U.S. patent application number 10/376460 was filed with the patent office on 2003-09-04 for apm1 biallelic markers and uses thereof.
This patent application is currently assigned to Genset, S.A.. Invention is credited to Bihain, Bernard, Bougueleret, Lydie, Denison, Blake, Yen-Potin, Frances.
Application Number | 20030165967 10/376460 |
Document ID | / |
Family ID | 26804398 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165967 |
Kind Code |
A1 |
Bougueleret, Lydie ; et
al. |
September 4, 2003 |
APM1 biallelic markers and uses thereof
Abstract
The invention provides novel APM1 genomic sequences,
polypeptides, antibodies, and polynucleotides including biallelic
markers derived from the APM1 locus. Primers hybridizing to regions
flanking these biallelic markers are also provided. This invention
also provides polynucleotides and methods suitable for genotyping a
nucleic acid containing sample for one or more biallelic markers of
the invention. Additionally, the invention provides methods to
detect a statistical correlation between a biallelic marker allele
and a phenotype and/or between a biallelic marker haplotype and a
phenotype. Further, the invention provides diagnostic methods for
early detection of obesity-related disorders.
Inventors: |
Bougueleret, Lydie;
(Petit-Lancy, CH) ; Bihain, Bernard; (Cancale,
FR) ; Denison, Blake; (San Diego, CA) ;
Yen-Potin, Frances; (San Diego, CA) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
2421 N.W. 41ST STREET
SUITE A-1
GAINESVILLE
FL
326066669
|
Assignee: |
Genset, S.A.
24, rue Royale
Paris
FR
|
Family ID: |
26804398 |
Appl. No.: |
10/376460 |
Filed: |
February 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10376460 |
Feb 28, 2003 |
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09569852 |
May 10, 2000 |
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6582909 |
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09569852 |
May 10, 2000 |
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PCT/IB99/01858 |
Nov 4, 1999 |
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09569852 |
May 10, 2000 |
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09434848 |
Nov 4, 1999 |
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60119593 |
Feb 10, 1999 |
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60107113 |
Nov 4, 1998 |
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60119593 |
Feb 10, 1999 |
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60107113 |
Nov 4, 1998 |
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Current U.S.
Class: |
435/6.11 ;
435/91.2 |
Current CPC
Class: |
Y10S 977/928 20130101;
C12Q 1/6883 20130101; C07K 14/47 20130101; C12Q 2600/156 20130101;
A01K 2217/05 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
We claim:
1. A method of genotyping comprising the steps of: a) obtaining a
biological sample; and b) determining the identity of a nucleotide
at a biallelic marker at position 15863 (biallelic marker A7
(99-14405/105)) of SEQ ID NO: 1 or the complements thereof within
said sample.
2. The method of claim 1, wherein said biological sample is
obtained from a single subject.
3. The method of claim 2, wherein the identity of the nucleotides
at said biallelic marker is determined for both copies of said
biallelic marker present in said subject's genome.
4. The method of claim 1, wherein said biological sample is
obtained from multiple subjects.
5. The method of claim 1, further comprising amplifying a portion
of said sequence comprising the biallelic marker prior to said
determining step.
6. The method of claim 1, wherein said determining is performed by
a method selected from the group consisting of a hybridization
assay, a sequencing assay, a microsequencing assay, and an
allele-specific amplification assay.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/569,852, filed May 10, 2000, which is a
continuation-in-part of International Patent Application No.
PCT/IB99/01858, filed Nov. 4, 1999 and U.S. Non-Provisional patent
application Ser. No. 09/434,848, filed Nov. 4, 1999, both of which
claim priority to U.S. Provisional Patent Application Serial No.
60/119,593, filed Feb. 10, 1999, and U.S. Provisional Patent
Application Serial No. 60/107,113, filed Nov. 4, 1998. All of the
above-referenced applications are hereby incorporated by reference
herein in their entireties, including any figures, tables, nucleic
acid sequences, amino acid sequences, or drawings.
FIELD OF THE INVENTION
[0002] The invention concerns the genomic and cDNA sequences of the
APM1 gene, as well as methods and kits for detecting these
polynucleotides. The invention also concerns the regulatory
regions, particularly the promoter region of the APM1 gene. The
invention comprises biallelic markers of the APM1 gene which can be
useful for diagnosis of obesity or disorders related to
obesity.
BACKGROUND
[0003] Obesity is a public health problem that is both serious and
widespread. One-third of the population in industrialized countries
has an excess weight of at least 20% relative to the ideal weight.
The phenomenon continues to worsen, particularly in regions of the
globe where economies are modernizing. In the United States, the
number of obese people has escalated from 25% at the end of the 70s
to 33% at the beginning of the 90s.
[0004] Obesity considerably increases the risk of developing
cardiovascular and metabolic diseases. It is estimated that if the
entire population had an ideal weight, the risk of coronary
insufficiency would decrease by 25% and that of cardiac
insufficiency and of cerebral vascular accidents by 35%. Coronary
insufficiency, atheromatous disease and cardiac insufficiency are
at the forefront of the cardiovascular complications induced by
obesity. For an excess weight greater than 30%, the incidence of
coronary diseases is doubled in subjects less than 50 years old.
Studies carried out for other diseases are equally significant. For
an excess weight of 20%, the risk of high blood pressure is
doubled. For an excess weight of 30%, the risk of developing
non-insulin-dependent diabetes is tripled and the risk of
hyperlipidemias is multiplied six fold.
[0005] The list of diseases having onsets promoted by obesity is
long: hyperuricemia (11.4% in obese subjects, compared with 3.4% in
the general population), digestive pathologies, abnormalities in
hepatic functions, and even certain cancers.
[0006] Whether the physiological changes in obesity are
characterized by an increase in the number of adipose cells, or by
an increase in the quantity of triglycerides stored in each adipose
cell, or by both, this excess weight results mainly from an
imbalance between the quantities of calories consumed and the
quantity of calories used by the body. Some studies on the causes
of this imbalance have focused on studying the mechanism of
absorption of foods, and therefore the molecules which control food
intake and the feeling of satiety. Other studies have characterized
the pathways through which the body uses its calories.
[0007] The treatments for obesity which have been proposed are of
four types. 1) Food restriction is the most frequently used. Obese
individuals are advised to change their dietary habits so as to
consume fewer calories. This type of treatment is effective in the
short-term. However, the recidivation rate is very high. 2)
Increased calorie use through physical exercise is also proposed.
This treatment is ineffective when applied alone, but it improves,
however, weight-loss in subjects on a low-calorie diet. 3)
Gastrointestinal surgery, which reduces the absorption of the
calories ingested, is effective but has been virtually abandoned
because of the side effects which it causes. 4) The medicinal
approach uses either the anorexigenic action of molecules involved
at the level of the central nervous system, or the effect of
molecules which increase energy use by increasing the production of
heat. The prototypes of this kind of molecule are the thyroid
hormones that uncouple oxidative phosphorylations of the
mitochondrial respiratory chain. The side effects and the toxicity
of this type of treatment make their use dangerous. An approach
that aims to reduce the absorption of dietary lipids by
sequestering them in the lumen of the digestive tube is also in
place. However, it induces physiological imbalances that are
difficult to tolerate: deficiency in the absorption of fat-soluble
vitamins, flatulence and steatorrhoea. Whatever the envisaged
therapeutic approach, the treatments of obesity are all
characterized by an extremely high recidivation rate.
