the analysis of a human methionine synthase nucleic
acid, said kit comprising a nucleic acid probe useful for detecting
in the nucleic acids of a human a mutation or polymorphism in said
methionine synthase nucleic acid, wherein said mutation or
polymorphism is selected from the group consisting of D919G, H920D,
and .DELTA.Ile881.
10. The kit of claim 9, wherein said probe comprises at least 40
nucleotides that hybridizes at high stringency to a sequence found
within the nucleic acid of SEQ ID NO:1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This invention claims priority from U.S. Provisional
Applications Serial Nos. 60/031,964 and 60/050,310, filed Nov. 27,
1996 and Jun. 20, 1997, respectively.
FIELD OF THE INVENTION
[0002] The invention relates to the diagnosis and treatment of
patients at risk for methionine synthase deficiency and associated
altered risk for diseases such as neural tube defects,
cardiovascular disease, and cancer.
BACKGROUND OF THE INVENTION
[0003] Methionine synthase (EC 2.1.1.13,
5-methyltetrahydrofolate-homocyst- eine methyltransferase)
catalyses the remethylation of homocysteine to methionine in a
reaction in which methylcobalamin serves as an intermediate methyl
carrier. This occurs by transfer of the methyl group of
5-methyltetrahydrofolate to the enzyme-bound cob(I)alamin to form
methylcobalamin with subsequent transfer of the methyl group to
homocysteine to form methionine. Over time, cob(I)alamin may become
oxidized to cob(II)alamin rendering the enzyme inactive.
Regeneration of the functional enzyme occurs through the methionine
synthase-mediated methylation of the cob(II)alamin in which
S-adenosylmethionine is utilized as methyl donor. In E. coli, two
flavodoxins have been implicated in the reductive activation of
methionine synthase (Fujii, K. and Huennekens, F. M. (1974) J.
Biol. Chem., 249, 6745-6753). A methionine synthase-linked reducing
system has yet to be identified in mammalian cells.
[0004] Deficiency of methionine synthase activity results in
hyperhomocysteinemia, homocystinuria, and megaloblastic anemia
without methylmalonic aciduria (Rosenblatt, D. S. (1995) The
Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill,
New York, pp. 3111-3128; Fenton, W. A. and Rosenberg, L. E. (1995)
The Metabolic and Molecular Bases of Inherited Disease.
McGraw-Hill, New York, pp. 3129-3149). Two classes of methionine
synthase-associated genetic diseases have been proposed based on
complementation experiments between patient fibroblast cell lines
(Watkins, D. and Rosenblatti D. S. (1988) J. Clin. Invest., 81,
1690-1694). One complementation group, cblE, has been postulated to
be due to deficiency of the reducing system required for methionine
synthesis (Rosenblatt, D. S., Cooper, B. A., Pottier, A.,
Lue-Shing, H., Matiaszuk, N. and Grauer, K. (1984) J. Clin.
Invest., 74, 2149-2156). Cells from patients in the cblE group fail
to incorporate .sup.14C-methyltetrahydrofolate into methionine in
whole cells but have significant methionine synthase activity in
cell extracts in the presence of a potent reducing agent. The
second complementation group, cblG group, is thought to result from
defects of the methionine synthase apoenzyme. Mutant cells from
this group show deficient methionine synthase activity in both
whole cells and cell extracts (Watkins, D. and Rosenblatt, D. S.
(1988) J. Clin. Invest., 81, 1690-1694; Watkins, D. and Rosenblatt,
D. S. (1989) Am. J Med. Genet., 34, 427-434). Moreover, some cblG
patients show defective binding of cobalamin to methionine synthase
in cells incubated with radiolabelled cyanocobalamin (Sillaots, S.
L., Hall, C. A., Hurteloup, V., and Rosenblatt, D. S. (1992)
Biochem. Med. Metab. Biol., 47, 242-249).
[0005] The cobalamin-dependent methionine synthase of E. coli has
been crystallized and the structure of its active site determined
(Luschinsky, C. L., Drummond, J. T., Matthews, R. G., and Ludwig,
M. L. (1992) J. Molec. Biol., 225, 557-560; Drennan, C. L., Huang,
S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994)
Science, 266, 1669-1674.). The gene encoding methionine synthase
has not been cloned from mammals.
SUMMARY OF THE INVENTION
[0006] We have cloned a gene for mammalian methionine synthase from
humans and discovered that mutations in this gene are associated
with hyperhomocysteinemia. Hyperhomocysteinemia is a condition that
has been implicated in cardiovascular disease and neural tube
defects. The presence of such mutations in methionine synthase gene
are, thus, associated with increased risk for cardiovascular
disease, altered risk for neural tube defects, and decreased risk
of colon cancer. The invention features methods for risk detection
and treatment of patients with hyperhomocysteinemia, cardiovascular
disease, neural tube defects, and cancer. The invention also
features compounds and kits which may be used to practice the
methods of the invention, methods and compounds for treating or
preventing these conditions and methods of identifying therapeutics
for the treatment and prevention of these conditions.
[0007] In the first aspect, the invention provides purified
wild-type mammalian methionine synthase gene, and mutated and
polymorphic versions of the mammalian methionine synthase gene,
fragments of the wild-type, mutated, and polymorphic gene, and
sense and antisense sequences which may be used in the methods of
the invention. Preferably, the gene is human. The proteins encoded
therefrom are also an aspect of the invention as is a methionine
synthase polypeptide having conservative substitutions. Preferably,
the protein is a recombinant or purified protein having a mutation
conferring hypeihomocysteinemia when present in a mammal. In
addition, nucleic acids, including genomic DNA, mRNA, and cDNA, and
the nucleic acid set forth in SEQ ID NO: 1, or degenerate variants
thereof, are provided. The shorter nucleic acid sequences are
appropriate for use in cloning, characterizing mutations, the
construction of mutations, and creating deletions. In one
embodiment, the nucleic acid set forth in SEQ ID NO: 1 is a probe
that hybridizes at high stringency to sequences found within the
nucleic acid of SEQ ID NO: 1. In further embodiments, the probe has
a sequence complementary to at least 50% of at least 60
nucleotides, or the sequence is complementary to at least 90% of at
least 18 nucleotides. Protein fragments also are provided. The
shorter peptides may be used, for example, in the generation of
antibodies to the methionine synthase protein. In some embodiments
of this aspect of the invention nucleic acid fragments useful for
detection of mutations in the region of the methionine synthase
gene which encodes the cobalamin binding domain, and for detecting
those mutations which indicate an increased likelihood of
hyperhomocysteinemia, are preferred. Most preferred fragments are
those useful for detecting the 2756 A.fwdarw.G, .DELTA.bp
2640-2642, and 2758 C.fwdarw.G mutations/polymorphisms. Given
Applicants' discovery, one skilled in the art may readily determine
which nucleic acids, detection methods, and mutations are most
useful. Mutant proteins encoded by these mutations, including, but
not limited to, H920D, .DELTA.Ile 881, and D919G are also provided
by the invention. Such mutant and polymorphic polypeptides may have
decreased or increased biological activity, relative to wild-type
methionine synthase.
[0008] In a related aspect, the invention provides antibodies that
specifically bind mammalian methionine synthase, and a method for
generating such an antibody. The antibody may specifically bind a
wild-type methionine synthase, or a mutant or polymorphic
methionine synthase. A method for detecting a wild-type, mutant, or
polymorphic methionine synthase using the antibody is also provided
by the invention.
[0009] In a second aspect, the invention provides a method for
detecting an increased or decreased risk for hyperhomocysteinemia
in a fetus or individual patient. Such a fetus or patient is at
increased or decreased risk for neural tube defects and/or
cardiovascular disease and at a decreased risk of developing colon
cancer. The method includes detection of mutations in the
methionine synthase gene present in the fetus, the individual
patient, and/or the blood relatives of the fetus and patient. The
presence of mutations, particularly in the cobalamin binding
domain, indicate an altered (e.g., increased or decreased) risk of
hyperhomocysteinemia, neural tube defects, cancer, and
cardiovascular disease.
[0010] In a related aspect, the invention provides kits for the
detection of mutations in the human methionine synthase gene. Such
kits may include, for example, nucleic acid sequences, including
probes, useful for PCR, SSCP, or RFLP detection of such mutations.
Antibodies specific for proteins having mutations, correlated with
an increased likelihood of hyperhomocysteinemia, may also be
included in the kits of the invention.
[0011] In a fourth aspect, the invention features a method for
screening for compounds which alter methionine synthase expression
or ameliorate or exacerbate conditions of hyperhomocysteinemia. In
various embodiments, the invention includes monitoring mutant or
wild-type mammalian methionine synthase biological activity by
monitoring methionine synthase enzymatic activity, or monitoring
methionine synthase gene expression levels, by monitoring
methionine synthase gene transcription, RNA stability, RNA
translation and/or protein stability. In preferred embodiments the
methionine synthase gene or protein being monitored is a gene or
protein having a mutation associated with hyperhomocysteinemia, and
samples are selected from purifed or partially purified methionine
synthase, cell lysate, a cell, or an animal. Standard assay
techniques known to those skilled in the art may be employed in the
various embodiments. Compounds detected using this screen can be
used to prevent or treat cardiovascular disease and neural tube
defects or, in the alternative, to prevent or treat colon cancer.
Kits for performing the above screens are also a part of the
invention.
[0012] In a related aspect, the invention provides nucleic acids
encoding wild-type, polymorphic, and mutated methionine synthase,
in which the nucleic acid is operably linked to regulatory
sequences, comprising a promoter, for the expression of the encoded
polypeptides. In one embodiment, the promoter is inducible. The
invention also provides cells, including prokaryotic and eukaryotic
cells, comprising the nucleic acids. The eukaryotic cells may be
yeast cells or mammalian cells.
[0013] In another related aspect, the invention features a
transgenic mammal having a methionine synthase transgene. The gene
may be wild-type, or may contain a mutation or polymorphism. The
mammal may have a mutation associated with hyperhomocysteinemia in
its methionine synthase gene in an expressible genetic construction
or may have a deletion or knockout mutation in one or both alleles
sufficient to abolish methionine synthase expression from the
locus. In addition, or as a replacement, the mammal may have the
methionine synthase gene from another species. For example, in one
preferred embodiment the transgenic mammal is a rodent such as a
mouse and the transgene is from a human. Cells from these
transgenic or knockout animals are also provided by the invention.
Such transgenic mammals may be used to screen for drugs for the
treatment of diseases related to hyperhomocysteinemia.
[0014] In a sixth aspect, the invention features a method for
treating patients with neural tube defects, colon cancer or related
cancers by the delivery of antisense methionine synthase nucleic
acid sufficient to lower the levels of methionine synthase
polypeptide biological activity.
[0015] In a related aspect, the invention provides a method for
treating or preventing cardiovascular disease, neural tube defects
and cancer. The method comprises detecting an altered risk of such
defects by analyzing methionine synthase nucleic acid, potential
test subjects being a mammal, a potential parent, either male or
female, a pregnant mammal, or a developing embryo or fetus, and
then by exposing the subject (e.g., patient or pregnant mammal) to
metabolites or cofactors such as, but not limited to, folate,
cobalamin, S-adenosyl methionine, betaine, or methionine. In
another related aspect, the invention features a method of
pretreating or treating colon cancer or neural tube defects by
inhibiting or activating methionine synthase biological activity in
a mammal, pregnant mammal, embryo, or fetus. In preferred
embodiments, this inhibiting or activating may be effected by
exposing the subject to nucleic acids, peptides or small
molecule-based inhibitors or activators of methionine synthase or
substrates. The exposure is to quantities of the compound
sufficient to reduce the probability of the subject developing the
disease or to confer an increased likelihood of a decrease in the
disease symptoms of the subject.
[0016] By "methionine synthase," "methionine synthase protein," or
"methionine synthase polypeptide" is meant a polypeptide, or
fragment thereof, which has at least 50% amino acid identity to
boxes 1-4 of the human methionine synthase polypeptide (SEQ ID NO:
2) (see FIG. 1). It is understood that polypeptide products from
splice variants of methionine synthase gene sequences are also
included in this definition. Preferably, the methionine synthase
protein is encoded by nucleic acid having a sequence which
hybridizes to a nucleic acid sequence present in SEQ ID NO: 1
(human methionine synthase cDNA) under stringent conditions. Even
more preferably the encoded polypeptide also has methionine
synthase biological activity.
[0017] By "methionine synthase nucleic acid" or "methionine
synthase gene" is meant a nucleic acid, such as genomic DNA, cDNA,
or mRNA, that encodes methionine synthase, a methionine synthase
protein, methionine synthase polypeptide, or portion thereof, as
defined above. A methionine synthase nucleic acid also may be a
methionine synthase primer or probe, or antisense nucleic acid that
is complementary to a methionine synthase nucleic acid.
[0018] By "wild-type methionine synthase" is meant a methionine
synthase nucleic acid or methionine synthase polypeptide having the
nucleic acid and/or amino acid sequence most often observed among
members of a given animal species and not statistically associated
with a disease phenotype. Wild-type methionine synthase is
biologically active methionine synthase. A wild-type methionine
synthase is, for example, a human methionine synthase polypeptide
having the sequence of SEQ ID NO: 1.
[0019] By "mutant methionine synthase," "methionine synthase
mutation(s)," "mutations in methionine synthase," "polymorphic
methionine synthase," "methionine synthase polymorphism(s),"
"polymorphisms in methionine synthase," is meant a methionine
synthase polypeptide or nucleic acid having a sequence that
deviates from the wild-type sequence in a manner sufficient to
confer an altered risk for a disease phenotype, or enhanced
protection against a disease, in at least some genetic and/or
environmental backgrounds. Such is mutations may be naturally
occurring or artificially induced. They may be, without limitation,
insertion, deletion, frameshift, or missense mutations. A mutant
methionine synthase protein may have one or more mutations, and
such mutations may affect different aspects of methionine synthase
biological activity (protein function), to various degrees.
Alternatively, a methionine synthase mutation may indirectly affect
methionine synthase biological activity by influencing, for
example, the transcriptional activity of a gene encoding methionine
synthase, or the stability of methionine synthase mRNA. For
example, a mutant methionine synthase gene may be a gene which
expresses a mutant methionine synthase protein or may be a gene
which alters the level of methionine synthase protein in a manner
sufficient to confer a disease phenotype in at least some genetic
and/or environmental backgrounds.
[0020] By "biologically active" methionine synthase is meant a
methionine synthase protein or methionine synthase gene that
provides at least one biological function equivalent to that of the
wild-type methionine synthase polypeptide or methionine synthase
gene. Biological activities of a methionine synthase polypeptide
include, and are not limited to, the ability to catalyze the
methylation of homocysteine to generate methionine. Preferably, a
biologically active methionine synthase will display activity
equivalent to at least 35% of wild-type activity, more preferably,
a biologically active methionine synthase will display at least
40-55% of wild-type activity, still more preferably, a biologically
active methionine synthase will display at least 60-75% of
wild-type activity, and most preferably, a biologically active
methionine synthase will display at least 80-90% of wild-type
activity. A biologically active methionine synthase also may
display more than 100% of wild-type activity. Preferably, the
biological activity of the wild-type methionine synthase is
determined using the methionine synthase nucleic acid of SEQ ID NO:
1 or methionine synthase polypeptide of SEQ ID NO: 2. The degree of
methionine synthase biological activity may be intrinsic to the
methionine synthase polypeptide itself, or may be modulated by
increasing or decreasing the number of methionine synthase
polypeptide molecules present intracellularly.