[0008] The molecular mechanisms responsible for obesity in man are
complex and involve genetic and environmental factors. Because of
the low efficiency of the treatments known up until now, it is
urgent to define the genetic mechanisms that determine obesity, so
as to be able to develop better targeted medicaments.
[0009] More than 20 genes have been studied as possible candidates,
either because they have been implicated in diseases of which
obesity is one of the clinical manifestations, or because they are
homologues of genes involved in obesity in animal models. Situated
in the 7q31 chromosomal region, the OB gene is one of the most
widely studied. Its product, leptin, is involved in the mechanisms
of satiety. Leptin is a plasma protein of 16 kDa produced by the
adipocytes under the action of various stimuli. Obese mice of the
ob/ob type exhibit a deficiency in the leptin gene; this protein is
undetectable in the plasma of these animals. The administration of
leptin obtained by genetic engineering to ob/ob mice corrects their
relative hyperphagia and allows normalization of their weight. This
anorexigenic effect of leptin calls into play a receptor of the
central nervous system: the ob receptor which belongs to the family
of class 1 cytokine receptors. The ob receptor is deficient in
obese mice of the db/db strain. The administration of leptin to
these mice has no effect on their food intake and does not allow
substantial reduction in their weight. The mechanisms by which the
ob receptors transmit the signal for satiety are not precisely
known. It is possible that neuropeptide Y is involved in this
signaling pathway. It is important to specify at this stage that
the ob receptors are not the only regulators of appetite. The
Melanocortin 4 receptor is also involved since mice made deficient
in this receptor are obese (Gura, (1997)).
[0010] The discovery of leptin and the characterization of the
leptin receptor at the level of the central nervous system opened a
new route for the search for medicaments against obesity. This
model, however, rapidly proved disappointing. Indeed, with only one
exception (Montague et al., (1997)), the genes encoding leptin or
its ob receptor have proved to be normal in obese human subjects.
Furthermore and paradoxically, the plasma concentrations of leptin,
the satiety hormone, are abnormally high in most obese human
subjects.
[0011] Clearly there remains a need for novel medicaments that are
useful for reducing body weight in humans. Such pharmaceutical
compositions advantageously would help to control obesity and
thereby alleviate many of the cardiovascular consequences
associated with this condition.
[0012] The human adipocyte-specific APM1 gene encodes a secretory
protein of the adipose tissue and is likely to play a role in the
pathogenesis of obesity. Knowledge of the APM1 genomic sequence,
and particularly of both promoter and splice junction sequences,
allows the design of novel diagnostics and therapeutic tools that
act on lipid metabolism, and are useful for diagnosing and treating
obesity disorders.
SUMMARY OF THE INVENTION
[0013] The present invention stems from the isolation and
characterization of the genomic sequence of APM1 gene including its
regulatory regions and of the complete CDNA sequence encoding the
APM1 protein. Oligonucleotide probes and primers hybridizing
specifically with a genomic sequence of APM1 are also part of the
invention. A further object of the invention consists of
recombinant vectors comprising any of the nucleic acid sequences
described in the present invention, and in particular of
recombinant vectors comprising the promoter region of APM1 or a
sequence encoding the APM1 protein, as well as cell hosts
comprising said nucleic acid sequences or recombinant vectors. The
invention also encompasses methods of screening of molecules which
modulate or inhibit the expression of the APM1 gene. The invention
is also directed to biallelic markers that are located within the
APM1 genomic sequence, these biallelic markers representing useful
tools in order to identify a statistically significant association
between specific alleles of APM1 gene and one or several disorders
related to obesity. Further, the invention relates to the use of
these biallelic marker associations to indicate people at risk for
diseases, including obesity-related diseases, as well as to
identify people who would be candidates or non-candidates for a
drug treatment, or a clinical trial.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows a map of the genomic organization of human Apm1
(Adipose Most Abundant Gene Transcript 1) and the location of the
biallelic markers identified in the application.
[0015] FIGS. 2A, 2B, and 2C are a graphical representation of the
effect of Apm1 polymorphisms on plasma lipid values in obese
adolescent girls. The mean and 99.99% confidence interval are
indicated as a solid and dotted line, respectively.
[0016] FIGS. 3A and 3B are a graphical representation of the effect
of APM1 polymorphism on leptin/BMI relationship in obese adolescent
girls.
[0017] FIGS. 4A and 4B are a graphical representation of the effect
of APM1 polymorphism on FFA in obese adolescents girls.
[0018] FIGS. 5A and 5B are a graphical representation of the effect
of APM1 polymorphism on respiratory quotient in obese
adolescents.
[0019] FIG. 6 is a graphical representation of the effect of APM1
on leptin/BMI ratio in obese adolescents girls.
[0020] FIG. 7 is a graphical representation of the effect of APM1
polymorphism on glucose tolerance in obese adolescent girls.
[0021] FIG. 8 shows Apm1 function predicted from polymorphism and
in vivo analysis.
BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE
LISTING
[0022] SEQ ID NO: 1 contains a genomic sequence of APM1 comprising
the 5' regulatory region (upstream untranscribed region), the exons
and introns, and the 3' regulatory region (downstream untranscribed
region).
[0023] SEQ ID NO: 2 contains a 5' regulatory region (upstream
untranscribed region) of the APM1 gene.
[0024] SEQ ID NO: 3 contains a 3' regulatory region (downstream
untranscribed region) of the APM1 gene.
[0025] SEQ ID NO: 4 contains a partial 5' cDNA of APM1.
[0026] SEQ ID NO: 5 contains a complete human APM1 cDNA.
[0027] SEQ ID NO: 6 contains the APM1 protein encoded by the cDNA
of SEQ ID NO 5.
[0028] SEQ ID NO: 7 contains a primer containing the additional PU
5' sequence described further in Example 2
[0029] SEQ ID NO: 8 contains a primer containing the additional RP
5' sequence described further in Example 2.
[0030] In accordance with the regulations relating to Sequence
Listings, the following codes have been used in the Sequence
Listing to indicate the locations of biallelic markers within the
sequences and to identify each of the alleles present at the
polymorphic base. The code "r" in the sequences indicates that one
allele of the polymorphic base is a guanine, while the other allele
is an adenine. The code "y" in the sequences indicates that one
allele of the polymorphic base is a thymine, while the other allele
is a cytosine. The code "m" in the sequences indicates that one
allele of the polymorphic base is an adenine, while the other
allele is an cytosine. The code "k" in the sequences indicates that
one allele of the polymorphic base is a guanine, while the other
allele is a thymine. The code "s" in the sequences indicates that
one allele of the polymorphic base is a guanine, while the other
allele is a cytosine. The code "w" in the sequences indicates that
one allele of the polymorphic base is an adenine, while the other
allele is an thymine. The nucleotide code of the original allele
for each biallelic marker is the following:
1 Biallelic marker Original allele 9-27-261 G 99-14387-129 A
9-12-48 T 9-12-124 T 9-12-355 G 9-12-428 A 99-14405-105 G 17-30-216
G 9-27-211 A 9-27-246 G 17-31-298 A 17-31-413 T 17-32-24 T
99-14387-50 C 99-14387-199 A 17-33-TGAGACT none 17-34-860 G
17-34-915 G 17-35-71 C 17-35-306 G 17-36-47 G 17-36-120 C 17-37-629
A 17-37-811 G 17-38-349 C
DETAILED DESCRIPTION
[0031] The aim of the present invention is to provide
polynucleotides derived from the APM1 gene, which are particularly
useful to design suitable means for detecting the presence of this
gene in a test sample or alternatively the APM1 mRNA molecules that
are present in a test sample. The present invention also deals with
polynucleotides involved in the expression of the APM1 gene and
which can be used for designing means capable of modulating the
expression of APM1. Other polynucleotides of the invention are
useful to design suitable means to express a desired polynucleotide
of interest. The present invention also encompasses biallelic
markers of the APM1 gene, and their use, based on biallelic marker
association studies, to indicate people at risk for diseases,
including obesity-related diseases, as well as to identify people
who would be candidates or non-candidates for a drug treatment, or
a clinical trial.
[0032] Definitions
[0033] Before describing the invention in greater detail, the
following definitions are set forth to illustrate and define the
meaning and scope of the terms used to describe the invention
herein.
[0034] The term "APM1 gene", when used herein, encompasses genomic,
mRNA and cDNA sequences encoding the APM1 protein, including the
untranslated regulatory regions of the genomic DNA.
[0035] The term "heterologous protein", when used herein, is
intended to designate any protein or polypeptide other than the
APM1 protein. More particularly, the heterologous protein is a
compound which can be used as a marker in further experiments with
a APM1 regulatory region.
[0036] The term "isolated" requires that the material be removed
from its original environment (e.g., the natural environment if it
is naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or DNA or polypeptide,
separated from some or all of the coexisting materials in the
natural system, is isolated. Such polynucleotide could be part of a
vector and/or such polynucleotide or polypeptide could be part of a
composition, and still be isolated in that the vector or
composition is not part of its natural environment.
[0037] Specifically excluded from the definition of "isolated" are:
naturally-occurring chromosomes (such as chromosome spreads),
artificial chromosome libraries, genomic libraries, and cDNA
libraries that exist either as an in vitro nucleic acid preparation
or as a transfected/transformed host cell preparation, wherein the
host cells are either an in vitro heterogeneous preparation or
plated as a heterogeneous population of single colonies. Also
specifically excluded are the above libraries wherein a specified
5' EST makes up less than 5% of the number of nucleic acid inserts
in the vector molecules. Further specifically excluded are whole
cell genomic DNA or whole cell RNA preparations (including said
whole cell preparations which are mechanically sheared or
enzymaticly digested). Further specifically excluded are the above
whole cell preparations as either an in vitro preparation or as a
heterogeneous mixture separated by electrophoresis (including blot
transfers of the same) wherein the polynucleotide of the invention
has not further been separated from the heterologous
polynucleotides in the electrophoresis medium (e.g., further
separating by excising a single band from a heterogeneous band
population in an agarose gel or nylon blot).
[0038] The term "purified" does not require absolute purity;
rather, it is intended as a relative definition. Purification of
starting material or natural material to at least one order of
magnitude, preferably two or three orders, and more preferably four
or five orders of magnitude is expressly contemplated. As an
example, purification from 0.1% concentration to 10% concentration
is two orders of magnitude.
[0039] The term "purified polynucleotide" or "purified
polynucleotide vector" is used herein to describe a polynucleotide
or polynucleotide vector of the invention which has been separated
from other compounds including, but not limited to other nucleic
acids, carbohydrates, lipids and proteins (such as the enzymes used
in the synthesis of the polynucleotide), or the separation of
covalently closed polynucleotides from linear polynucleotides. A
polynucleotide is substantially pure when at least about 50%,
preferably 60 to 75% of a sample exhibits a single polynucleotide
sequence and conformation (linear versus covalently closed). A
substantially pure polynucleotide typically comprises about 50%,
preferably 60 to 90% weight/weight of a nucleic acid sample, more
usually about 95%, and preferably is over about 99% pure.
Polynucleotide purity or homogeneity is indicated by a number of
means well known in the art, such as agarose or polyacrylamide gel
electrophoresis of a sample, followed by visualizing a single
polynucleotide band upon staining the gel. For certain purposes
higher resolution can be provided by using HPLC or other means well
known in the art.
[0040] The term "polypeptide" refers to a polymer of amino without
regard to the length of the polymer; thus, peptides, oligopeptides,
and proteins are included within the definition of polypeptide.
This term also does not specify or exclude post-translation
modifications of polypeptides. For example, polypeptides that
include the covalent attachment of glycosyl groups, acetyl groups,
phosphate groups, lipid groups and the like are expressly
encompassed by the term polypeptide. Also included within the
definition are polypeptides which contain one or more analogs of an
amino acid (including, for example, non-naturally occurring amino
acids, amino acids which only occur naturally in an unrelated
biological system, modified amino acids from mammalian systems,
etc.), polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring.
[0041] The term "recombinant polypeptide" is used herein to refer
to polypeptides that have been artificially designed and which
comprise at least two polypeptide sequences that are not found as
contiguous polypeptide sequences in their initial natural
environment, or to refer to polypeptides which have been expressed
from a recombinant polynucleotide.
[0042] The term "purified polypeptide" is used herein to describe a
polypeptide of the invention that has been separated from other
compounds including, but not limited to nucleic acids, lipids,
carbohydrates and other proteins. A polypeptide is substantially
pure when a sample contains at least about 50%, preferably 60 to
75%, of a single polypeptide sequence. A substantially pure
polypeptide typically comprises about 50%, preferably 60 to 90%,
more preferably 95 to 99% weight/weight of a protein sample.
Polypeptide purity or homogeneity is indicated by a number of means
well known in the art, such as agarose or polyacrylamide gel
electrophoresis of a sample, followed by visualizing polypeptide
bands upon staining the gel. For certain purposes higher resolution
can be provided by using HPLC or other means well known in the
art.
[0043] As used herein, the term "non-human animal" refers to any
non-human vertebrate, birds and more usually mammals, preferably
primates, farm animals such as swine, goats, sheep, donkeys, and
horses, rabbits or rodents, more preferably rats or mice. As used
herein, the term "animal" is used to refer to any vertebrate,
preferable a mammal. Both the terms "animal" and "mammal" expressly
embrace human subjects unless preceded with the term
"non-human".
[0044] As used herein, the term "antibody" refers to a polypeptide
or group of polypeptides which are comprised of at least one
binding domain, where an antibody binding domain is formed from the
folding of variable domains of an antibody molecule to form
three-dimensional binding spaces with an internal surface shape and
charge distribution complementary to the features of an antigenic
determinant of an antigen., which allows an immunological reaction
with the antigen. Antibodies include recombinant proteins
comprising the binding domains, as wells as fragments, including
Fab, Fab', F(ab).sub.2, and F(ab').sub.2 fragments.