[0021] By "high stringency conditions" is meant hybridization in
2.times.SSC at 40.degree. C. with a DNA probe length of at least 40
nucleotides. For other definitions of high stringency conditions,
see F. Ausubel et al., Current Protocols in Molecular Biology, pp.
6.3.1-6.3.6, John Wiley & Sons, New York, N.Y., 1994, hereby
incorporated by reference.
[0022] By "analyzing" or "analysis" is meant subjecting a
methionine synthase nucleic acid or methionine synthase polypeptide
to a test procedure that allows the determination of whether a
methionine synthase gene is wild-type or mutant. For example, one
could analyze the methionine synthase genes of an animal by
amplifying genomic DNA using the polymerase chain reaction, and
then determining the DNA sequence of the amplified DNA.
[0023] By "probe" or "primer" is meant a single- or double-stranded
DNA or RNA molecule of defined sequence that can base pair to a
second DNA or RNA molecule that contains a complementary sequence
(the "target"). The stability of the resulting hybrid depends upon
the extent of the base pairing that occurs. The extent of
base-pairing is affected by parameters such as the degree of
complementarity between the probe and target molecules, and the
degree of stringency of the hybridization conditions. The degree of
hybridization stringency is affected by parameters such as
temperature, salt concentration, and the concentration of organic
molecules such as formamide, and is determined by methods known to
one skilled in the art Probes or primers specific for methionine
synthase nucleic acid preferably will have at least 35% sequence
identity, more preferably at least 45-55% sequence identity, still
more preferably at least 60-75% sequence identity, still more
preferably at least 80-90% sequence identity, and most preferably
100% sequence identity. Probes may be detectably-labelled, either
radioactively, or non-radioactively, by methods well-known to those
skilled in the art. Probes are used for methods involving nucleic
acid hybridization, such as: nucleic acid sequencing, nucleic acid
amplification by the polymerase chain reaction, single stranded
conformational polymorphism (SSCP) analysis, restriction fragment
polymorphism (RFLP) analysis, Southern hybridization, Northern
hybridization, in situ hybridization, electrophoretic mobility
shift assay (EMSA).
[0024] By "pharmaceutically acceptable carrier" means a carrier
which is physiologically acceptable to the treated mammal while
retaining the therapeutic properties of the compound with which it
is administered. One exemplary pharmaceutically acceptable carrier
is physiological saline. Other physiologically acceptable carriers
and their formulations are known to one skilled in the art and
described, for example, in Remington's Pharmaceutical Sciences,
(18.sup.th edition), ed. A. Gennaro, 1990,, Mack Publishing
Company, Easton, Pa.
[0025] By "substantially identical" is meant a polypeptide or
nucleic acid exhibiting at least 50%, preferably 85%, more
preferably 90%, and most preferably 95% identity to a reference
amino acid or nucleic acid sequence. For polypeptides, the length
of comparison sequences will generally be at least 16 amino acids,
preferably at least 20 amino acids, more preferably at least 25
amino acids, and most preferably 35 amino acids. For nucleic acids,
the length of comparison sequences will generally be at least 50
nucleotides, preferably at least 60 nucleotides, more preferably at
least 75 nucleotides, and most preferably 110 nucleotides.
[0026] Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and other modifications.
Conservative nucleotide substitutions typically include
substitutions which generate changes within the following groups:
glycine, alanine, valine, isoleucine, leucine; aspartic acid,
glutamic acid, asparagine, glutamine; serine, threonine; lysine,
arginine; and phenylalanine, tyrosine.
[0027] By "substantially pure polypeptide" is meant a polypeptide
that has been separated from the components that naturally
accompany it. Typically, the polypeptide is substantially pure when
it is at least 60%, by weight, free from the proteins and
naturally-occurring organic molecules with which it is naturally
associated. Preferably, the polypeptide is a methionine synthase
polypeptide that is at least 75%, more preferably at least 90%, and
most preferably at least 99%, by weight, pure. A substantially pure
methionine synthase polypeptide may be obtained, for example, by
extraction from a natural source (e.g., a fibroblast or liver cell)
by expression of a recombinant nucleic acid encoding a methionine
synthase polypeptide, or by chemically synthesizing the protein.
Purity can be measured by any appropriate method, e.g., by column
chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0028] A protein is substantially free of naturally associated
components when it is separated from those contaminants which
accompany it in its natural state. Thus, a protein which is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides not only includes
those derived from eukaryotic organisms but also those synthesized
in E. coli or other prokaryotes.
[0029] By "substantially pure DNA" is meant DNA that is free of the
genes which, in the naturally-occurring genome of the organism from
which the DNA of the invention is derived, flank the gene. The term
therefore includes, for example, a recombinant DNA which is
incorporated into a vector; into an autonomously replicating
plasmid or virus; or into the genomic DNA of a prokaryote or
eukaryote; or which exists as a separate molecule (e.g., a cDNA or
a genomic or cDNA fragment produced by PCR or restriction
endonuclease digestion) independent of other sequences. It also
includes a recombinant DNA which is part of a hybrid gene encoding
additional polypeptide sequence.
[0030] By "transgene" is meant any piece of DNA which is inserted
by artifice into a cell, and becomes part of the genome of the
organism which develops from that cell. Such a transgene may
include a gene which is partly or entirely heterologous (i.e.,
foreign) to the transgenic organism, or may represent a gene
homologous to an endogenous gene of the organism.
[0031] By "transgenic" is meant any cell which includes a DNA
sequence which is inserted by artifice into a cell and becomes part
of the genome of the organism which develops from that cell. As
used herein, the transgenic organisms are generally transgenic
mammals (e.g., rodents such as rats or mice) and the DNA
(transgene) is inserted by artifice into the nuclear genome.
Preferably the inserted DNA encodes a protein in at least some
cells of the organism.
[0032] By "knockout mutation" is meant an alteration in the nucleic
acid sequence that reduces the biological activity of the
polypeptide normally encoded therefrom by at least 80% relative to
the unmutated gene. The mutation may, without limitation, be an
insertion, deletion, frameshift mutation, or a missense mutation.
Preferably, the mutation is an insertion or deletion, or is a
frameshift mutation that creates a stop codon.
[0033] By "transformation" is meant any method for introducing
foreign molecules into a cell. Lipofection, DEAE-dextran-mediated
transfection, microinjection, protoplast fusion, calcium phosphate
precipitation, retroviral delivery, electroporation, and biolistic
transformation are just a few of the methods known to those skilled
in the art which may be used. For example, biolistic transformation
is a method for introducing foreign molecules into a cell using
velocity driven microprojectiles such as tungsten or gold
particles. Such velocity-driven methods originate from pressure
bursts which include, but are not limited to, helium-driven,
air-driven, and gunpowder-driven techniques. Biolistic
transformation may be applied to the transformation or transfection
of a wide variety of cell types and intact tissues including,
without limitation, intracellular organelles (e.g., and
mitochondria and chloroplasts), bacteria, yeast, fungi, algae,
animal tissue, and cultured cells.
[0034] By "transformed cell" is meant a cell into which (or into an
ancestor of which) has been introduced, by means of recombinant DNA
techniques, a DNA molecule encoding (as used herein) a methionine
synthase polypeptide.
[0035] By "positioned for expression" is meant that the DNA
molecule is positioned adjacent to a DNA sequence which directs
transcription and translation of the sequence (i.e., facilitates
the production of, e.g., a methionine synthase polypeptide, a
recombinant protein or a RNA molecule).
[0036] By "promoter" is meant a minimal sequence sufficient to
direct transcription. Also included in the invention are those
promoter elements which are sufficient to render promoter-dependent
gene expression controllable for cell type-specific,
tissue-specific, temporal-specific, or inducible by external
signals or agents; such elements may be located in the 5' or 3' or
intron sequence regions of the native gene.
[0037] By "operably linked" is meant that a gene and one or more
regulatory sequences are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the regulatory sequences.
[0038] By "conserved region" is meant any stretch of six or more
contiguous amino acids exhibiting at least 30%, preferably 50%, and
most preferably 70% amino acid sequence identity between two or
more of the methionine synthase family members, (e.g., between
human and bacterial methionine synthase). Examples of conserved
regions within methionine synthase are Boxes 1-4 (FIG. 1).
[0039] By "detectably-labeled" is meant any means for marking and
identifying the presence of a molecule, e.g., an oligonucleotide
probe or primer, a gene or fragment thereof, or a cDNA molecule.
Methods for detectably-labeling a molecule are well known in the
art and include, without limitation, radioactive labeling (e.g.,
with an isotope such as .sup.32P or .sup.35S) and nonradioactive
labeling (e.g., chemiluminescent labeling, e.g., fluorescein
labeling).
[0040] By "antisense" as used herein in reference to nucleic acids,
is meant a nucleic acid sequence that is complementary to the
coding strand of a gene, preferably, a methionine synthase gene. An
antisense nucleic acid is capable of preferentially lowering the
activity of a mutant methionine synthase polypeptide encoded by a
mutant methionine synthase gene.
[0041] By "purified antibody" is meant antibody which is at least
60%, by weight, free from proteins and naturally occurring organic
molecules with which it is naturally associated. Preferably, the
preparation is at least 75%, more preferably 90%, and most
preferably at least 99%, by weight, antibody, e.g., a methionine
synthase amino-terminus-specific antibody. A purified antibody may
be obtained, for example, by affinity chromatography using
recombinantly-produced protein or conserved motif peptides and
standard techniques.
[0042] By "specifically binds" is meant an antibody that recognizes
and binds a human methionine synthase polypeptide but that does not
substantially recognize and bind other non-methionine synthase
molecules in a sample, e.g., a biological sample, that naturally
includes protein. A preferred antibody binds to the methionine
synthase polypeptide sequence of SEQ ID NO: 2 (FIG. 3).
[0043] By "neutralizing antibodies" is meant antibodies that
interfere with any of the biological activities of a wild-type or
mutant methionine synthase polypeptide, for example, the ability of
methionine synthase to catalyze the transfer of a methyl group to
homocysteine. The neutralizing antibody may reduce the ability of a
methionine synthase polypeptide to catalyze the transfer preferably
by 10% or more, more preferably by 25% or more, still more
preferably by 50% or more, yet preferably by 70% or more, and most
preferably by 90% or more.
[0044] Any standard assay for the biological activity of methionine
synthase, may be used to assess potentially neutralizing antibodies
that are specific for methionine synthase.
[0045] By "expose" is meant to allow contact between an animal,
cell, lysate or extract derived from a cell, or molecule derived
from a cell, and a test compound.
[0046] By "treat" is meant to submit or subject an animal (e.g. a
human), cell, lysate or extract derived from a cell, or molecule
derived from a cell to a test compound.
[0047] By "test compound" is meant a chemical, be it
naturally-occurring or artificially-derived, that is surveyed for
its ability to modulate an alteration in reporter gene activity or
protein levels, by employing one of the assay methods described
herein. Test compounds may include, for example, peptides,
polypeptides, synthesized organic molecules, naturally occurring
organic molecules, nucleic acid molecules, and components
thereof.
[0048] By "assaying" is meant analyzing the effect of a treatment,
be it chemical or physical, administered to whole animals or cells
derived therefrom. The material being analyzed may be an animal, a
cell, a lysate or extract derived from a cell, or a molecule
derived from a cell. The analysis may be for the purpose of
detecting altered protein biological activity, altered protein
stability, altered protein levels, altered gene expression, or
altered RNA stability. The means for analyzing may include, for
example, for example, the detection of the product of an enzymatic
reaction, (e.g., the formation of methionine as a result of
methionine synthase activity), antibody labeling,
immunoprecipitation, and methods known to those skilled in the art
for detecting nucleic acids.
[0049] By "modulating" is meant changing, either by decrease or
increase, in biological activity.
[0050] By "a decrease" is meant a lowering in the level of
biological activity, as measured by a lowering/increasing of: a)
the formation of methionine as a result of methionine synthase
activity; b) protein, as measured by ELISA; c) reporter gene
activity, as measured by reporter gene assay, for example,
lacZ/.beta.-galactosidase, green fluorescent protein, luciferase,
etc.; or d) mRNA, levels of at least 30%, as measured by PCR
relative to an internal control, for example, a "housekeeping" gene
product such as .beta.-actin or glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) or an externally added nucleic acid standard.
In all cases, the lowering is preferably by at least 10% more
preferably by at least 25%, still more preferably by at least 50%,
and even more preferably by at least 70%.
[0051] By "an increase" is meant a rise in the level of biological
activity, as measured by a lowering/increasing of: a) the formation
of methionine as a result of methionine synthase activity; b)
protein, as measured by ELISA; c) reporter gene activity, as
measured by reporter gene assay, for example,
lacZ/.beta.-galactosidase, green fluorescent protein, luciferase,
etc.; or d) mRNA, levels of at least 30%, as measured by PCR
relative to an internal control, for example, a "housekeeping" gene
product such as .beta.-actin or glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) or an externally added nucleic acid standard.
Preferably, the increase is by 10% or more, more preferably by 25%
or more, even more preferably by 2-fold, and most preferably by at
least 3-fold.
[0052] By "alteration in the level of gene expression" is meant a
change in gene activity such that the amount of a product of the
gene, i.e., mRNA or polypeptide, is increased or decreased, or that
the stability of the mRNA or the polypeptide is increased or
decreased.
[0053] By "reporter gene" is meant any gene which encodes a product
whose expression is detectable and/or quantitatable by
immunological, chemical, biochemical or biological assays. A
reporter gene product may, for example, have one of the following
attributes, without restriction: fluorescence (e.g., green
fluorescent protein), enzymatic activity (e.g.,
lacZ/.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase), toxicity (e.g., ricin A), or an ability to be
specifically bound by a second molecule (e.g., biotin or a
detectably labelled antibody). It is understood that any engineered
variants of reporter genes, which are readily available to one
skilled in the art, are also included, without restriction, in the
forgoing definition.
[0054] By "protein" or "polypeptide" or "polypeptide fragment" is
meant any chain of more than two amino acids, regardless of
post-translational modification (e.g., glycosylation or
phosphorylation), constituting all or part of a naturally-occurring
polypeptide or peptide, or constituting a non-naturally occurring
polypeptide or peptide.
[0055] By "missense mutation" is meant the substitution of one
purine or pyrimidine base (i.e. A, T, G, or C) by another within a
nucleic acid sequence, such that the resulting new codon encodes an
amino acid distinct from the amino acid originally encoded by the
reference (e.g. wild-type) codon.
[0056] By "deletion mutation" is meant the deletion of at least one
nucleotide within a polynucleotide coding sequence. A deletion
mutation alters the reading frame of a coding region unless the
deletion consists of one or more contiguous 3-nucleotide stretches
(i.e. "codons"). Deletion of a codon from a nucleotide coding
region results in the deletion of an amino acid from the resulting
polypeptide.