[0045] As used herein, an "antigenic determinant" is the portion of
an antigen molecule, in this case an APM1 polypeptide, that
determines the specificity of the antigen-antibody reaction. An
"epitope" refers to an antigenic determinant of a polypeptide. An
epitope can comprise as few as 3 amino acids in a spatial
conformation which is unique to the epitope. Generally an epitope
consists of at least 6 such amino acids, and more usually at least
8-10 such amino acids. Methods for determining the amino acids
which make up an epitope include x-ray crystallography,
2-dimensional nuclear magnetic resonance, and epitope mapping e.g.
he Pepscan method described by H. Mario Geysen et al. 1984. Proc.
Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO
84/03564; and PCT Publication No. WO 84/03506.
[0046] Throughout the present specification, the expression
"nucleotide sequence" may be employed to designate indifferently a
polynucleotide or a nucleic acid. More precisely, the expression
"nucleotide sequence" encompasses the nucleic material itself and
is thus not restricted to the sequence information (i.e. the
succession of letters chosen among the four base letters) that
biochemically characterizes a specific DNA or RNA molecule.
[0047] As used interchangeably herein, the terms "nucleic acids",
"oligonucleotides", and "polynucleotides" include RNA, DNA, or
RNA/DNA hybrid sequences of more than one nucleotide in either
single chain or duplex form. The term "nucleotide" as used herein
as an adjective to describe molecules comprising RNA, DNA, or
RNA/DNA hybrid sequences of any length in single-stranded or duplex
form. The term "nucleotide" is also used herein as a noun to refer
to individual nucleotides or varieties of nucleotides, meaning a
molecule, or individual unit in a larger nucleic acid molecule,
comprising a purine or pyrimidine, a ribose or deoxyribose sugar
moiety, and a phosphate group, or phosphodiester linkage in the
case of nucleotides within an oligonucleotide or polynucleotide.
The term "nucleotide" is also used herein to encompass "modified
nucleotides" which comprise at least one of the following
modifications: (a) an alternative linking group, (b) an analogous
form of purine, (c) an analogous form of pyrimidine, or (d) an
analogous sugar. For examples of analogous linking groups, purine,
pyrimidines, and sugars, see for example PCT publication No. WO
95/04064. The polynucleotide sequences of the invention may be
prepared by any known method, including synthetic, recombinant, ex
vivo generation, or a combination thereof, as well as utilizing any
purification methods known in the art.
[0048] A "promoter" refers to a DNA sequence recognized by the
synthetic machinery of the cell required to initiate the specific
transcription of a gene.
[0049] A sequence which is "operably linked" to a regulatory
sequence such as a promoter means that said regulatory element is
in the correct location and orientation in relation to the nucleic
acid to control RNA polymerase initiation and expression of the
nucleic acid of interest. As used herein, the term "operably
linked" refers to a linkage of polynucleotide elements in a
functional relationship. For instance, a promoter or enhancer is
operably linked to a coding sequence if it affects the
transcription of the coding sequence. More precisely, two DNA
molecules (such as a polynucleotide containing a promoter region
and a polynucleotide encoding a desired polypeptide or
polynucleotide) are said to be "operably linked" if the nature of
the linkage between the two polynucleotides does not (1) result in
the introduction of a frame-shift mutation or (2) interfere with
the ability of the polynucleotide containing the promoter to direct
the transcription of the coding polynucleotide.
[0050] The term "primer" denotes a specific oligonucleotide
sequence which is complementary to a target nucleotide sequence and
used to hybridize to the target nucleotide sequence. A primer
serves as an initiation point for nucleotide polymerization
catalyzed by either DNA polymerase, RNA polymerase or reverse
transcriptase.
[0051] The term "probe" denotes a defined nucleic acid segment (or
nucleotide analog segment, e.g., polynucleotide as defined
hereinbelow) which can be used to identify a specific
polynucleotide sequence present in samples, said nucleic acid
segment comprising a nucleotide sequence complementary to the
specific polynucleotide sequence to be identified.
[0052] The terms "trait" and "phenotype" are used interchangeably
herein and refer to any visible, detectable or otherwise measurable
property of an organism such as symptoms of, or susceptibility to a
disease for example. Typically the terms "trait" or "phenotype" are
used herein to refer to symptoms of, or susceptibility to, either
obesity or disorders related to obesity, more particularly
atherosclerosis, insulin resistance, hypertension, hyperlipidemia,
hypertriglyceridemia, cardiovascular disease, microangiopathic in
obese individuals with Type II diabetes, ocular lesions associated
with microangiopathy in obese individuals with Type II diabetes,
renal lesions associated with microangiopathy in obese individuals
with Type II diabetes, and Syndrome X
[0053] The term "allele" is used herein to refer to variants of a
nucleotide ssequence. A biallelic polymorphism has two forms.
Typically the first identified allele is designated as the original
allele whereas other alleles are designated as alternative alleles.
Diploid organisms may be homozygous or heterozygous for an allelic
form.
[0054] The term "heterozygosity rate" is used herein to refer to
the incidence of individuals in a population which are heterozygous
at a particular allele. In a biallelic system, the heterozygosity
rate is on average equal to 2P.sub.a(1-P.sub.a), where P.sub.a is
the frequency of the least common allele. In order to be useful in
genetic studies, a genetic marker should have an adequate level of
heterozygosity to allow a reasonable probability that a randomly
selected person will be heterozygous.
[0055] The term "genotype" as used herein refers to the identity of
the alleles present in an individual or a sample. In the context of
the present invention, a genotype preferably refers to the
description of the biallelic marker alleles present in an
individual or a sample. The term "genotyping" a sample or an
individual for a biallelic marker consists of determining the
specific allele or the specific nucleotide carried by an individual
at a biallelic marker.
[0056] The term "mutation" as used herein refers to a difference in
DNA sequence between or among different genomes or individuals
which has a frequency below 1%.
[0057] The term "haplotype" refers to a combination of alleles
present in an individual or a sample. In the context of the present
invention, a haplotype preferably refers to a combination of
biallelic marker alleles found in a given individual and which may
be associated with a phenotype.
[0058] The term "polymorphism" as used herein refers to the
occurrence of two or more alternative genomic sequences or alleles
between or among different genomes or individuals. "Polymorphic"
refers to the condition in which two or more variants of a specific
genomic sequence can be found in a population. A "polymorphic site"
is the locus at which the variation occurs. A single nucleotide
polymorphism is the replacement of one nucleotide by another
nucleotide at the polymorphic site. Deletion of a single nucleotide
or insertion of a single nucleotide also gives rise to single
nucleotide polymorphisms. In the context of the present invention,
"single nucleotide polymorphism" preferably refers to a single
nucleotide substitution. Typically, between different individuals,
the polymorphic site may be occupied by two different
nucleotides.
[0059] The term "biallelic polymorphism" and "biallelic marker" are
used interchangeably herein to refer to a single nucleotide
polymorphism having two alleles at a fairly high frequency in the
population. A "biallelic marker allele" refers to the nucleotide
variants present at a biallelic marker site. Typically, the
frequency of the less common allele of the biallelic markers of the
present invention has been validated to be greater than 1%,
preferably the frequency is greater than 10%, more preferably the
frequency is at least 20% (i.e. heterozygosity rate of at least
0.32), even more preferably the frequency is at least 30% (i.e.
heterozygosity rate of at least 0.42). A biallelic marker wherein
the frequency of the less common allele is 30% or more is termed a
"high quality biallelic marker".
[0060] The location of nucleotides in a polynucleotide with respect
to the center of the polynucleotide are described herein in the
following manner. When a polynucleotide has an odd number of
nucleotides, the nucleotide at an equal distance from the 3' and 5'
ends of the polynucleotide is considered to be "at the center" of
the polynucleotide, and any nucleotide immediately adjacent to the
nucleotide at the center, or the nucleotide at the center itself is
considered to be "within 1 nucleotide of the center." With an odd
number of nucleotides in a polynucleotide any of the five
nucleotides positions in the middle of the polynucleotide would be
considered to be within 2 nucleotides of the center, and so on.
When a polynucleotide has an even number of nucleotides, there
would be a bond and not a nucleotide at the center of the
polynucleotide. Thus, either of the two central nucleotides would
be considered to be "within 1 nucleotide of the center" and any of
the four nucleotides in the middle of the polynucleotide would be
considered to be "within 2 nucleotides of the center", and so on.
For polymorphisms which involve the substitution, insertion or
deletion of 1 or more nucleotides, the polymorphism, allele or
biallelic marker is "at the center" of a polynucleotide if the
difference between the distance from the substituted, inserted, or
deleted polynucleotides of the polymorphism and the 3' end of the
polynucleotide, and the distance from the substituted, inserted, or
deleted polynucleotides of the polymorphism and the 5' end of the
polynucleotide is zero or one nucleotide. If this difference is 0
to 3, then the polymorphism is considered to be "within 1
nucleotide of the center." If the difference is 0 to 5, the
polymorphism is considered to be "within 2 nucleotides of the
center." If the difference is 0 to 7, the polymorphism is
considered to be "within 3 nucleotides of the center," and so
on.
[0061] The term "upstream" is used herein to refer to a location
which is toward the 5' end of the polynucleotide from a specific
reference point.
[0062] The terms "base paired" and "Watson & Crick base paired"
are used interchangeably herein to refer to nucleotides which can
be hydrogen bonded to one another by virtue of their sequence
identities in a manner like that found in double-helical DNA with
thymine or uracil residues linked to adenine residues by two
hydrogen bonds and cytosine and guanine residues linked by three
hydrogen bonds (See Stryer, L., Biochemistry, 4.sup.th edition,
1995).
[0063] The terms "complementary" or "complement thereof" are used
herein to refer to the sequences of polynucleotides that are
capable of forming Watson & Crick base pairing with another
specified polynucleotide throughout the entirety of the
complementary region. For the purpose of the present invention, a
first polynucleotide is deemed to be complementary to a second
polynucleotide when each base in the first polynucleotide is paired
with its complementary base. Complementary bases are, generally, A
and T (or A and U), or C and G. "Complement" is used herein as a
synonym from "complementary polynucleotide", "complementary nucleic
acid" and "complementary nucleotide sequence". These terms are
applied to pairs of polynucleotides based solely upon their
sequences and not any particular set of conditions under which the
two polynucleotides would actually bind.
[0064] The term "non-genic" is used herein to describe APM1-related
biallelic markers, as well as polynucleotides and primers which
occur outside the nucleotide positions shown in the human APM1
genomic sequence of SEQ ID NO: 1. The term "genic" is used herein
to describe APM1-related biallelic markers as well as
polynucleotides and primers which do occur in the nucleotide
positions shown in the human APM1 genomic sequence of SEQ ID NO:
1.
[0065] Variants and Fragments
[0066] The invention also relates to variants and fragments of the
polynucleotides described herein, particularly of a APM1 gene
containing one or more biallelic markers according to the
invention.
[0067] Variants of polynucleotides, as the term is used herein, are
polynucleotides that differ from a reference polynucleotide. A
variant of a polynucleotide may be a naturally occurring variant
such as a naturally occurring allelic variant, or it may be a
variant that is not known to occur naturally. Such non-naturally
occurring variants of the polynucleotide may be made by mutagenesis
techniques, including those applied to polynucleotides, cells or
organisms. Generally, differences are limited so that the
nucleotide sequences of the reference and the variant are closely
similar overall and, in many regions, identical.
[0068] Variants of polynucleotides according to the invention
include, without being limited to, nucleotide sequences which are
at least 95% identical to a polynucleotide selected from the group
consisting of SEQ ID Nos 1-4, or to any polynucleotide fragment of
at least 8 consecutive nucleotides of a polynucleotide selected
from the group consisting of SEQ ID Nos 1-3, and preferably at
least 99% identical, more preferably at least 99.5% identical, and
most preferably at least 99.8% identical to a polynucleotide
selected from the group consisting of SEQ ID Nos 1-5 or to any
polynucleotide fragment of at least 8 consecutive nucleotides of a
polynucleotide selected from the group consisting of SEQ ID Nos
1-3.
[0069] Nucleotide changes present in a variant polynucleotide may
be silent, which means that they do not alter the amino acids
encoded by the polynucleotide. However, nucleotide changes may also
result in amino acid substitutions, additions, deletions, fusions
and truncations in the polypeptide encoded by the reference
sequence. The substitutions, deletions or additions may involve one
or more nucleotides. The variants may be altered in coding or
non-coding regions or both. Alterations in the coding regions may
produce conservative or non-conservative amino acid substitutions,
deletions or additions.
[0070] In the context of the present invention, particularly
preferred embodiments are those in which the polynucleotides encode
polypeptides that increase or retain substantially the same
biological function or activity as the mature APM1 protein, or
those in which the polynucleotides encode polypeptides that
maintain or increase a particular biological activity, while
reducing or maintaining a second biological activity
[0071] A polynucleotide fragment is a polynucleotide having a
sequence that is entirely the same as part but not all of a given
nucleotide sequence, preferably the nucleotide sequence of a APM1
gene, and variants thereof. The fragment can be a portion of an
intron of a APM1 gene. It can also be a portion of the regulatory
regions of APM1, preferably of the promoter sequence of the APM1
gene. Preferably, such fragments comprise at least one of the
biallelic markers A1 to A26 or the complements thereto or a
biallelic marker in linkage disequilibrium with one or more of the
biallelic markers A1 to A26.
[0072] Such fragments may be "free-standing", i.e. not part of or
fused to other polynucleotides, or they may be comprised within a
single larger polynucleotide of which they form a part or region.
Indeed, several of these fragments may be present within a single
larger polynucleotide.
[0073] Optionally, such fragments may consist of, or consist
essentially of a contiguous span of at least 8, 10, 12, 15, 18, 20,
25, 35, 40, 50, 70, 80, 100, 250, 500 or 1000 nucleotides in
length. A set of preferred fragments contain at least one of the
biallelic markers A1 to A26 of the APM1 gene which are described
herein or the complements thereto.