[0057] By "frameshift mutation" is meant the insertion or deletion
of at least one nucleotide within a polynucleotide coding sequence.
A frameshift mutation alters the codon reading frame at and/or
downstream from the mutation site. Such a mutation results either
in the substitution of the encoded wild-type amino acid sequence by
a novel amino acid sequence, or a premature termination of the
encoded polypeptide due to the creation of a stop codon, or
both.
DETAILED DESCRIPTION OF THE DRAWINGS
[0058] The drawings will first be briefly described.
[0059] FIG. 1 is a diagram showing four homologous regions among
methionine synthases. Boxes 1 to 4 were used to design degenerate
oligonucleotides for the initial cloning experiments. Ec:
Escherichia coli, accession number J04975; Ss: Synechocystis sp.,
accession number D64002; Ml1 and Ml2: Mycobacterium leprae,
accession number U000175 (see Drennan et al., 1994); Hi:
Haemophilus influenzae, accession number U32730; Ce: Caenorhabditis
elegans, accession number Z46828; Hs: Homo sapiens, this work.
Identical residues are indicated by a star above the alignment.
Amino acid position for each protein is shown at left.
[0060] FIG. 2 is a diagram showing overlapping PCR fragments
generated to clone human methionine synthase. Oligonucleotides are
described in Table 1.
[0061] Primers in parentheses designate mispriming outcomes that
generated valid internal sequence. iPCRc: inverse PCR on cDNA,
iPCRg: inverse PCR on genomic DNA.
[0062] FIG. 3 is a diagram showing nucleotide sequence (SEQ ID NO:
1) and deduced amino acid sequence (SEQ ID NO: 2) of human
methionine synthase. The nucleotide residue numbering is shown in
the left margin, and the amino acid residue numbering is shown in
the right margin.
[0063] FIG. 4 is a photograph showing mapping of the human
methionine synthase gene using FISH. Signals are clearly visible at
1q43 (arrows).
[0064] FIGS. 5A-5C is a series of photographs showing diagnostic
tests for mutations in the methionine synthase gene. Numbers above
the gel lanes correspond to patients cell lines whereas the letter
"c" identifies wild-type controls. FIG. 5A: HaeIII restriction
analysis of genomic DNA PCR products using primers #1796 and #305A.
The 2756A-G change creates a HaeIII site. Expected fragments, 2756A
allele: 189 bp, 2756G allele: 159 and 30 bp (the 30 bp fragment was
run off the gel). FIG. 5B: Heteroduplex analysis of PCR products
amplified from RT reactions of patient 1892 and 3 controls. RT-PCR
was done with primers #1772 and #1773. Expected PCR product: 338
bp, heteroduplexes can be seen above this band in patient 1892
(heterozygous for .DELTA.2640-2642). C. Sau96I restriction analysis
of genomic DNA PCR products. PCR was done as in (A). The
2758C.fwdarw.G mutation abolishes a Sau96I restriction endonuclease
site in patient 2290. Expected fragments, control allele: 159, 30
bp, mutant allele: 189 bp (the 30 bp fragment has been run off the
gel).
[0065] FIG. 6. shows an amino acid sequence comparison among
methionine synthases in the Box 2 region. Identical residues are
indicated by a star above the alignment. Dots show partially
conserved residues, for which at least {fraction (6/7)} identical
or similar residues can be aligned (A,G,S,T; D,E,N,Q; V,L,I,M; K,R;
and F,W,Y (Bordo,D. and Argos,P. (1991) J. Molec. Biol., 217,
721-729)). Mutations identified in this work are shown below the
alignment. For abbreviations, see FIG. 1; Mm: Mus musculus. The
seven amino acids conserved in cobalamin-binding proteins (Drennan,
C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M.
L. (1994) Science, 266, 1669-1674) are underlined.
DETAILED DESCRIPTION
[0066] We used specific regions of homology within the methionine
synthase sequences of several lower organisms to clone a human
methionine synthase cDNA (SEQ ID NO:1) by a combination of RT-PCR
and inverse PCR. The enzyme (SEQ ID NO:2) is 1265 amino acids in
length and contains the seven residue structure-based sequence
fingerprint identified for cobalamin-containing enzymes. The gene
was localized to chromosome 1q43 by the FISH technique. We have
identified one missense mutation and a 3 base pair deletion in
patients of the cblG complementation group of inherited
homocysteine/folate disorders by SSCP and sequence analysis, as
well as an amino acid substitution present in high frequency in the
general population.
[0067] We conclude that the cDNA that we have identified
corresponds to human methionine synthase on the basis of homology
to known methionine synthases and by the identification of
mutations in patients with a deficiency of enzyme activity. The
most striking sequence conservation was found in four boxes of 9-13
amino acids. Box 2 has been proposed to correspond to part of the
cobalamin binding domain (Drennan, C. L., Huang, S., Drummond, J.
T., Matthews, R. G., and Ludwig, M. L. (1994) Science, 266,
1669-1674). It contains 13 consecutive residues that are identical
in all known methionine synthases. Three amino acids within box 2
and four others C-terminal to it correspond to residues proposed by
Drennan et al. (Drennan, C. L., Huang, S., Drummond, J. T.,
Matthews, R. G., and Ludwig, M. L. (1994) Science, 266, 1669-1674)
as a structure-based sequence fingerprint for cobalamin binding.
The three amino acids appear to make direct contact with the lower
face of the corrin ring and dimethylbenzimidazole tail of
cobalamin, determined from the crystal structure of the E. coli
enzyme at 3 .ANG. resolution (Drennan, C. L., Huang, S., Drummond,
J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science, 266,
1669-1674). All seven residues are identical in the human sequence
(FIG. 6).
[0068] A survey of the NCBI databases for homology to the human
methionine synthase using BLASTP yielded the various methionine
synthases listed in FIG. 1, as well as the glutamate mutase
(S41332, Q05488) and methylmalonyl-CoA mutase
(P11653)(adenosyl-cobalamin dependent mutases) used to deduce the
sequence fingerprint for cobalamin binding (Drennan, C. L., Huang,
S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994)
Science, 266, 1669-1674). Homology was also found with the
cobalamin binding region of the corrinoid: coenzyme M
methyltransferase of Methanosarcina barkeri (U36337), the
5-methyltetrahydrofolate corrinoid/iron sulfur protein
methyltransferase of Clostridium thermoaceticum (L34780) and the
B12-dependent 2-methyleneglutarate mutase of Clostridium barkeri
(S43552, S43237). Further, homology was found with the B12-binding
site domain of the recently identified putative methionine synthase
of Agrobacterium tumefaciens (U48718; partial N-terminal sequence
is given, up to region of box 4). Significantly, homology with the
B12-binding site domain was also found in the Hg resistance protein
of Myxococcus xanthus (Z21955). This protein has not been described
as having a cobalamin prosthetic group.
[0069] The two mutations we have identified as candidates for
causing cblG disease are located in the vicinity of the cobalamin
binding domain by comparison with E. coli methionine synthase (FIG.
6). Ile881 corresponds by sequence alignment to Val855 in the E.
coli enzyme. Val855 is within a beta sheet strand that is part of
an .alpha./.beta. domain that is a variant of the Rossmann
nucleotide binding fold. The H920D substitution is found in a
region which, in the E. coli enzyme, is in an .alpha. helix at the
C-terminal end of the .alpha./.beta. domain. It is interesting that
the polymorphism we have identified is at the adjacent residue
(D919G). The functional role of the polymorphism and deleterious
mutations will have to be examined in expression experiments to
confirm their precise effect on the protein.
[0070] Through the cloning of a cDNA for human methionine synthase
and mutations therein, we can now determine the properties of the
human enzyme and complete the characterization of mutations in
patients with severe synthase deficiency. This analysis has allowed
us to tie mutations in the gene to disturbances in homocysteine
metabolism which are known to result in hyperhomocysteinemia is a
risk factor for cardiovascular disease (Boushey, C. J., Beresford,
S. A., Omenn, G. S., and Motulsky, A. G. (1995) JAMA, 274,
1049-1057) and neural tube defects (Steegers-Theunissen, R. P.,
Boers, G. H., Trijbels, F. J., Finkelstein, J. D., Blom, H. J.,
Thomas, C. M., Borm, G. F., Wouters, M. G., and Eskes, T. K. (1994)
Metab. Clin. Exp., 43, 1475-1480; and Mills, J. L., McPartlin, J.
M., Kirke, P. N., Lee, Y. J., Conley, M. R., Weir, D. G. and Scott,
J. M. (1995) Lancet, 345, 149-151).
[0071] Our observations indicate the importance of methionine
synthase as one of several genes involved in homocysteine
metabolism. Results with other pathway genes underscores the
significance of our findings. For example, a recently-identified
mutation in methylenetetrahydrofolate reductase, the enzyme that
synthesizes the 5-methyltetrahydrofolate substrate for the
methionine synthase reaction, results in mild hyperhomocysteinemia
(Frosst,P., Blom,H. J., Milos,R., Goyette,P., Sheppard,C. A.,
Matthews,R. G., Boers,G. J., den Heijer,M., Kluijtmans,L. A., van
den Heuvel,L. P., et al. (1995) Nat. Genet., 10, 111-113). Evidence
is accumulating that this mutation, present in 35-40% of alleles,
is a risk factor in both cardiovascular disease and neural tube
defects (Rozen,R. (1996) Clin. Invest. Med., 19, 171-178). We
believe that genetic variants of methionine synthase similarly lead
to mild hyperhomocysteinemia with consequent impact on these two
multifactorial disorders.
[0072] We used specific regions of homology within the methionine
synthase sequences, including a portion of the cobalamin binding
site determined from the E. coli enzyme, to design degenerate
oligonucleotides for RT-PCR-dependent cloning of human methionine
synthase. We confirmed the identification of the cDNA sequences for
human methionine synthase by the high degree of homology to the
enzymes in other species and the identification of mutations in
patients from the cblG complementation group. We also mapped the
gene to human chromosome 1.
[0073] The assays described herein can be used to test for
compounds that modulate methione synthase activity and hence may
have therapeutic value in the prevention of neural tube defects,
prevention and/or treatment of colon cancer, cardiovascular
disease, hyperhomocysteinemia, and megaloblastic anemia without
methylmalonic aciduria.
Screens for Compounds that Modulate Methionine Synthase Enzymatic
Activity
[0074] Screens for potentially useful therapeutic compounds that
modulate methionine synthase activity may be readily performed, for
example, by assays that measure the incorporation of
[14C]5-methyltetrahydrofolate into methionine or protein, or assays
that measure the conversion of [14C]-homocysteine into methionine
or protein. Examples of such assays, which employ whole cells or
cell lysates, are well-known to those skilled in the art (see,
e.g., Schuh, S., et al., N. Engl. J. Med. 1984, 310:686-69;
Rosenblatt, D. S., et al., J. Clin. Invest. 1984, 74:2149-2156;
Watkins, D., and Rosenblatt, D. S., J. Clin. Invest. 1988,
81:1690-1694; and Watkins, D., and Rosenblatt, D. S., Am. J. Med.
Genet. 1989, 34:427-434), and may be readily adapted for high
throughput screening.
ELISA for the Detection of Compounds that Modulate Methionine
Synthase Expression
[0075] Enzyme-linked immunosorbant assays (ELISAs) are easily
incorporated into high-throughput screens designed to test large
numbers of compounds for their ability to modulate levels of a
given protein. When used in the methods of the invention, changes
in a given protein level of a sample, relative to a control,
reflect changes in the methionine synthase expression status of the
cells within the sample. Protocols for ELISA may be found, for
example, in Ausubel et al.,Current Protocols in Molecular Biology,
John Wiley & Sons, New York, N.Y., 1997. Lysates from cells
treated with potential modulators of methionine synthase expression
are prepared (see, for example, Ausubel et al., supra), and are
loaded onto the wells of microtiter plates coated with "capture"
antibodies specific for methionine synthase. Unbound antigen is
washed out, and a methionine synthase-specific antibody, coupled to
an agent to allow for detection, is added. Agents allowing
detection include alkaline phosphatase (which can be detected
following addition of colorimetric substrates such as
p-nitrophenolphosphate), horseradish peroxidase (which can be
detected by chemiluminescent substrates such as ECL, commercially
available from Amersham) or fluorescent compounds, such as FITC
(which can be detected by fluorescence polarization or
time-resolved fluorescence). The amount of antibody binding, and
hence the level of a methionine synthase polypeptide within a
lysate sample, is easily quantitated on a microtiter plate
reader.
[0076] As a baseline control for methionine synthase expression, a
sample that is not exposed to test compound is included.
Housekeeping proteins are used as internal standards for absolute
protein levels. A positive assay result, for example,
identification of a compound that decreases methionine synthase
expression, is indicated by a decrease in methionine synthase
polypeptide within a sample, relative to the methionine synthase
level observed in cells which are not treated with a test
compound.
Reporter Gene Assays for Compounds that Modulate Methionine
Synthase Expression
[0077] Assays employing the detection of reporter gene products are
extremely sensitive and readily amenable to automation, hence
making them ideal for the design of high-throughput screens. Assays
for reporter genes may employ, for example, colorimetric,
chemiluminescent, or fluorometric detection of reporter gene
products. Many varieties of plasmid and viral vectors containing
reporter gene cassettes are easily obtained. Such vectors contain
cassettes encoding reporter genes such as
lacZ/.beta.-galactosidase, green fluorescent protein, and
luciferase, among others. Cloned DNA fragments encoding
transcriptional control regions of interest (e.g. that of the
mammalian methionine synthase gene) are easily inserted, by DNA
subcloning, into such reporter vectors, thereby placing a
vector-encoded reporter gene under the transcriptional control of
any gene promoter of interest. The transcriptional activity of a
promoter operatively linked to a reporter gene can then be directly
observed and quantitated as a function of reporter gene activity in
a reporter gene assay.
[0078] Cells are transiently- or stably-transfected with methionine
synthase control region/reporter gene constructs by methods that
are well known to those skilled in the art. Transgenic mice
containing methionine synthase control region/reporter gene
constructs are used for late-stage screens in vivo. Cells
containing methionine synthase/reporter gene constructs are exposed
to compounds to be tested for their potential ability to modulate
methionine synthase expression. At appropriate timepoints, cells
are lysed and subjected to the appropriate reporter assays, for
example, a colorimetric or chemiluminescent enzymatic assay for
lacZ/.beta.-galactosidase activity, or fluorescent detection of
GFP. Changes in reporter gene activity of samples treated with test
compounds, relative to reporter gene activity of appropriate
control samples, indicate the presence of a compound that modulates
methionine synthase expression.
Quantitative PCR of Methionine Synthase mRNA as an Assay for
Compounds that Modulate Methionine Synthase Expression
[0079] The polymerase chain reaction (PCR), when coupled to a
preceding reverse transcription step (rtPCR), is a commonly used
method for detecting vanishingly small quantities of a target mRNA.
When performed within the linear range, with an appropriate
internal control target (employing, for example, a housekeeping
gene such as actin), such quantitative PCR provides an extremely
precise and sensitive means of detecting slight modulations in mRNA
levels. Moreover, this assay is easily performed in a 96-well
format, and hence is easily incorporated into a high-throughput
screening assay. Cells are treated with test compounds for the
appropriate time course, lysed, the mRNA is reverse-transcribed,
and the PCR is performed according to commonly used methods, (such
as those described in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y., 1997),
using oligonucleotide primers that specifically hybridize with
methionine synthase nucleic acid. Changes in product levels of
samples exposed to test compounds, relative to control samples,
indicate test compounds that modulate methionine synthase
expression.
Secondary Screens of Test Compounds that Appear to Modulate
Methionine Synthase Activity
[0080] After test compounds that appear to have methionine
synthase-modulating activity are identified, it may be necessary or
desirable to subject these compounds to further testing. At late
stages testing will be performed in vivo to confirm that the
compounds initially identified to affect methionine synthase
activity will have the predicted effect in vivo.
Test Compounds
[0081] In general, novel drugs for prevention of neural tube
defects, or prevention and/or treatment of colon cancer or
cardiovascular disease are identified from large libraries of both
natural product or synthetic (or semi-synthetic) extracts or
chemical libraries according to methods known in the art. Those
skilled in the field of drug discovery and development will
understand that the precise source of test extracts or compounds is
not critical to the screening procedure(s) of the invention.
Accordingly, virtually any number of chemical extracts or compounds
can be screened using the exemplary methods described herein.
Examples of such extracts or compounds include, but are not limited
to, plant-, fungal-, prokaryotic- or animal-based extracts,
fermentation broths, and synthetic compounds, as well as
modification of existing compounds. Numerous methods are also
available for generating random or directed synthesis (e.g.,
semi-synthesis or total synthesis) of any number of chemical
compounds, including, but not limited to, saccharide-, lipid-,
peptide-, and nucleic acid-based compounds. Synthetic compound
libraries are commercially available from Brandon Associates
(Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In
addition, natural and synthetically produced libraries are
produced, if desired, according to methods known in the art, e.g.,
by standard extraction and fractionation methods. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0082] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their therapeutic activities for homocysteinemia, megaloblastic
anemia without methylmalonic aciduria, cardiovasular disease, colon
cancer, and neural tube defects should be employed whenever
possible.
[0083] When a crude extract is found to modulate methionine
synthase biological activity, further fractionation of the positive
lead extract is necessary to isolate chemical constituents
responsible for the observed effect. Thus, the goal of the
extraction, fractionation, and purification process is the careful
characterization and identification of a chemical entity within the
crude extract that modulates methionine synthase biological
activity. The same assays described herein for the detection of
activities in mixtures of compounds can be used to purify the
active component and to test derivatives thereof. Methods of
fractionation and purification of such heterogenous extracts are
known in the art. If desired, compounds shown to be useful agents
for treatment are chemically modified according to methods known in
the art. Compounds identified as being of therapeutic value may be
subsequently analyzed using mammalian models of homocysteinemia,
megaloblastic anemia without methylmalonic aciduria, cardiovasular
disease, colon cancer, and neural tube defects.
Therapy
[0084] Compounds identified using any of the methods disclosed
herein, may be administered to patients or experimental animals
with a pharmaceutically-acceptable diluent, carrier, or excipient,
in unit dosage form. Conventional pharmaceutical practice may be
employed to provide suitable formulations or compositions to
administer such compositions to patients or experimental animals.
Although intravenous administration is preferred, any appropriate
route of administration may be employed, for example, parenteral,
subcutaneous, intramuscular, intracranial, intraorbital,
ophthalmic, intraventricular, intracapsular, intraspinal,
intracisternal, intraperitoneal, intranasal, aerosol, or oral
administration. Therapeutic formulations may be in the form of
liquid solutions or suspensions; for oral administration,
formulations may be in the form of tablets or capsules; and for
intranasal formulations, in the form of powders, nasal drops, or
aerosols.
[0085] Methods well known in the art for making formulations are
found in, for example, "Remington's Pharmaceutical Sciences."
Formulations for parenteral administration may, for example,
contain excipients, sterile water, or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, or
hydrogenated naphthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Other potentially useful parenteral
delivery systems for antagonists or agonists of the invention
include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycocholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel.
EXAMPLES
[0086] The following examples are to illustrate, not limit the
invention.
Example 1
Cloning Human Methionine Synthase cDNA
[0087] An initial survey of the NCBI databases yielded several
sequences corresponding to methionine synthase from different
organisms. Comparison of these sequences generated four very
conserved regions identified as Boxes 1-4 in FIG. 1 (SEQ ID
Nos:3-25). Degenerate oligonucleotides (SEQ ID Nos:26-66) were
synthesized corresponding to these conserved sequences (Table 1).
These were used as primers for RT-PCR with human and mouse mRNA.
These experiments yielded PCR products which were subcloned,
sequenced and aligned as shown in FIG. 2. In subsequent
experiments, oligonucleotide primers were specified from the
non-degenerate internal sequences of the subclones and additional
PCR products encompassing the conserved boxes were obtained. In
later experiments, additional sequences were obtained by inverse
PCR ("PCR", FIG. 2) to obtain upstream or downstream sequences from
those already determined. At the 3' end, a mouse sequence was
obtained from the dbEST database (Accession Number W33307). This
sequence was used as the source of primers for additional PCR
experiments. Throughout the experiments, the sequences of the PCR
products were considered provisionally authentic if they were
homologous to the methionine synthase sequences obtained from the
databases. The sequences were taken as error free by comparison of
the sequences of at least two, and usually three, independent PCR
reactions. Sequences were linked into a common sequence if RT-PCRs
bridging independently isolated sequences were successful. Through
this approach the complete coding sequence was determined through
exclusive use of PCR reactions.
[0088] The coding sequence of human methionine synthase contains
3795 bp (SEQ ID NO:1) encoding a polypeptide of 1265 amino acids in
length (SEQ ID NO:2) (FIG. 3), exceeding the length of published
methionine synthases by 11-29 residues. The putative initiation
codon is in a sequence of good context for the initiation of
translation in eukaryotic cells (GACAACATGT, underlined nucleotides
matching Kozak consensus (Kozak,M. (1991) J. Biol. Chem., 266,
19867-19870)). The predicted MW of methionine synthase is 141,000,
comparing favorably with the published size of 151,000 based on
SDS-polyacrylamide electrophoresis of the pig enzyme (Chen, Z.,
Crippen, K., Gulati, S., and Baneijee, R. (1994) J. Biol. Chem.,
269, 27193-27197). It shares 58% identity with the E. coli and 65%
identity with the C. elegans enzyme.
Example 2
Chromosomal Location
[0089] Using FISH, the gene encoding methionine synthase was mapped
to chromosome band 1q43, close to the telomeric region of the long
arm (FIG. 4). A total of 50 cells with at least one signal were
observed. A signal was seen on 1 chromatid in 26 cells, on two
chromatids in 14 cells, on 3 chromatids in 7 cells, and on 4
chromatids in 3 cells. These results confirm the previous
assignment of the gene to chromosome 1 by Mellman et al. (Mellman,
I. S., Lin, P. F., Ruddle, F. H., and Rosenberg, L. E. (1979) Proc.
Natl. Acad. Sci. USA, 76, 405-409), who used cobalamin binding as a
marker for the enzyme in human-hamster hybrids.
Example 3
Mutations in the cblG Complementation Group
[0090] Patients with deficiency of methionine synthase activity
have been grouped into the cblG complementation group in cell
fusion experiments (Watkins, D. and Rosenblatt, D. S. (1988) J.
Clin. Invest., 81, 1690-1694). Fibroblast cultures from patients
assigned to cblG were examined by RT-PCR based SSCP analysis. Three
mutations were identified by sequencing PCR fragments showing band
shifts by SSCP (FIG. 5). In each case, the change was confirmed by
an independent diagnostic test on genomic DNA or a separate
preparation of cDNA from patient fibroblasts. One of the mutations,
2756A.fwdarw.G (D919G), was confirmed by a diagnostic test that
monitored the presence of a HaeIII site created by the mutation
(FIG. 5A). Using this test, it was identified as a polymorphism
since it was seen in 8 of 52 control alleles (15%). In two other
cases, candidate deleterious mutations were identified. One is a 3
bp deletion, bp 2640-2642, that results in the deletion of an
isoleucine codon (.DELTA.Ile881). It was confirmed by heteroduplex
analysis of cDNA generated by RT-PCR (FIG. 5B). The second is a
point mutation, 2758C.fwdarw.G. It results in the amino acid
substitution H920D. It was confirmed in genomic DNA by the loss of
a Sau96I site (FIG. 5C). The latter two mutations were heterozygous
in the patient cell lines. Their second mutation has not been
identified. The candidate deleterious mutations were not seen in
panels of 68 or 52 control alleles, respectively.
Example 4
Additional Roles for Methionine Synthase Polymorphism (Asp919Gly or
D919G) in Disease
[0091] The following data suggest that the D919G polymorphism
contributes to altered metabolism of homocysteine, methionine,
folates, Vit. B12, and S-adenosylmethionine.
[0092] First in a Montreal study (n=303), in which mother-child
pairs (cases and controls) were examined, we observed that infants
who were homozygous for the polymorphism (Gly/Gly; Table 2) were at
decreased risk for NTD.
[0093] Measurements of serum folate, RBC folate, plasma
homocysteine and serum cobalamin did not give any statistically
significant differences, except the trend was toward low folate
levels in Gly/Gly individuals (cases and controls).
[0094] A second study (n=255) in California also examined the
methionine synthase polymorphism as a risk factor for neural tube
defects (Table 3). This study shows a similar decreased risk of
neural tube defects in children homozygous for Gly/Gly. Since the
study encompassed a mix of whites and Hispanics, the data were
reexamined stratified according to ethnic origin. Both groups
showed a protective effect of Gly/Gly.
[0095] In summary, two independent studies suggest a protective
effect of Gly/Gly against the risk of neural tube defects. This is
likely to be mediated by a mild reduction in methionine synthase
activity.
[0096] Next, in a study of colon cancer, (212 cases and 345
controls), we observed a decreased risk for colon cancer in the
individuals who were homozygous for the polymorphism (relative
risk=0.62); see Table 4. In the same study, we observed
significantly decreased levels of plasma folate in individuals who
were homozygous for the polymorphism; see Table 5.
[0097] The Boston study described in Tables 4 and 5 is presented
again in Table 6 with the data stratified according to alcohol
intake. As shown in the table, Gly/Gly individuals with a low to
medium alcohol intake had a relative risk associated with colon
cancer of 0.11. The combined data (low+high alcohol) gave a risk
level of 0.62 (Table 6).
[0098] In summary, drug therapy targeted to a reduction in
methionine synthase activity may be protective in individuals at
risk for colon cancer or at risk for neural tube defects.
Additional polymorphisms or mutations may also exert a protective
effect against the risk of neural tube defects or colon cancer.
Conversely, it is understood that some polymorphisms and/or
mutations may enhance the risk of neural tube defects or colon
cancer, for example, by increasing methionine synthase
activity.
Example 5
Role of Polymorphism on Homocysteine and Folate Levels
[0099] Third, in a study of individuals participating in the U.S.
NHLBI Family Heart Study, we observed both an increase in plasma
homocysteine following a methionine load and a decrease in plasma
folate in individuals who were homozygous for this polymorphism;
see Table 7.
Example 6
Methionine Synthase Assays for the Detection of Compounds that
Modulate Methionine Synthase Activity and Expression
[0100] Potentially useful therapeutic compounds that modulate (e.g.
increase or decrease) methionine synthase activity or expression
may be isolated by various screens that are well-known to those
skilled in the art. Such compounds may modulate methionine synthase
expression at the pre- or post-transcriptional level, or at the
pre- or post-translational level.
Example 7
Materials and Methods
[0101] Cell lines. The skin fibroblast lines are from patients with
methionine synthase deficiency. They were assigned to the cblG
complementation group in cell fusion experiments assayed by
.sup.14C-methyltetrahydrofolate incorporation into cellular
macromolecules (Watkins, D. and Rosenblatt, D. S. (1988) J. Clin.
Invest., 81, 1690-1694). Control fibroblasts were from other
laboratory stocks or the Montreal Children's Hospital Cell
Repository for Mutant Human Cell Strains. Of the patients for which
non-polymorphic mutations were found, WG 1892, a Caucasian male,
was diagnosed at the age of 4 years with developmental delay,
tremors, gait instability, megaloblastic anemia and homocystinuria;
and WG2290, also a Caucasian male, was diagnosed at age 3 months
with failure to thrive, severe eczema, megaloblastic anemia and
surprisingly both homocystinuria and methylmalonic aciduria.
[0102] Materials. The T/A cloning kit was from Invitrogen. The
Geneclean III Kit was obtained from Bio 101 Inc. and the Wizard
Mini-Preps were from Promega. The random-primed DNA labelling kit
was from Boehringer-Mannheim. Taq polymerase, Superscript II
reverse transcriptase, AMV reverse transcriptase, Trizol reagent,
DNAzol reagent, T4 DNA ligase, and restriction enzymes were
purchased from Gibco BRL. The Sequenase kit for manual sequencing
was from United States Biochemicals. The .alpha.-[.sup.35S]dATP
(12.5 Ci/mole) was from Dupont or ICN. The oligonucleotide primers
were synthesized by R. Clarizio of the Montreal Children's Hospital
Research Institute Oligonucleotide Synthesis Facility or the
Sheldon Biotechnology Centre, McGill University.
[0103] Homology matches. Comparisons were made between the
published E. coli cobalamin-dependent methionine synthase sequence
and sequences in the NCBI databases (dbEST and GenBank) using the
BLAST programs.
[0104] PCR cloning and DNA sequencing. DNA was prepared from
fibroblast pellets by the method of Hoar et al. (Hoar,D. I.,
Haslam,D. B., and Starozik,D. M. (1984) Prenat. Diag., 4, 241-247).
Total cellular RNA was isolated by the method of Chirgwin et al.
(Chirgwin,J. M., Przybyla,A. E., MacDonald,R. J., and Rutter,W. J.
(1979) Biochemistry, 18, 5294-5299) and is reverse-transcribed
using oligo-dT.sub.15 as primer. PCR was conducted using degenerate
oligonucleotides as primers, paired so as to link the sequences of
different homology boxes. The PCRs were conducted as described
previously (Triggs-Raine,B. L., Akerman,B. R., Clarke,J. T., and
Gravel,R. A. (1991) Am. J. Hum. Genet., 49, 1041-1054) except that
the temperature of incubation was modified to accommodate the use
of reduced temperatures in the annealing step or by step-down PCR
(Hecker,K. H. and Roux,K. H. (1996) Biotechniques, 20, 478-485.
(Abstract)). In some experiments, inverse PCR was used to determine
sequence upstream or downstream of known sequence (Ochman,H.,
Medhora,M. M., Garza,D., and Hartl,D. L. (1990) PCR Protocols: A
Guide to Methods and Applications, Academic Press, San Diego, pp.