[0074] Identity Between Nucleic Acids Or Polypeptides
[0075] The terms "percentage of sequence identity" and "percentage
homology" are used interchangeably herein to refer to comparisons
among polynucleotides and polypeptides, and are determined by
comparing two optimally aligned sequences over a comparison window,
wherein the portion of the polynucleotide or polypeptide sequence
in the comparison window may comprise additions or deletions (i.e.,
gaps) as compared to the reference sequence (which does not
comprise additions or deletions, although gaps may result where the
other sequence contains additions) for optimal alignment of the two
sequences. The percentage is calculated by determining the number
of positions at which the identical nucleic acid base or amino acid
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison and multiplying the
result by 100 to yield the percentage of sequence identity.
Homology is evaluated using any of the variety of sequence
comparison algorithms and programs known in the art. Such
algorithms and programs include, but are by no means limited to,
TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,
1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al.,
1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic
Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol.
266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410;
Altschul et al., 1993, Nature Genetics 3:266-272). In a
particularly preferred embodiment, protein and nucleic acid
sequence homologies are evaluated using the Basic Local Alignment
Search Tool ("BLAST") which is well known in the art (see, e.g.,
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268;
Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,
1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids
Res. 25:3389-3402). In particular, five specific BLAST programs are
used to perform the following task:
[0076] (1) BLASTP and BLAST3 compare an amino acid query sequence
against a protein sequence database;
[0077] (2) BLASTN compares a nucleotide query sequence against a
nucleotide sequence database;
[0078] (3) BLASTX compares the six-frame conceptual translation
products of a query nucleotide sequence (both strands) against a
protein sequence database;
[0079] (4) TBLASTN compares a query protein sequence against a
nucleotide sequence database translated in all six reading frames
(both strands); and
[0080] (5) TBLASTX compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0081] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic acid
sequence and a test sequence which is preferably obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are preferably identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992,
Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins
17:49-61). Less preferably, the PAM or PAM250 matrices may also be
used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for
Detecting Distance Relationships: Atlas of Protein Sequence and
Structure, Washington: National Biomedical Research Foundation).
The BLAST programs evaluate the statistical significance of all
high-scoring segment pairs identified, and preferably selects those
segments which satisfy a user-specified threshold of significance,
such as a user-specified percent homology. Preferably, the
statistical significance of a high-scoring segment pair is
evaluated using the statistical significance formula of Karlin
(see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA
87:2267-2268).
[0082] Stringent Hybridization Conditions
[0083] By way of example and not limitation, procedures using
conditions of high stringency are as follows: Prehybridization of
filters containing DNA is carried out for 8 h to overnight at
65.degree. C. in buffer composed of 6.times. SSC, 50 mM Tris-HCl
(pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500
.mu.g/ml denatured salmon sperm DNA. Filters are hybridized for 48
h at 65.degree. C., the preferred hybridization temperature, in
prehybridization mixture containing 100 .mu.g/ml denatured salmon
sperm DNA and 5-20.times.10.sup.6 cpm of .sup.32P-labeled probe.
Alternatively, the hybridization step can be performed at
65.degree. C. in the presence of SSC buffer, 1.times. SSC
corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently,
filter washes can be done at 37.degree. C. for 1 h in a solution
containing 2.times. SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA,
followed by a wash in 0.1.times. SSC at 50.degree. C. for 45 min.
Alternatively, filter washes can be performed in a solution
containing 2.times. SSC and 0.1% SDS, or 0.5.times. SSC and 0.1%
SDS, or 0.1.times. SSC and 0.1% SDS at 68.degree. C. for 15 minute
intervals. Following the wash steps, the hybridized probes are
detectable by autoradiography. Other conditions of high stringency
which may be used are well known in the art and as cited in
Sambrook et al., 1989; and Ausubel et al., 1989, are incorporated
herein in their entirety. These hybridization conditions are
suitable for a nucleic acid molecule of about 20 nucleotides in
length. The hybridization conditions described above are adapted
according to the length of the desired nucleic acid, following
techniques well known to the one skilled in the art. Hybridization
conditions may, for example, be adapted according to the teachings
disclosed in the book of Hames and Higgins (1985) or in Sambrook et
al. (1989).
[0084] Genomic Sequences of APM1
[0085] The present invention concerns the genomic sequence of APM1.
The present invention encompasses polynucleotides, APM1 genes, or
APM1 genomic sequences consisting of, consisting essentially of, or
comprising the sequence of SEQ ID No 1, a sequence complementary
thereto, as well as fragments and variants thereof. These
polynucleotides may be purified, isolated, or recombinant. This
genomic sequence of APM1 has been localized on locus 3p27 by
FISH.
[0086] The invention also encompasses purified, isolated, or
recombinant polynucleotides comprising a nucleotide sequence having
at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a
nucleotide sequence of SEQ ID No 1 or a complementary sequence
thereto or a fragment thereof. The nucleotide differences as
regards to the nucleotide sequence of SEQ ID No 1 may be generally
randomly distributed throughout the entire nucleic acid.
Nevertheless, preferred nucleic acids are those wherein the
nucleotide differences as regards to the nucleotide sequence of SEQ
ID No 1 are predominantly located outside the coding sequences
contained in the exons. These nucleic acids, as well as their
fragments and variants, may be used as oligonucleotide primers or
probes in order to detect the presence of a copy of the APM1 gene
in a test sample, or alternatively in order to amplify a target
nucleotide sequence within the APM1 sequences.
[0087] Another object of the invention consists of a purified,
isolated, or recombinant nucleic acids that hybridizes with the
nucleotide sequence of SEQ ID No 1 or a complementary sequence
thereto or a variant thereof, under the stringent hybridization
conditions as defined above.
[0088] Particularly preferred nucleic acids of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 1 or the complements thereof, wherein said contiguous
span comprises at least 1, 2, 3, 5, or 10 of the following
nucleotide positions of SEQ ID No 1: 1 to 3528, 4852 to 15143,
15366 to 16276, and 20560 to 20966. Other preferred nucleic acids
of the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises positions 4150 to 4154, or
17169 to 17170 of SEQ ID No: 1. Additional preferred nucleic acids
of the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises a G at position 3787, a G at
position 3809, a T at position 4311, an A at position 4328, an A at
position 4683, or an A at position 15319 of SEQ ID No: 1.
Additional preferred nucleic acids of the invention include
isolated, purified, or recombinant polynucleotides comprising a
contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No: 1
or the complements thereof, wherein said contiguous span comprises
a G at position 15196, a deletion of an A at position 17170, a G at
position 17829, an A at position 18011, and a T at position 18489.
It should be noted that nucleic acid fragments of any size and
sequence may also be comprised by the polynucleotides described in
this section. Other particularly preferred nucleic acids of the
invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises at least 1, 2, 3, 5, or 10
of nucleotide positions 1 to 4833 of SEQ ID No 1.