219-227). In these instances, genomic DNA or cDNA prepared by
reverse transcription of RNA was digested with different four base
restriction endonucleases, ligated with T4 DNA ligase, and
amplified by PCR using adjacent oligonucleotides priming in
opposite directions. Templates for inverse PCR at the cDNA level
were generated with 12.5 .mu.g RNA reversed transcribed using
AMV-RT. Second strand synthesis was carried out using the
random-primed DNA labelling kit adding 1 .mu.l of each dNTP.
Samples were incubated 30 min. at 37.degree. C. Template was then
treated as genomic DNA for digestion and ligation. Inverse PCR was
used to obtain the 5' and 3' ends of the cDNA and to define an
intron sequence adjacent to a splice junction for the design of a
mutation diagnostic test. The PCR products were purified with
Geneclean and were subcloned in the pCR2.1 vector and transformed
into E. coli as per the supplier's protocol (TA Cloning Kit). The
candidate clones were sequenced manually or by the DNA Core
Facility of the Canadian Genetic Diseases Network or the McGill
University Sheldon Biotechnology Centre.
[0105] Mutation analysis. Genomic DNA and RNA were isolated from
control or patient fibroblast pellets using the DNAzol or Trizol
reagents, respectively, as per the manufacturer. The cDNA template
for PCR was prepared by reverse transcription of 3-5 .mu.g total
RNA in reactions containing 400 U of Superscript II reverse
transcriptase and 100 ng random hexamers in a total reaction volume
of 20 ul. SSCP analysis was performed as described previously
(Triggs-Raine,B. L., Akerman,B. R., Clarke,J. T., and Gravel,R. A.
(1991) Am. J. Hum. Genet., 49, 1041-1054) in reactions containing 4
.mu.l of template, 1 .mu.l of each dTTP, dCTP, dGTP (0.625 mM), 0.5
.mu.l of dATP (0.625 mM), 1 .mu.l .alpha.-[.sup.35S]-dATP (12.5
Ci/mole). The radio labelled PCR products mixed with sequencing
stop solution were heat denatured and quick chilled on ice prior to
loading (Triggs-Raine,B. L., Akerman,B. R., Clarke,J. T., and
Gravel,R. A. (1991) Am. J. Hum. Genet., 49, 1041-1054). As well, an
aliquot of each sample was run without prior heating to identify
the duplex product. The fragments were subjected to electrophoresis
in a 6% acrylamide/10% glycerol gel in 1.times.TBE for 18 hrs at 8
watts at room temperature. The gel was dried and exposed to Biomax
film (Kodak). Fragments that displayed band shifts were sequenced
directly.
[0106] Two mutations were confirmed directly in PCR amplification
products from genomic DNA and one mutation was confirmed in
reversed transcribed mRNA. The PCR reactions for mutation
confirmation were performed using 4 .mu.l of cDNA template or 500
ng genomic DNA, 500 ng of specific primers, 2.5 U Taq polymerase
and 1.5 mM MgCl2 in a 50 .mu.l volume. Heteroduplex analysis was
accomplished by preheating PCR products to 95.degree. C. for five
minutes and subjecting the samples to electrophoresis in a 9%
polyacrylamide gel (Triggs-Raine,B. L., Akerman,B. R., Clarke,J.
T., and Gravel,R. A. (1991) Am. J. Hum. Genet., 49, 1041-1054).
Other diagnostic assays were accomplished by digesting a 15 .mu.l
sample of the PCR products with the indicated restriction
endonuclease prior to electrophoresis.
[0107] Chromosomal localization. Human metaphase spreads were
obtained from short-term cultures of phytohemaglutinin-stimulated
peripheral blood lymphocytes. The cells were synchronized with
thymidine and treated with BrdU during the late S-phase before
harvesting for simultaneous observation of the hybridized sites and
chromosome banding. The protocol for FISH was essentially as
described previously (Lemieux, N., Malfoy, B., and Forrest, G. L.
(1993) Genomics, 15, 169-172; Zhang, X. X., Rozen, R., Hediger, M.
A., Goodyer, P., and Eydoux, P. (1994) Genomics, 24, 413-414).
Briefly, a 5 kb DNA fragment of the methionine synthase genomic DNA
(generated by PCR using oligonucleotides #1782 and #1780) was
labelled by nick translation with biotin-16-dUTP
(Boehringer-Mannheim), ethanol precipitated and dissolved in
hybridization buffer at a final concentration of 8 ng/.mu.l. The
slides were denatured in 70% formamide, 2.times.SSC at 70.degree.
C. for 2 min. The biofinylated probe was denatured in the
hybridization buffer at 95.degree. C. for 10 min, quickly cooled on
ice, then applied on slides. Post-washing was done by rinsing in
50% formamide, 2.times.SSC at 37.degree. C. The slides were
incubated with rabbit antibiotin antibody (Enzo Biochemicals),
biotinylated goat anti-rabbit antibodies (BRL) and
streptavidin-FITC. They were stained with propidium iodide and
mounted in p-phenylenediamine, pH 11. Cells were observed under the
microscope (Zeiss), then captured through a CCD camera and
processed using a FISH software (Applied Imaging).
1TABLE 1 Oligonucleotides used for cDNA cloning, chromosome mapping
and mutation detection. Oligonucleotides.sup.a Sequence
Location.sup.b D1729 5'-GAYGGNGCNATGGGNACNATGATHCA (SEQ ID NO:26)
100-125 D1730 5'-GCNACNGTNAARGGNGAYGTNCAYGAYAT (SEQ ID NO:27)
2332-2360 D1731 5'-RTTYTTNCCDATRTCRTGNACRTCNCCYTT (SEQ ID NO:28)
2370-2341 D1733 5'-RTGNAGRTAYTCNGCRAANGCYTCNGC (SEQ ID NO:29)
3426-3400 D1754 5'-ATRTGRTCNGGNGTNGTNCCRCARCANCCNCC (SEQ ID NO:30)
992-961 D1755 5'-GGNGGNTGYTGYGGNACNACNCCNGAYCAY- AT (SEQ ID NO:31)
961-992 M1806A 5'-GTCTGTGTCATAGCCCAGAATG- GG (SEQ ID NO:32)
3795-3772 M1806B 5'-TCAGTCTGTGTCATAGCCCA- GAAT (SEQ ID NO:33)
3798-3775 305A 5'-GAACTAGAAGACAGAAATTC- TCTA (SEQ ID NO:34)
(intronic) 407A 5'-TTCCGAGGTCAGGAATTTAAAGATCA (SEQ ID NO:35)
151-176 407B 5'-GTGTTCTTCGTTTAGCTTCTCCCG (SEQ ID NO:36) 150-127
407D 5'-CCCCAGCCAGCAAGTATTCCTTAT (SEQ ID NO:37) 268-245 1107A
5'-CTAGGTTGTATTTCCTTGAGGATC (SEQ ID NO:38) 3856-3833 1406D
5'-GGAGCTGGAAAAATGTTTCTACCTC (SEQ ID NO:39) 2170-2194 1406E
5'-ACAGGAGGGAAGAAAGTCATTCAG (SEQ ID NO:40) 1963-1986 1706A
5'-CCTTCAATTATATTGAGAGGTCGGG (SEQ ID NO:41) 2129-2105 1707A
5'-CAACCCGAAGGTCTGAAGAAAACC (SEQ ID NO:42) 28-51 1707B
5'-CCCGCGCTCCAAGACCTGTCG (SEQ ID NO:43) 7-27 1707C
5'-CGACAGGTCTTGGAGCGCGGG (SEQ ID NO:44) 27-7 1758
5'-GGAGTCATGACTCCTAAATCAATAACTC (SEQ ID NO:45) 2432-2405 1760
5'-GACGACTACAGCAGCATCATGGT (SEQ ID NO:46) 3355-3377 1766
5'-AAAAATCATTTCATCCAGGGAA (SEQ ID NO:47) 2526-2505 1772
5'-ATAGGCAAGAACATAGTTGGAGTAGT (SEQ ID NO:48) 2359-2384 1773
5'-TTTCATCTAACAGCTGGGAACACAC (SEQ ID NO:49) 2698-2674 1774
5'-TGCCTCTCAGACTTCATCGCTCCC (SEQ ID NO:50) 3241-3264 1780
5'-TGCAGCCTGGGGCACAGCAGC (SEQ ID NO:51) 3168-3148 1782
5'-ATGGATTGGCTGTCTGAACCTCAC (SEQ ID NO:52) 2824-2847 1796
5'-CATGGAAGAATATGAAGATATTAGAC (SEQ ID NO:53) 2727-2752 1803
5'-ACCATCATCCTCATAGGCCTTGCT (SEQ ID NO:54) 3354-3331 1806C
5'-CAGACCTGCGAAGGTTGCGGTAC (SEQ ID NO:55) 3482-3504 1806F
5'-GAAGTGGTTGCTCCTCCAATCAAC (SEQ ID NO:56) 2591-2568 1808
5'-GAGCAGCTTTCAGTATCTTATCACAT (SEQ ID NO:57) 2458-2433 1827
5'-ACAAGTTGTGTTCCTCCATTCCAGT (SEQ ID NO:58) 1657-1633 1828
5'-AGAGCGCTGTAATGTTGCAGGATCA (SEQ ID NO:59) 1125-1149 1907B
5'-TGTTTTTCAATGCCCTTCACAAGGG (SEQ ID NO:60) 2057-2033 1907C
5'-TAAAAAGTATGGAGCTGCTATGGTG (SEQ ID NO:61) 1464-1488 2606A
5'-GACCAGACAGTAACATATGTCCTTC (SEQ ID NO:62) 1078-1054 2606B
5'-ACATTACAGCGCTCTCCAATGTTAAC (SEQ ID NO:63) 1139-1114 2706A
5'-TGAGGTTGAGAAATGGCTTGGACC (SEQ ID NO:64) 3750-3773 2706B
5'-GCCACAGATATGTTCTTCCTCAATG (SEQ ID NO:65) 3749-3725 3107A
5'-TGTGGAGAGCACGTCTTCTCTGCC (SEQ ID NO:66) -55--32 .sup.aNumbers
with the prefix "D" refer to oligonucleotides with degenerate bases
shown as N (any base), H (A, C, or T), D (A, G, or T), Y (T or C),
or R (A or G); those with the prefix "M" refer to mouse sequences
(see FIG. 3). .sup.bFrom the first methionine codon, see FIG.
3.
[0108]
2TABLE 2 MS Polymorphism in Neural Tube Defects - Montreal Study
Case Con- Control Cases mothers trols mothers Odds Genotype N % N %
N % N % ratio* 95% C.I. Asp/Asp 38 69 40 66 59 61 55 61 Asp/Gly 17
31 20 33 28 29 34 38 Gly/Gly 0 0.9 1 2 10 10 1 1 0.07 0.004-1.29 N
55 61 97 90 *Odds ratio calculated for genotypes Asp/Asp vs Gly/Gly
(to permit the calculation, the 0 cell was increased to 0.5)
[0109]
3TABLE 3 MS Polymorphisms in Neural Tube Defects - California Study
Genotype Cases Controls Odds Ethnic Group 2756A-G N % N % ratio*
95% C.I. Overall Asp/Asp 64 67 104 64 1.0 Asp/Gly 30 32 49 30 0.99
0.56-1.72 Gly/Gly 1 1 7 4 0.23 0.05-1.92 White only Asp/Asp 21 66
38 66 2.0 Asp/Gly 10 31 16 28 1.1 0.44-2.9 Gly/Gly 1 3 3 5 0.60
0.11-5.6 Hispanic only Asp/Asp 43 68 66 63 1.0 Asp/Gly 20 32 33 31
0.9 0.45-1.8 Gly/Gly 0 0 4 4 0 *Odds ration calculated for
genotypes Asp/Asp vs Gly/Gly
[0110]
4TABLE 4 Frequency of MS genotype and relative risk (RR) of
colorectal cancer by MS genotype Cases Controls MS Genotype n % n %
RR 95% CI Asp/Asp 145 (68) 234 (68) 1.0 Asp/Gly 61 (29) 95 (28)
1.02 0.69-1.50 Gly/Gly 6 (3) 16 (5) 0.62 0.24-1.64 Total 212
345
[0111]
5TABLE 5 Mean of homocysteine and folate (geometric) by case
control status and MS Genotype in a colon cancer study Cases &
Cases Controls Controls MS genotype n mean n mean n mean Folate
(Bio-Kit) ng/ml Asp/Asp 115 3.8 201 3.9 * 316 3.9 ** Asp/Gly 49 4.1
* 80 3.8 * 129 3.9 ** Gly/Gly 6 2.1 12 2.3 18 2.2 Homocysteine
(.mu.M) Asp/Asp 66 12.5 160 12.1 226 12.3 Asp/Gly 30 10.8 50 11.6
80 11.2 Gly/Gly 4 13.4 9 11.7 13 12.5 * = p < 0.05 ** = p <
0.01
[0112]
6TABLE 6 Age Adjusted Relative Risk of Colon Cancer According to MS
Polymorphism and Alcohol Intake Status Among US Physicians Genotype
2756A->G Cases Controls Odds Alcohol intake Asp919Gly N N ratio
95% C.I. Low-Medium Asp/Asp 1013 2e+09 1.0 0-0.8 drinks/day Asp/Gly
7113 0.87 0.54-1.4 Gly/Gly 9 0.11 0.01-0.82 N High Asp/Asp 3721
7e+06 0.74 0.46-1.19 1-2+ drinks/day Asp/Gly 563 1.15 0.60-2.18
Gly/Gly 3.83 0.72-20.47 N