[0089] The APM1 genomic nucleic acid comprises 3 exons. Exon 1
starts at the nucleotide in position 4812 and ends at the
nucleotide in position 4851 of the nucleotide sequence of SEQ ID No
1; Exon 2 starts at the nucleotide in position 15144 and ends at
the nucleotide in position 15365 of the nucleotide sequence of SEQ
ID No 1; Exon 3 starts at the nucleotide in position 16277 and ends
at the nucleotide in position 20559 of the nucleotide sequence of
SEQ ID No 1. Thus, the invention embodies purified, isolated, or
recombinant polynucleotides comprising a nucleotide sequence
selected from the group consisting of the three exons of the APM1
gene, or a sequence complementary thereto. The invention also deals
with purified, isolated, or recombinant nucleic acids comprising a
combination of at least two exons of the APM1 gene, wherein the
polynucleotides are arranged within the nucleic acid, from the
5'-end to the 3'-end of said nucleic acid, in the same order as in
SEQ ID No 1.
[0090] Intron 1 (nucleotide sequence located between Exon 1 and
Exon 2) starts at the nucleotide in position 4852 of the nucleotide
sequence of SEQ ID No 1 and ends at the nucleotide in position
15143 of the nucleotide sequence of SEQ ID No 1; Intron 2
(nucleotide sequence located between Exon 2 and Exon 3) starts at
the nucleotide in position 15366 and ends at the nucleotide in
position 16276 of the nucleotide sequence of SEQ ID No 1. Thus, the
invention embodies purified, isolated, or recombinant
polynucleotides comprising a nucleotide sequence selected from the
group consisting of Intron 1 and Intron 2 of the APM1 gene, and
sequences complementary thereto.
[0091] While this section is entitled "Genomic Sequences of APM1,"
it should be noted that nucleic acid fragments of any size and
sequence may also be comprised by the polynucleotides described in
this section, including those flanking the genomic sequences of
APM1 on either side and/or between two or more such genomic
sequences.
[0092] APM1 cDNA Sequences
[0093] The expression of the APM1 gene has been shown to lead to
the production of at least one mRNA species with the nucleic acid
sequence set forth in SEQ ID No 5.
[0094] Another object of the invention is a purified, isolated, or
recombinant nucleic acid comprising the nucleotide sequence of SEQ
ID No 5, complementary sequences thereto, as well as allelic
variants, and fragments thereof. Moreover, preferred
polynucleotides of the invention include purified, isolated, or
recombinant APM1 cDNAs consisting of, consisting essentially of, or
comprising the sequence of SEQ ID No: 5. Particularly preferred
embodiments of the invention include isolated, purified, or
recombinant polynucleotides comprising a contiguous span of at
least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
200, 500, or 1000 nucleotides of SEQ ID No: 5 or the complements
thereof, wherein said contiguous span comprises positions selected
from the group consisting of a nucleotide T at the position 93,
positions 1154-1157, a nucleotide G at the position 1997, positions
2083-2086, a nucleotide C at the position 2367, 2456, 2467, 2475,
or 2631, an nucleotide A at the position 2778, positions 2785-2788,
positions 2797-2801, a nucleotide T at the position 3594, a
nucleotide G at the position 3684, positions 3697-3701, positions
4026-4027, a nucleotide T at the position 4053, 4078, 4533 or 4536
of SEQ ID No 5. Alternative preferred embodiments of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 5 or the complements thereof, wherein said contiguous
span comprises positions selected from the group consisting of a
nucleotide G at the position 93, a nucleotide G at the position
1815, a nucleotide A at the position 1997, and a nucleotide T at
position 2475 of SEQ ID NO: 5. Additional particularly preferred
embodiments of the invention include isolated, purified, or
recombinant polynucleotides comprising a contiguous span of at
least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
200, 500, or 1000 nucleotides of SEQ ID No: 5 or the complements
thereof, wherein said contiguous span comprises at least 1, 2, 3,
5, or 10 of nucleotide positions 1 to 22 of SEQ ID No 5.
[0095] The cDNA of SEQ ID No 5 includes a 5'-UTR region starting
from the nucleotide at position 1 and ending at the nucleotide in
position 48 of SEQ ID No 5. The cDNA of SEQ ID No 5 includes a
3'-UTR region starting from the nucleotide at position 785 and
ending at the nucleotide at position 4545 of SEQ ID No 5. At least
two polyadenylation sites are present at position 2937 to 2942 and
position 4525 to 4530 of SEQ ID No 5.
[0096] Consequently, the invention concerns a purified, isolated,
and recombinant nucleic acids comprising a nucleotide sequence of
the 5'UTR of the APM1 cDNA, a sequence complementary thereto, or an
allelic variant thereof.
[0097] The sequence at the 5'-end of this cDNA, more particularly
the nucleotide sequence comprising 1 to 367 of SEQ ID No 5,
corresponds to the nucleotide sequence of a 5'-EST that was
obtained from a human dystrophic muscle cDNA library, and
characterized following the teachings of the PCT Application No WO
96/34981 and of the U.S. patent application Ser. No. 08/905,134
filed on Aug. 1, 1997. Polynucleotides comprising this 5'-EST are
also part of the invention. This 5' EST is set forth in SEQ ID No
4.
[0098] While this section is entitled "APM1 cDNA Sequences," it
should be noted that nucleic acid fragments of any size and
sequence may also be comprised by the polynucleotides described in
this section, including those flanking the genomic sequences of
APM1 and/or between two or more such genomic sequences.
[0099] Regulatory Sequences of APM1
[0100] The genomic sequence of the APM1 gene contains regulatory
sequences both in the non-coding 5'-flanking region and in the
non-coding 3'-flanking region that border the APM1 coding region
containing the three exons of this gene, as well as in the
introns.
[0101] The 5'-regulatory sequence of the APM1 gene comprises the
nucleotide sequence of SEQ ID No 2, and from 1 to 4811 of SEQ ID No
1. This polynucleotide contains the promoter site.
[0102] The 3'-regulatory sequence of the APM1 gene comprises the
nucleotide sequence of SEQ ID No 3, and from 20560 to 20966 of SEQ
ID No 1.
[0103] Polynucleotides derived from the SEQ ID Nos 2 or 3 are
useful in order to detect the presence of at least a copy of a
nucleotide sequence of SEQ ID No 1 or a fragment thereof in a test
sample. They are also useful to express APM1 or a heterologous
protein in cells.
[0104] The promoter activity of the regulatory regions of APM1 can
be assessed as described below.
[0105] Methods to identify relevant biologically active
polynucleotide fragments or variants of SEQ ID Nos 2 and 3, are
known to one with skill in the art, and exemplary methods are
described in Sambrook et al. (Sambrook, 1989). For example, the
presence of a promoter (or other regulatory sequences) in test
sequences can be determined by splicing the test sequences
(fragments or variants of SEQ ID Nos 2 and 3, for example) into a
recombinant vector carrying a marker gene (i.e. beta galactosidase,
chloramphenicol acetyl transferase, etc.) that is expressed only if
a promoter (or other regulatory sequences) is present in the test
sequences. Genomic sequences located upstream of the first exon of
the APM1 gene can be cloned into a suitable promoter reporter
vector, such as the pSEAP-Basic, pSEAP-Enhancer, p.beta.gal-Basic,
p.beta.gal-Enhancer, or pEGFP-1 Promoter Reporter vectors available
from Clontech, or pGL2-basic or pGL3-basic promoterless luciferase
reporter gene vector from Promega. Briefly, each of these promoter
reporter vectors include multiple cloning sites positioned upstream
of a reporter gene encoding a readily assayable protein such as
secreted alkaline phosphatase, luciferase, .beta. galactosidase, or
green fluorescent protein. The sequences upstream from the APM1
coding region are inserted into the cloning sites upstream of the
reporter gene in both orientations and introduced into an
appropriate host cell. The level of reporter protein is assayed and
compared to the level obtained from a vector which lacks an insert
in the cloning site. The presence of an elevated expression level
in the vector containing the insert with respect to the control
vector indicates the presence of a promoter in the insert. If
necessary, the upstream sequences can be cloned into vectors which
contain an enhancer for increasing transcription levels from weak
promoter sequences. A significant level of expression above that
observed with the vector lacking an insert indicates that a
promoter sequence is present in the inserted upstream sequence.