[0113]
7TABLE 7 Mean Homocysteine and Folate Status by MS Genotype (Date
of Analysis: Feb. 18, 1997) Methionine Synthase Genotype Asp/Asp
Asp/Gly Gly/Gly Result Result P Result P N 252 111 17 Fasting Hcy
(.mu.M) 8.5 8.4 0.76 8.7 post-methionine load 17.9 19.6 0.74 Hcy
(.mu.M) 7.4 0.05* 6.9 22.3 Folate (microb test) 0.37 0.03* 6.3
0.37
[0114]
Sequence CWU 1
1
76 1 3919 DNA Homo sapiens Other (1)...(3919) Entire cloned cDNA
encoding wild type methionine synthase. 1 ggtcacctgt ggagagcacg
tcttctctgc cgcgccctct gcgcaaggag gagactcgac 60 aacatgtcac
ccgcgctcca agacctgtcg caacccgaag gtctgaagaa aaccctgcgg 120
gatgagatca atgccattct gcagaagagg attatggtgc tggatggagg gatggggacc
180 atgatccagc gggagaagct aaacgaagaa cacttccgag gtcaggaatt
taaagatcat 240 gccaggccgc tgaaaggcaa caatgacatt ttaagtataa
ctcagcctga tgtcatttac 300 caaatccata aggaatactt gctggctggg
gcagatatca ttgaaacaaa tacttttagc 360 agcactagta ttgcccaagc
tgactatggc cttgaacact tggcctaccg gatgaacatg 420 tgctctgcag
gagtggccag aaaagctgcc gaggaggtaa ctctccagac aggaattaag 480
aggtttgtgg caggggctct gggtccgact aataagacac tctctgtgtc cccatctgtg
540 gaaaggccgg attataggaa catcacattt gatgagcttg ttgaagcata
ccaagagcag 600 gccaaaggac ttctggatgg cggggttgat atcttactca
ttgaaactat ttttgatact 660 gccaatgcca aggcagcctt gtttgcactc
caaaatcttt ttgaggagaa atatgctccc 720 cggcctatct ttatttcagg
gacgatcgtt gataaaagtg ggcggactct ttccggacag 780 acaggagagg
gatttgtcat cagcgtgtct catggagaac cactctgcat tggattaaat 840
tgtgctttgg gtgcagctga gatgagacct tttattgaaa taattggaaa atgtacaaca
900 gcctatgtcc tctgttatcc caatgcaggt cttcccaaca cctttggtga
ctatgatgaa 960 acgccttcta tgatggccaa gcacctaaag gattttgcta
tggatggctt ggtcaatata 1020 gttggaggat gctgtgggtc aacaccagat
catatcaggg aaattgctga agctgtgaaa 1080 aattgtaagc ctagagttcc
acctgccact gcttttgaag gacatatgtt actgtctggt 1140 ctagagccct
tcaggattgg accgtacacc aactttgtta acattggaga gcgctgtaat 1200
gttgcaggat caaggaagtt tgctaaactc atcatggcag gaaactatga agaagccttg
1260 tgtgttgcca aagtgcaggt ggaaatggga gcccaggtgt tggatgtcaa
catggatgat 1320 ggcatgctag atggtccaag tgcaatgacc agattttgca
acttaattgc ttccgagcca 1380 gacatcgcaa aggtaccttt gtgcatcgac
tcctccaatt ttgctgtgat tgaagctggg 1440 ttaaagtgct gccaagggaa
gtgcattgtc aatagcatta gtctgaagga aggagaggac 1500 gacttcttgg
agaaggccag gaagattaaa aagtatggag ctgctatggt ggtcatggct 1560
tttgatgaag aaggacaggc aacagaaaca gacacaaaaa tcagagtgtg cacccgggcc
1620 taccatctgc ttgtgaaaaa actgggcttt aatccaaatg acattatttt
tgaccctaat 1680 atcctaacca ttgggactgg aatggaggaa cacaacttgt
atgccattaa ttttatccat 1740 gcaacaaaag tcattaaaga aacattacct
ggagccagaa taagtggagg tctttccaac 1800 ttgtccttct ccttccgagg
aatggaagcc attcgagaag caatgcatgg ggttttcctt 1860 taccatgcaa
tcaagtctgg catggacatg gagatagtga atgctggaaa cctccctgtg 1920
tatgatgata tccataagga acttctgcag ctctgtgaag atctcatctg gaataaagac
1980 cctgaggcca ctgagaagct cttacgttat gcccagactc aaggcacagg
agggaagaaa 2040 gtcattcaga ctgatgagtg gagaaatggc cctgtcgaag
aacgccttga gtatgccctt 2100 gtgaagggca ttgaaaaaca tattattgag
gatactgagg aagccaggtt aaaccaaaaa 2160 aaatatcccc gacctctcaa
tataattgaa ggacccctga tgaatggaat gaaaattgtt 2220 ggtgatcttt
ttggagctgg aaaaatgttt ctacctcagg ttataaagtc agcccgggtt 2280
atgaagaagg ctgttggcca ccttatccct ttcatggaaa aagaaagaga agaaaccaga
2340 gtgcttaacg gcacagtaga agaagaggac ccttaccagg gcaccatcgt
gctggccact 2400 gttaaaggcg acgtgcacga cataggcaag aacatagttg
gagtagtcct tggctgcaat 2460 aatttccgag ttattgattt aggagtcatg
actccatgtg ataagatact gaaagctgct 2520 cttgaccaca aagcagatat
aattggcctg tcaggactca tcactccttc cctggatgaa 2580 atgatttttg
ttgccaagga aatggagaga ttagctataa ggattccatt gttgattgga 2640
ggagcaacca cttcaaaaac ccacacagca gttaaaatag ctccgagata cagtgcacct
2700 gtaatccatg tcctggacgc gtccaagagt gtggtggtgt gttcccagct
gttagatgaa 2760 aatctaaagg atgaatactt tgaggaaatc atggaagaat
atgaagatat tagacaggac 2820 cattatgagt ctctcaagga gaggagatac
ttacccttaa gtcaagccag aaaaagtggt 2880 ttccaaatgg attggctgtc
tgaacctcac ccagtgaagc ccacgtttat tgggacccag 2940 gtctttgaag
actatgacct gcagaagctg gtggactaca ttgactggaa gcctttcttt 3000
gatgtctggc agctccgggg caagtacccg aatcgaggct tccccaagat atttaacgac
3060 aaaacagtag gtggagaggc caggaaggtc tacgatgatg cccacaatat
gctgaacaca 3120 ctgattagtc aaaagaaact ccgggcccgg ggtgtggttg
ggttctggcc agcacagagt 3180 atccaagacg acattcacct gtacgcagag
gctgctgtgc cccaggctgc agagcccata 3240 gccactttct atgggttaag
gcaacaggct gagaaggact ctgccagcac ggagccatac 3300 tactgcctct
cagacttcat cgctcccttg cattctggca tccgtgacta cctgggcctg 3360
tttgccgttg cctgctttgg ggtagaagag ctgagcaagg cctatgagga tgatggtgac
3420 gactacagca gcatcatggt caaggcgctg ggggaccggc tggcagaggc
ctttgcagaa 3480 gagctccatg aaagagttcg ccgagaactg tgggcctact
gtggcagtga gcagctggac 3540 gtcgcagacc tgcgaaggtt gcggtacaag
ggcatccgcc cggctcctgg ctaccccagc 3600 cagcccgacc acaccgagaa
gctcaccatg tggagactcg cagacatcga gcagtctaca 3660 ggcattaggt
taacagaatc attagcaatg gcacctgctt cagcagtctc aggcctctac 3720
ttctccaatt tgaagtccaa atattttgct gtggggaaga tttccaagga tcaggttgag
3780 gattatgcat tgaggaagaa catatctgtg gctgaggttg agaaatggct
tggacccatt 3840 ttgggatatg atacagacta actttttttt ttttttttgc
cttttttatc ttgatgatcc 3900 tcaaggaaat acaacctag 3919 2 1265 PRT
Homo sapiens VARIANT (1)...(1265) Wild type methionine synthase
polypeptide. 2 Met Ser Pro Ala Leu Gln Asp Leu Ser Gln Pro Glu Gly
Leu Lys Lys 1 5 10 15 Thr Leu Arg Asp Glu Ile Asn Ala Ile Leu Gln
Lys Arg Ile Met Val 20 25 30 Leu Asp Gly Gly Met Gly Thr Met Ile
Gln Arg Glu Lys Leu Asn Glu 35 40 45 Glu His Phe Arg Gly Gln Glu
Phe Lys Asp His Ala Arg Pro Leu Lys 50 55 60 Gly Asn Asn Asp Ile
Leu Ser Ile Thr Gln Pro Asp Val Ile Tyr Gln 65 70 75 80 Ile His Lys
Glu Tyr Leu Leu Ala Gly Ala Asp Ile Ile Glu Thr Asn 85 90 95 Thr
Phe Ser Ser Thr Ser Ile Ala Gln Ala Asp Tyr Gly Leu Glu His 100 105
110 Leu Ala Tyr Arg Met Asn Met Cys Ser Ala Gly Val Ala Arg Lys Ala
115 120 125 Ala Glu Glu Val Thr Leu Gln Thr Gly Ile Lys Arg Phe Val
Ala Gly 130 135 140 Ala Leu Gly Pro Thr Asn Lys Thr Leu Ser Val Ser
Pro Ser Val Glu 145 150 155 160 Arg Pro Asp Tyr Arg Asn Ile Thr Phe
Asp Glu Leu Val Glu Ala Tyr 165 170 175 Gln Glu Gln Ala Lys Gly Leu
Leu Asp Gly Gly Val Asp Ile Leu Leu 180 185 190 Ile Glu Thr Ile Phe
Asp Thr Ala Asn Ala Lys Ala Ala Leu Phe Ala 195 200 205 Leu Gln Asn
Leu Phe Glu Glu Lys Tyr Ala Pro Arg Pro Ile Phe Ile 210 215 220 Ser
Gly Thr Ile Val Asp Lys Ser Gly Arg Thr Leu Ser Gly Gln Thr 225 230
235 240 Gly Glu Gly Phe Val Ile Ser Val Ser His Gly Glu Pro Leu Cys
Ile 245 250 255 Gly Leu Asn Cys Ala Leu Gly Ala Ala Glu Met Arg Pro
Phe Ile Glu 260 265 270 Ile Ile Gly Lys Cys Thr Thr Ala Tyr Val Leu
Cys Tyr Pro Asn Ala 275 280 285 Gly Leu Pro Asn Thr Phe Gly Asp Tyr
Asp Glu Thr Pro Ser Met Met 290 295 300 Ala Lys His Leu Lys Asp Phe
Ala Met Asp Gly Leu Val Asn Ile Val 305 310 315 320 Gly Gly Cys Cys
Gly Ser Thr Pro Asp His Ile Arg Glu Ile Ala Glu 325 330 335 Ala Val
Lys Asn Cys Lys Pro Arg Val Pro Pro Ala Thr Ala Phe Glu 340 345 350
Gly His Met Leu Leu Ser Gly Leu Glu Pro Phe Arg Ile Gly Pro Tyr 355
360 365 Thr Asn Phe Val Asn Ile Gly Glu Arg Cys Asn Val Ala Gly Ser
Arg 370 375 380 Lys Phe Ala Lys Leu Ile Met Ala Gly Asn Tyr Glu Glu
Ala Leu Cys 385 390 395 400 Val Ala Lys Val Gln Val Glu Met Gly Ala
Gln Val Leu Asp Val Asn 405 410 415 Met Asp Asp Gly Met Leu Asp Gly
Pro Ser Ala Met Thr Arg Phe Cys 420 425 430 Asn Leu Ile Ala Ser Glu
Pro Asp Ile Ala Lys Val Pro Leu Cys Ile 435 440 445 Asp Ser Ser Asn
Phe Ala Val Ile Glu Ala Gly Leu Lys Cys Cys Gln 450 455 460 Gly Lys
Cys Ile Val Asn Ser Ile Ser Leu Lys Glu Gly Glu Asp Asp 465 470 475
480 Phe Leu Glu Lys Ala Arg Lys Ile Lys Lys Tyr Gly Ala Ala Met Val
485 490 495 Val Met Ala Phe Asp Glu Glu Gly Gln Ala Thr Glu Thr Asp
Thr Lys 500 505 510 Ile Arg Val Cys Thr Arg Ala Tyr His Leu Leu Val
Lys Lys Leu Gly 515 520 525 Phe Asn Pro Asn Asp Ile Ile Phe Asp Pro
Asn Ile Leu Thr Ile Gly 530 535 540 Thr Gly Met Glu Glu His Asn Leu
Tyr Ala Ile Asn Phe Ile His Ala 545 550 555 560 Thr Lys Val Ile Lys
Glu Thr Leu Pro Gly Ala Arg Ile Ser Gly Gly 565 570 575 Leu Ser Asn
Leu Ser Phe Ser Phe Arg Gly Met Glu Ala Ile Arg Glu 580 585 590 Ala
Met His Gly Val Phe Leu Tyr His Ala Ile Lys Ser Gly Met Asp 595 600
605 Met Glu Ile Val Asn Ala Gly Asn Leu Pro Val Tyr Asp Asp Ile His
610 615 620 Lys Glu Leu Leu Gln Leu Cys Glu Asp Leu Ile Trp Asn Lys
Asp Pro 625 630 635 640 Glu Ala Thr Glu Lys Leu Leu Arg Tyr Ala Gln
Thr Gln Gly Thr Gly 645 650 655 Gly Lys Lys Val Ile Gln Thr Asp Glu
Trp Arg Asn Gly Pro Val Glu 660 665 670 Glu Arg Leu Glu Tyr Ala Leu
Val Lys Gly Ile Glu Lys His Ile Ile 675 680 685 Glu Asp Thr Glu Glu
Ala Arg Leu Asn Gln Lys Lys Tyr Pro Arg Pro 690 695 700 Leu Asn Ile
Ile Glu Gly Pro Leu Met Asn Gly Met Lys Ile Val Gly 705 710 715 720
Asp Leu Phe Gly Ala Gly Lys Met Phe Leu Pro Gln Val Ile Lys Ser 725
730 735 Ala Arg Val Met Lys Lys Ala Val Gly His Leu Ile Pro Phe Met
Glu 740 745 750 Lys Glu Arg Glu Glu Thr Arg Val Leu Asn Gly Thr Val
Glu Glu Glu 755 760 765 Asp Pro Tyr Gln Gly Thr Ile Val Leu Ala Thr
Val Lys Gly Asp Val 770 775 780 His Asp Ile Gly Lys Asn Ile Val Gly
Val Val Leu Gly Cys Asn Asn 785 790 795 800 Phe Arg Val Ile Asp Leu
Gly Val Met Thr Pro Cys Asp Lys Ile Leu 805 810 815 Lys Ala Ala Leu
Asp His Lys Ala Asp Ile Ile Gly Leu Ser Gly Leu 820 825 830 Ile Thr
Pro Ser Leu Asp Glu Met Ile Phe Val Ala Lys Glu Met Glu 835 840 845
Arg Leu Ala Ile Arg Ile Pro Leu Leu Ile Gly Gly Ala Thr Thr Ser 850
855 860 Lys Thr His Thr Ala Val Lys Ile Ala Pro Arg Tyr Ser Ala Pro
Val 865 870 875 880 Ile His Val Leu Asp Ala Ser Lys Ser Val Val Val
Cys Ser Gln Leu 885 890 895 Leu Asp Glu Asn Leu Lys Asp Glu Tyr Phe
Glu Glu Ile Met Glu Glu 900 905 910 Tyr Glu Asp Ile Arg Gln Asp His
Tyr Glu Ser Leu Lys Glu Arg Arg 915 920 925 Tyr Leu Pro Leu Ser Gln
Ala Arg Lys Ser Gly Phe Gln Met Asp Trp 930 935 940 Leu Ser Glu Pro
His Pro Val Lys Pro Thr Phe Ile Gly Thr Gln Val 945 950 955 960 Phe
Glu Asp Tyr Asp Leu Gln Lys Leu Val Asp Tyr Ile Asp Trp Lys 965 970
975 Pro Phe Phe Asp Val Trp Gln Leu Arg Gly Lys Tyr Pro Asn Arg Gly
980 985 990 Phe Pro Lys Ile Phe Asn Asp Lys Thr Val Gly Gly Glu Ala
Arg Lys 995 1000 1005 Val Tyr Asp Asp Ala His Asn Met Leu Asn Thr
Leu Ile Ser Gln Lys 1010 1015 1020 Lys Leu Arg Ala Arg Gly Val Val
Gly Phe Trp Pro Ala Gln Ser Ile 1025 1030 1035 1040 Gln Asp Asp Ile
His Leu Tyr Ala Glu Ala Ala Val Pro Gln Ala Ala 1045 1050 1055 Glu
Pro Ile Ala Thr Phe Tyr Gly Leu Arg Gln Gln Ala Glu Lys Asp 1060
1065 1070 Ser Ala Ser Thr Glu Pro Tyr Tyr Cys Leu Ser Asp Phe Ile
Ala Pro 1075 1080 1085 Leu His Ser Gly Ile Arg Asp Tyr Leu Gly Leu
Phe Ala Val Ala Cys 1090 1095 1100 Phe Gly Val Glu Glu Leu Ser Lys
Ala Tyr Glu Asp Asp Gly Asp Asp 1105 1110 1115 1120 Tyr Ser Ser Ile
Met Val Lys Ala Leu Gly Asp Arg Leu Ala Glu Ala 1125 1130 1135 Phe
Ala Glu Glu Leu His Glu Arg Val Arg Arg Glu Leu Trp Ala Tyr 1140
1145 1150 Cys Gly Ser Glu Gln Leu Asp Val Ala Asp Leu Arg Arg Leu
Arg Tyr 1155 1160 1165 Lys Gly Ile Arg Pro Ala Pro Gly Tyr Pro Ser
Gln Pro Asp His Thr 1170 1175 1180 Glu Lys Leu Thr Met Trp Arg Leu
Ala Asp Ile Glu Gln Ser Thr Gly 1185 1190 1195 1200 Ile Arg Leu Thr
Glu Ser Leu Ala Met Ala Pro Ala Ser Ala Val Ser 1205 1210 1215 Gly
Leu Tyr Phe Ser Asn Leu Lys Ser Lys Tyr Phe Ala Val Gly Lys 1220
1225 1230 Ile Ser Lys Asp Gln Val Glu Asp Tyr Ala Leu Arg Lys Asn
Ile Ser 1235 1240 1245 Val Ala Glu Val Glu Lys Trp Leu Gly Pro Ile
Leu Gly Tyr Asp Thr 1250 1255 1260 Asp 1265 3 9 PRT Escherichia
coli 3 Asp Gly Gly Met Gly Thr Met Ile Gln 1 5 4 9 PRT
Cyanobacterium synechocystis 4 Asp Gly Ala Met Gly Thr Asn Leu Gln
1 5 5 9 PRT Mycobacterium leprae 5 Asp Gly Ala Met Gly Thr Gln Leu
Gln 1 5 6 9 PRT Hemophilus influenzae 6 Asp Gly Ala Met Gly Thr Met
Ile Gln 1 5 7 9 PRT Caenorrhabditis elegans 7 Asp Gly Ala Met Gly
Thr Met Ile Gln 1 5 8 9 PRT Homo sapiens 8 Asp Gly Gly Met Gly Thr
Met Ile Gln 1 5 9 13 PRT Escherichia coli 9 Ala Thr Val Lys Gly Asp
Val His Asp Ile Gly Lys Asn 1 5 10 10 13 PRT Cyanobacterium
synechocystis 10 Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys
Asn 1 5 10 11 13 PRT Mycobacterium leprae 11 Ala Thr Val Lys Gly
Asp Val His Asp Ile Gly Lys Asn 1 5 10 12 13 PRT Hemophilus
influenzae 12 Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn 1
5 10 13 13 PRT Caenorrhabditis elegans 13 Ala Thr Val Lys Gly Asp
Val His Asp Ile Gly Lys Asn 1 5 10 14 13 PRT Homo sapiens 14 Ala
Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn 1 5 10 15 10 PRT
Escherichia coli 15 Leu Ala Glu Ala Phe Ala Glu Tyr Leu His 1 5 10
16 10 PRT Cyanobacterium synechocystis 16 Met Ala Glu Ala Leu Ala
Glu Trp Thr His 1 5 10 17 10 PRT Mycobacterium leprae 17 Leu Thr
Glu Ala Leu Ala Glu Tyr Trp His 1 5 10 18 10 PRT Hemophilus
influenzae 18 Leu Ala Glu Ala Met Ala Glu Tyr Leu His 1 5 10 19 10
PRT Caenorrhabditis elegans 19 Leu Ala Glu Ala Tyr Ala Glu Tyr Leu
His 1 5 10 20 10 PRT Homo sapiens 20 Leu Ala Glu Ala Phe Ala Glu
Glu Leu His 1 5 10 21 11 PRT Escherichia coli 21 Gly Gly Cys Cys
Gly Thr Thr Pro Gln His Ile 1 5 10 22 11 PRT Cyanobacterium
synechocystis 22 Gly Gly Cys Cys Gly Thr Arg Pro Asp His Ile 1 5 10
23 11 PRT Mycobacterium leprae 23 Gly Gly Cys Cys Gly Thr Thr Pro
Asp His Ile 1 5 10 24 11 PRT Caenorrhabditis elegans 24 Gly Gly Cys
Cys Gly Thr Thr Pro Asp His Ile 1 5 10 25 11 PRT Homo sapiens 25
Gly Gly Cys Cys Gly Ser Thr Pro Asp His Ile 1 5 10 26 26 DNA Homo
sapiens variation (1)...(26) n is a, t, g, or c; h is a, c, or t; d
is a, g, or t; and r is a or g; 26 gayggngcna tgggnacnat gathca 26
27 29 DNA Homo sapiens variation (1)...(29) n is a, t, g, or c; h
is a, c, or t; d is a, g, or t; and r is a or g; 27 gcnacngtna
arggngaygt ncaygayat 29 28 30 DNA Homo sapiens variation (1)...(30)
n is a, t, g, or c; h is a, c, or t; d is a, g, or t; and r is a or
g; 28 rttyttnccd atrtcrtgna crtcnccytt 30 29 27 DNA Homo sapiens
variation (1)...(27) n is a, t, g, or c; h is a, c, or t; d is a,
g, or t; and r is a or g; 29 rtgnagrtay tcngcraang cytcngc 27 30 32
DNA Homo sapiens variation (1)...(32) n is a, t, g, or c; h is a,
c, or t; d is a, g, or t; and r is a or g; 30 atrtgrtcng gngtngtncc
rcarcanccn cc 32 31 32 DNA Homo sapiens variation (1)...(32) n is
a, t, g, or c; h is a, c, or t; d is a, g, or t; and r is a or g;
31 ggnggntgyt gyggnacnac nccngaycay at 32 32 24 DNA Mus musculus 32
gtctgtgtca
tagcccagaa tggg 24 33 24 DNA Mus musculus 33 tcagtctgtg tcatagccca
gaat 24 34 24 DNA Homo sapiens 34 gaactagaag acagaaattc tcta 24 35
26 DNA Homo sapiens 35 ttccgaggtc aggaatttaa agatca 26 36 24 DNA
Homo sapiens 36 gtgttcttcg tttagcttct cccg 24 37 24 DNA Homo
sapiens 37 ccccagccag caagtattcc ttat 24 38 24 DNA Homo sapiens 38
ctaggttgta tttccttgag gatc 24 39 25 DNA Homo sapiens 39 ggagctggaa
aaatgtttct acctc 25 40 24 DNA Homo sapiens 40 acaggaggga agaaagtcat
tcag 24 41 25 DNA Homo sapiens 41 ccttcaatta tattgagagg tcggg 25 42
24 DNA Homo sapiens 42 caacccgaag gtctgaagaa aacc 24 43 21 DNA Homo
sapiens 43 cccgcgctcc aagacctgtc g 21 44 21 DNA Homo sapiens 44
cgacaggtct tggagcgcgg g 21 45 28 DNA Homo sapiens 45 ggagtcatga
ctcctaaatc aataactc 28 46 23 DNA Homo sapiens 46 gacgactaca
gcagcatcat ggt 23 47 22 DNA Homo sapiens 47 aaaaatcatt tcatccaggg
aa 22 48 26 DNA Homo sapiens 48 ataggcaaga acatagttgg agtagt 26 49
25 DNA Homo sapiens 49 tttcatctaa cagctgggaa cacac 25 50 24 DNA
Homo sapiens 50 tgcctctcag acttcatcgc tccc 24 51 21 DNA Homo
sapiens 51 tgcagcctgg ggcacagcag c 21 52 24 DNA Homo sapiens 52
atggattggc tgtctgaacc tcac 24 53 26 DNA Homo sapiens 53 catggaagaa
tatgaagata ttagac 26 54 24 DNA Homo sapiens 54 accatcatcc
tcataggcct tgct 24 55 23 DNA Homo sapiens 55 cagacctgcg aaggttgcgg
tac 23 56 24 DNA Homo sapiens 56 gaagtggttg ctcctccaat caac 24 57
26 DNA Homo sapiens 57 gagcagcttt cagtatctta tcacat 26 58 25 DNA
Homo sapiens 58 acaagttgtg ttcctccatt ccagt 25 59 25 DNA Homo
sapiens 59 agagcgctgt aatgttgcag gatca 25 60 25 DNA Homo sapiens 60
tgtttttcaa tgcccttcac aaggg 25 61 25 DNA Homo sapiens 61 taaaaagtat
ggagctgcta tggtg 25 62 25 DNA Homo sapiens 62 gaccagacag taacatatgt
ccttc 25 63 26 DNA Homo sapiens 63 acattacagc gctctccaat gttaac 26
64 24 DNA Homo sapiens 64 tgaggttgag aaatggcttg gacc 24 65 25 DNA
Homo sapiens 65 gccacagata tgttcttcct caatg 25 66 24 DNA Homo
sapiens 66 tgtggagagc acgtcttctc tgcc 24 67 50 PRT Homo sapiens 67
Leu Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val 1 5
10 15 Gly Val Val Leu Gly Cys Asn Asn Phe Arg Val Ile Asp Leu Gly
Val 20 25 30 Met Thr Pro Cys Asp Lys Ile Leu Lys Ala Ala Leu Asp
His Lys Ala 35 40 45 Asp Ile 50 68 50 PRT Mus musculus 68 Leu Ala
Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val 1 5 10 15
Gly Val Val Leu Ala Cys Asn Asn Phe Arg Val Ile Asp Leu Gly Val 20
25 30 Met Thr Pro Cys Asp Lys Ile Leu Gln Ala Ala Leu Asp His Lys
Ala 35 40 45 Asp Ile 50 69 50 PRT Cyanobacterium synechocystis 69
Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Leu Val 1 5
10 15 Asp Ile Ile Leu Ser Asn Asn Gly Tyr Arg Val Val Asn Leu Gly
Ile 20 25 30 Lys Gln Pro Val Glu Asn Ile Ile Glu Ala Tyr Lys Lys
His Arg Pro 35 40 45 Asp Cys 50 70 50 PRT Mycobacterium leprae 70
Leu Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Leu Val 1 5
10 15 Asp Ile Ile Leu Ser Asn Asn Gly Tyr Glu Val Val Asn Leu Gly
Ile 20 25 30 Lys Gln Pro Ile Thr Asn Ile Leu Glu Val Ala Glu Asp
Lys Ser Ala 35 40 45 Asp Val 50 71 50 PRT Caenorrhabditis elegans
71 Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val
1 5 10 15 Ser Val Val Leu Gly Cys Asn Asn Phe Lys Val Val Asp Leu
Gly Val 20 25 30 Met Thr Pro Cys Glu Asn Ile Ile Lys Ala Ala Ile
Glu Glu Lys Ala 35 40 45 Asp Phe 50 72 50 PRT Hemophilus influenzae
72 Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val
1 5 10 15 Ser Val Val Met Gln Cys Asn Asn Phe Glu Val Ile Asp Leu
Gly Val 20 25 30 Met Val Pro Ala Asp Lys Ile Ile Gln Thr Ala Ile
Asn Gln Lys Thr 35 40 45 Asp Ile 50 73 50 PRT Escherichia coli 73
Ile Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys Asn Ile Val 1 5
10 15 Gly Val Val Leu Gln Cys Asn Asn Tyr Glu Ile Val Asp Leu Gly
Val 20 25 30 Met Val Pro Ala Glu Lys Ile Leu Arg Thr Ala Lys Glu
Val Asn Ala 35 40 45 Asp Leu 50 74 1265 PRT Homo sapiens VARIANT
(1)...(1265) Xaa at position 881 is either Ile or no amino acid;
Xaa at position 919 is either Asp or Gly; Xaa at position 920 is
either His or Asp. 74 Met Ser Pro Ala Leu Gln Asp Leu Ser Gln Pro
Glu Gly Leu Lys Lys 1 5 10 15 Thr Leu Arg Asp Glu Ile Asn Ala Ile
Leu Gln Lys Arg Ile Met Val 20 25 30 Leu Asp Gly Gly Met Gly Thr
Met Ile Gln Arg Glu Lys Leu Asn Glu 35 40 45 Glu His Phe Arg Gly
Gln Glu Phe Lys Asp His Ala Arg Pro Leu Lys 50 55 60 Gly Asn Asn
Asp Ile Leu Ser Ile Thr Gln Pro Asp Val Ile Tyr Gln 65 70 75 80 Ile
His Lys Glu Tyr Leu Leu Ala Gly Ala Asp Ile Ile Glu Thr Asn 85 90
95 Thr Phe Ser Ser Thr Ser Ile Ala Gln Ala Asp Tyr Gly Leu Glu His
100 105 110 Leu Ala Tyr Arg Met Asn Met Cys Ser Ala Gly Val Ala Arg
Lys Ala 115 120 125 Ala Glu Glu Val Thr Leu Gln Thr Gly Ile Lys Arg
Phe Val Ala Gly 130 135 140 Ala Leu Gly Pro Thr Asn Lys Thr Leu Ser
Val Ser Pro Ser Val Glu 145 150 155 160 Arg Pro Asp Tyr Arg Asn Ile
Thr Phe Asp Glu Leu Val Glu Ala Tyr 165 170 175 Gln Glu Gln Ala Lys
Gly Leu Leu Asp Gly Gly Val Asp Ile Leu Leu 180 185 190 Ile Glu Thr
Ile Phe Asp Thr Ala Asn Ala Lys Ala Ala Leu Phe Ala 195 200 205 Leu
Gln Asn Leu Phe Glu Glu Lys Tyr Ala Pro Arg Pro Ile Phe Ile 210 215
220 Ser Gly Thr Ile Val Asp Lys Ser Gly Arg Thr Leu Ser Gly Gln Thr
225 230 235 240 Gly Glu Gly Phe Val Ile Ser Val Ser His Gly Glu Pro
Leu Cys Ile 245 250 255 Gly Leu Asn Cys Ala Leu Gly Ala Ala Glu Met
Arg Pro Phe Ile Glu 260 265 270 Ile Ile Gly Lys Cys Thr Thr Ala Tyr
Val Leu Cys Tyr Pro Asn Ala 275 280 285 Gly Leu Pro Asn Thr Phe Gly
Asp Tyr Asp Glu Thr Pro Ser Met Met 290 295 300 Ala Lys His Leu Lys
Asp Phe Ala Met Asp Gly Leu Val Asn Ile Val 305 310 315 320 Gly Gly
Cys Cys Gly Ser Thr Pro Asp His Ile Arg Glu Ile Ala Glu 325 330 335
Ala Val Lys Asn Cys Lys Pro Arg Val Pro Pro Ala Thr Ala Phe Glu 340
345 350 Gly His Met Leu Leu Ser Gly Leu Glu Pro Phe Arg Ile Gly Pro
Tyr 355 360 365 Thr Asn Phe Val Asn Ile Gly Glu Arg Cys Asn Val Ala
Gly Ser Arg 370 375 380 Lys Phe Ala Lys Leu Ile Met Ala Gly Asn Tyr
Glu Glu Ala Leu Cys 385 390 395 400 Val Ala Lys Val Gln Val Glu Met
Gly Ala Gln Val Leu Asp Val Asn 405 410 415 Met Asp Asp Gly Met Leu
Asp Gly Pro Ser Ala Met Thr Arg Phe Cys 420 425 430 Asn Leu Ile Ala
Ser Glu Pro Asp Ile Ala Lys Val Pro Leu Cys Ile 435 440 445 Asp Ser
Ser Asn Phe Ala Val Ile Glu Ala Gly Leu Lys Cys Cys Gln 450 455 460
Gly Lys Cys Ile Val Asn Ser Ile Ser Leu Lys Glu Gly Glu Asp Asp 465
470 475 480 Phe Leu Glu Lys Ala Arg Lys Ile Lys Lys Tyr Gly Ala Ala
Met Val 485 490 495 Val Met Ala Phe Asp Glu Glu Gly Gln Ala Thr Glu
Thr Asp Thr Lys 500 505 510 Ile Arg Val Cys Thr Arg Ala Tyr His Leu
Leu Val Lys Lys Leu Gly 515 520 525 Phe Asn Pro Asn Asp Ile Ile Phe
Asp Pro Asn Ile Leu Thr Ile Gly 530 535 540 Thr Gly Met Glu Glu His
Asn Leu Tyr Ala Ile Asn Phe Ile His Ala 545 550 555 560 Thr Lys Val
Ile Lys Glu Thr Leu Pro Gly Ala Arg Ile Ser Gly Gly 565 570 575 Leu
Ser Asn Leu Ser Phe Ser Phe Arg Gly Met Glu Ala Ile Arg Glu 580 585
590 Ala Met His Gly Val Phe Leu Tyr His Ala Ile Lys Ser Gly Met Asp
595 600 605 Met Glu Ile Val Asn Ala Gly Asn Leu Pro Val Tyr Asp Asp
Ile His 610 615 620 Lys Glu Leu Leu Gln Leu Cys Glu Asp Leu Ile Trp
Asn Lys Asp Pro 625 630 635 640 Glu Ala Thr Glu Lys Leu Leu Arg Tyr
Ala Gln Thr Gln Gly Thr Gly 645 650 655 Gly Lys Lys Val Ile Gln Thr
Asp Glu Trp Arg Asn Gly Pro Val Glu 660 665 670 Glu Arg Leu Glu Tyr
Ala Leu Val Lys Gly Ile Glu Lys His Ile Ile 675 680 685 Glu Asp Thr
Glu Glu Ala Arg Leu Asn Gln Lys Lys Tyr Pro Arg Pro 690 695 700 Leu
Asn Ile Ile Glu Gly Pro Leu Met Asn Gly Met Lys Ile Val Gly 705 710
715 720 Asp Leu Phe Gly Ala Gly Lys Met Phe Leu Pro Gln Val Ile Lys
Ser 725 730 735 Ala Arg Val Met Lys Lys Ala Val Gly His Leu Ile Pro
Phe Met Glu 740 745 750 Lys Glu Arg Glu Glu Thr Arg Val Leu Asn Gly
Thr Val Glu Glu Glu 755 760 765 Asp Pro Tyr Gln Gly Thr Ile Val Leu
Ala Thr Val Lys Gly Asp Val 770 775 780 His Asp Ile Gly Lys Asn Ile
Val Gly Val Val Leu Gly Cys Asn Asn 785 790 795 800 Phe Arg Val Ile
Asp Leu Gly Val Met Thr Pro Cys Asp Lys Ile Leu 805 810 815 Lys Ala
Ala Leu Asp His Lys Ala Asp Ile Ile Gly Leu Ser Gly Leu 820 825 830
Ile Thr Pro Ser Leu Asp Glu Met Ile Phe Val Ala Lys Glu Met Glu 835
840 845 Arg Leu Ala Ile Arg Ile Pro Leu Leu Ile Gly Gly Ala Thr Thr
Ser 850 855 860 Lys Thr His Thr Ala Val Lys Ile Ala Pro Arg Tyr Ser
Ala Pro Val 865 870 875 880 Xaa His Val Leu Asp Ala Ser Lys Ser Val
Val Val Cys Ser Gln Leu 885 890 895 Leu Asp Glu Asn Leu Lys Asp Glu
Tyr Phe Glu Glu Ile Met Glu Glu 900 905 910 Tyr Glu Asp Ile Arg Gln
Xaa Xaa Tyr Glu Ser Leu Lys Glu Arg Arg 915 920 925 Tyr Leu Pro Leu
Ser Gln Ala Arg Lys Ser Gly Phe Gln Met Asp Trp 930 935 940 Leu Ser
Glu Pro His Pro Val Lys Pro Thr Phe Ile Gly Thr Gln Val 945 950 955
960 Phe Glu Asp Tyr Asp Leu Gln Lys Leu Val Asp Tyr Ile Asp Trp Lys
965 970 975 Pro Phe Phe Asp Val Trp Gln Leu Arg Gly Lys Tyr Pro Asn
Arg Gly 980 985 990 Phe Pro Lys Ile Phe Asn Asp Lys Thr Val Gly Gly
Glu Ala Arg Lys 995 1000 1005 Val Tyr Asp Asp Ala His Asn Met Leu
Asn Thr Leu Ile Ser Gln Lys 1010 1015 1020 Lys Leu Arg Ala Arg Gly
Val Val Gly Phe Trp Pro Ala Gln Ser Ile 1025 1030 1035 1040 Gln Asp
Asp Ile His Leu Tyr Ala Glu Ala Ala Val Pro Gln Ala Ala 1045 1050
1055 Glu Pro Ile Ala Thr Phe Tyr Gly Leu Arg Gln Gln Ala Glu Lys
Asp 1060 1065 1070 Ser Ala Ser Thr Glu Pro Tyr Tyr Cys Leu Ser Asp
Phe Ile Ala Pro 1075 1080 1085 Leu His Ser Gly Ile Arg Asp Tyr Leu
Gly Leu Phe Ala Val Ala Cys 1090 1095 1100 Phe Gly Val Glu Glu Leu
Ser Lys Ala Tyr Glu Asp Asp Gly Asp Asp 1105 1110 1115 1120 Tyr Ser
Ser Ile Met Val Lys Ala Leu Gly Asp Arg Leu Ala Glu Ala 1125 1130
1135 Phe Ala Glu Glu Leu His Glu Arg Val Arg Arg Glu Leu Trp Ala
Tyr 1140 1145 1150 Cys Gly Ser Glu Gln Leu Asp Val Ala Asp Leu Arg
Arg Leu Arg Tyr 1155 1160 1165 Lys Gly Ile Arg Pro Ala Pro Gly Tyr
Pro Ser Gln Pro Asp His Thr 1170 1175 1180 Glu Lys Leu Thr Met Trp
Arg Leu Ala Asp Ile Glu Gln Ser Thr Gly 1185 1190 1195 1200 Ile Arg
Leu Thr Glu Ser Leu Ala Met Ala Pro Ala Ser Ala Val Ser 1205 1210
1215 Gly Leu Tyr Phe Ser Asn Leu Lys Ser Lys Tyr Phe Ala Val Gly
Lys 1220 1225 1230 Ile Ser Lys Asp Gln Val Glu Asp Tyr Ala Leu Arg
Lys Asn Ile Ser 1235 1240 1245 Val Ala Glu Val Glu Lys Trp Leu Gly
Pro Ile Leu Gly Tyr Asp Thr 1250 1255 1260 Asp 1265 75 3856 DNA
Homo sapiens variation (1)...(3856) nnn at positions 2640-2642 is
either AAT or no nucleotides; n at position 2756 is either A or G;
n at position 2758 is either C or G. 75 atgtcacccg cgctccaaga
cctgtcgcaa cccgaaggtc tgaagaaaac cctgcgggat 60 gagatcaatg
ccattctgca gaagaggatt atggtgctgg atggagggat ggggaccatg 120
atccagcggg agaagctaaa cgaagaacac ttccgaggtc aggaatttaa agatcatgcc
180 aggccgctga aaggcaacaa tgacatttta agtataactc agcctgatgt
catttaccaa 240 atccataagg aatacttgct ggctggggca gatatcattg
aaacaaatac ttttagcagc 300 actagtattg cccaagctga ctatggcctt
gaacacttgg cctaccggat gaacatgtgc 360 tctgcaggag tggccagaaa
agctgccgag gaggtaactc tccagacagg aattaagagg 420 tttgtggcag
gggctctggg tccgactaat aagacactct ctgtgtcccc atctgtggaa 480
aggccggatt ataggaacat cacatttgat gagcttgttg aagcatacca agagcaggcc
540 aaaggacttc tggatggcgg ggttgatatc ttactcattg aaactatttt
tgatactgcc 600 aatgccaagg cagccttgtt tgcactccaa aatctttttg
aggagaaata tgctccccgg 660 cctatcttta tttcagggac gatcgttgat
aaaagtgggc ggactctttc cggacagaca 720 ggagagggat ttgtcatcag
cgtgtctcat ggagaaccac tctgcattgg attaaattgt 780 gctttgggtg
cagctgagat gagacctttt attgaaataa ttggaaaatg tacaacagcc 840
tatgtcctct gttatcccaa tgcaggtctt cccaacacct ttggtgacta tgatgaaacg
900 ccttctatga tggccaagca cctaaaggat tttgctatgg atggcttggt
caatatagtt 960 ggaggatgct gtgggtcaac accagatcat atcagggaaa
ttgctgaagc tgtgaaaaat 1020 tgtaagccta gagttccacc tgccactgct
tttgaaggac atatgttact gtctggtcta 1080 gagcccttca ggattggacc
gtacaccaac tttgttaaca ttggagagcg ctgtaatgtt 1140 gcaggatcaa
ggaagtttgc taaactcatc atggcaggaa actatgaaga agccttgtgt 1200
gttgccaaag tgcaggtgga aatgggagcc caggtgttgg atgtcaacat ggatgatggc
1260 atgctagatg gtccaagtgc aatgaccaga ttttgcaact taattgcttc
cgagccagac 1320 atcgcaaagg tacctttgtg catcgactcc tccaattttg
ctgtgattga agctgggtta 1380 aagtgctgcc aagggaagtg cattgtcaat
agcattagtc tgaaggaagg agaggacgac 1440 ttcttggaga aggccaggaa
gattaaaaag tatggagctg ctatggtggt catggctttt 1500 gatgaagaag
gacaggcaac agaaacagac acaaaaatca gagtgtgcac ccgggcctac 1560
catctgcttg tgaaaaaact gggctttaat ccaaatgaca ttatttttga ccctaatatc
1620 ctaaccattg ggactggaat ggaggaacac aacttgtatg ccattaattt
tatccatgca 1680
acaaaagtca ttaaagaaac attacctgga gccagaataa gtggaggtct ttccaacttg
1740 tccttctcct tccgaggaat ggaagccatt cgagaagcaa tgcatggggt
tttcctttac 1800 catgcaatca agtctggcat ggacatggag atagtgaatg
ctggaaacct ccctgtgtat 1860 gatgatatcc ataaggaact tctgcagctc
tgtgaagatc tcatctggaa taaagaccct 1920 gaggccactg agaagctctt
acgttatgcc cagactcaag gcacaggagg gaagaaagtc 1980 attcagactg
atgagtggag aaatggccct gtcgaagaac gccttgagta tgcccttgtg 2040
aagggcattg aaaaacatat tattgaggat actgaggaag ccaggttaaa ccaaaaaaaa
2100 tatccccgac ctctcaatat aattgaagga cccctgatga atggaatgaa
aattgttggt 2160 gatctttttg gagctggaaa aatgtttcta cctcaggtta
taaagtcagc ccgggttatg 2220 aagaaggctg ttggccacct tatccctttc
atggaaaaag aaagagaaga aaccagagtg 2280 cttaacggca cagtagaaga
agaggaccct taccagggca ccatcgtgct ggccactgtt 2340 aaaggcgacg
tgcacgacat aggcaagaac atagttggag tagtccttgg ctgcaataat 2400
ttccgagtta ttgatttagg agtcatgact ccatgtgata agatactgaa agctgctctt
2460 gaccacaaag cagatataat tggcctgtca ggactcatca ctccttccct
ggatgaaatg 2520 atttttgttg ccaaggaaat ggagagatta gctataagga
ttccattgtt gattggagga 2580 gcaaccactt caaaaaccca cacagcagtt
aaaatagctc cgagatacag tgcacctgtn 2640 nnccatgtcc tggacgcgtc
caagagtgtg gtggtgtgtt cccagctgtt agatgaaaat 2700 ctaaaggatg
aatactttga ggaaatcatg gaagaatatg aagatattag acaggncnat 2760
tatgagtctc tcaaggagag gagatactta cccttaagtc aagccagaaa aagtggtttc
2820 caaatggatt ggctgtctga acctcaccca gtgaagccca cgtttattgg
gacccaggtc 2880 tttgaagact atgacctgca gaagctggtg gactacattg
actggaagcc tttctttgat 2940 gtctggcagc tccggggcaa gtacccgaat
cgaggcttcc ccaagatatt taacgacaaa 3000 acagtaggtg gagaggccag
gaaggtctac gatgatgccc acaatatgct gaacacactg 3060 attagtcaaa
agaaactccg ggcccggggt gtggttgggt tctggccagc acagagtatc 3120
caagacgaca ttcacctgta cgcagaggct gctgtgcccc aggctgcaga gcccatagcc
3180 actttctatg ggttaaggca acaggctgag aaggactctg ccagcacgga
gccatactac 3240 tgcctctcag acttcatcgc tcccttgcat tctggcatcc
gtgactacct gggcctgttt 3300 gccgttgcct gctttggggt agaagagctg
agcaaggcct atgaggatga tggtgacgac 3360 tacagcagca tcatggtcaa
ggcgctgggg gaccggctgg cagaggcctt tgcagaagag 3420 ctccatgaaa
gagttcgccg agaactgtgg gcctactgtg gcagtgagca gctggacgtc 3480
gcagacctgc gaaggttgcg gtacaagggc atccgcccgg ctcctggcta ccccagccag
3540 cccgaccaca ccgagaagct caccatgtgg agactcgcag acatcgagca
gtctacaggc 3600 attaggttaa cagaatcatt agcaatggca cctgcttcag
cagtctcagg cctctacttc 3660 tccaatttga agtccaaata ttttgctgtg
gggaagattt ccaaggatca ggttgaggat 3720 tatgcattga ggaagaacat
atctgtggct gaggttgaga aatggcttgg acccattttg 3780 ggatatgata
cagactaact tttttttttt tttttgcctt ttttatcttg atgatcctca 3840
aggaaataca acctag 3856 76 10 DNA Homo sapiens 76 gacaacatgt 10
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