[0106] A promoter sequence within the upstream genomic DNA may be
further defined by constructing nested 5' and/or 3' deletions in
the upstream DNA using conventional techniques such as Exonuclease
III or appropriate restriction endonuclease digestion. The
resulting deletion fragments can be inserted into the promoter
reporter vector to determine whether the deletion has reduced or
obliterated promoter activity, such as described, for example, by
Coles et al. (1998). In this way, the boundaries of the promoters
may be defined. If desired, potential individual regulatory sites
within the promoter may be identified using site directed
mutagenesis or linker scanning to obliterate potential
transcription factor binding sites within the promoter individually
or in combination. The effects of these mutations on transcription
levels may be determined by inserting the mutations into cloning
sites in promoter reporter vectors. This type of assay is
well-known to those skilled in the art and is described in WO
97/17359, U.S. Pat. No. 5,374,544, EP 582 796, U.S. Pat. No.
5,698,389, U.S. Pat. No. 5,643,746, U.S. Pat. No. 5,502,176, and
U.S. Pat. No. 5,266,488, incorporated herein by reference in their
entirety including any drawings, figures, or tables.
[0107] The strength and the specificity of the promoter of the APM1
gene can be assessed through the expression levels of a detectable
polynucleotide operably linked to the APM1 promoter in different
types of cells and tissues. The detectable polynucleotide may be
either a polynucleotide that specifically hybridizes with a
predefined oligonucleotide probe, or a polynucleotide encoding a
detectable protein, including a APM1 polypeptide or a fragment or a
variant thereof. This type of assay is well-known to those skilled
in the art and is described in U.S. Pat. No. 5,502,176, and U.S.
Pat. No. 5,266,488, incorporated herein by reference in their
entirety including any drawings, figures, or tables. In addition,
some of the methods are discussed in more detail below.
[0108] Polynucleotides carrying the regulatory elements located at
the 5' end and at the 3' end of the APM1 coding region may be
advantageously used to control the transcriptional and
translational activity of an heterologous polynucleotide of
interest.
[0109] Thus, the present invention also concerns a purified or
isolated nucleic acid comprising a polynucleotide which is selected
from the group consisting of the nucleotide sequences of SEQ ID Nos
2 and 3, or a sequence complementary thereto or a biologically
active fragment or variant thereof.
[0110] The invention also pertains to a purified or isolated
nucleic acid comprising a polynucleotide having at least 95%
nucleotide identity with a polynucleotide selected from the group
consisting of the nucleotide sequences of SEQ ID Nos 2 and 3,
advantageously 99% nucleotide identity, preferably 99.5% nucleotide
identity and most preferably 99.8% nucleotide identity with a
polynucleotide selected from the group consisting of the nucleotide
sequences of SEQ ID Nos 2 and 3, or a sequence complementary
thereto or a variant thereof or a biologically active fragment
thereof.
[0111] Another object of the invention consists of purified,
isolated or recombinant nucleic acids comprising a polynucleotide
that hybridizes, under the stringent hybridization conditions
defined herein, with a polynucleotide selected from the group
consisting of the nucleotide sequences of SEQ ID Nos 2 and 3, or a
sequence complementary thereto or a variant thereof or a
biologically active fragment thereof.
[0112] Preferred fragments of the nucleic acid of SEQ ID No 2 have
a length of about 1500 or 1000 nucleotides, preferably of about 500
nucleotides, more preferably about 400 nucleotides, even more
preferably 300 nucleotides and most preferably about 200
nucleotides. Preferably the fragments of SEQ ID No 2 are within
positions 1 to 3528.
[0113] Preferred fragments of the nucleic acid of SEQ ID No 3 are
at least 50, 100, 150, 200, 300 or 400 bases in length.
[0114] By "biologically active" polynucleotide derivatives of SEQ
ID Nos 1, 2 and 3 are polynucleotides comprising or alternatively
consisting of a fragment of said polynucleotide which is functional
as a regulatory region for expressing a recombinant polypeptide or
a recombinant polynucleotide in a recombinant cell host. It could
act either as an enhancer or as a repressor.
[0115] For the purpose of the invention, a nucleic acid or
polynucleotide is "functional" as a regulatory region for
expressing a recombinant polypeptide or a recombinant
polynucleotide if said regulatory polynucleotide contains
nucleotide sequences which contain transcriptional and
translational regulatory information, and such sequences are
"operably linked" to nucleotide sequences which encode the desired
polypeptide or the desired polynucleotide.
[0116] The regulatory polynucleotides of the invention may be
prepared from any of the nucleotide sequence of SEQ ID Nos 1, 2,
and 3 by cleavage using suitable restriction enzymes, as described
for example in Sambrook et al. (1989).
[0117] The regulatory polynucleotides may also be prepared by
digestion of any of SEQ ID Nos 1, 2, and 3 by an exonuclease
enzyme, such as Bal31 (Wabiko et al., 1986).
[0118] These regulatory polynucleotides can also be prepared by
nucleic acid chemical synthesis, as described elsewhere in the
specification.
[0119] The regulatory polynucleotides according to the invention
may be part of a recombinant expression vector that may be used to
express a coding sequence in a desired host cell or host organism.
The recombinant expression vectors according to the invention are
described elsewhere in the specification.
[0120] A preferred 5'-regulatory polynucleotide of the invention
includes the 5'-untranslated region (5'-UTR) of the APM1 cDNA, or a
biologically active fragment or variant thereof.
[0121] A preferred 3'-regulatory polynucleotide of the invention
includes the 3'-untranslated region (3'-UTR) of the APM1 cDNA, or a
biologically active fragment or variant thereof.
[0122] A further object of the invention consists of a purified or
isolated nucleic acid comprising:
[0123] a) a nucleic acid comprising a regulatory nucleotide
sequence selected from the group consisting of:
[0124] (i) a nucleotide sequence comprising a polynucleotide of SEQ
ID No 2 or a complementary sequence thereto;
[0125] (ii) a nucleotide sequence comprising a polynucleotide
having at least 95% of nucleotide identity with the nucleotide
sequence of SEQ ID No 2 or a complementary sequence thereto;
[0126] (iii) a nucleotide sequence comprising a polynucleotide that
hybridizes under stringent hybridization conditions with the
nucleotide sequence of SEQ ID No 2 or a complementary sequence
thereto; and
[0127] (iv) a biologically active fragment or variant of the
polynucleotides in (i), (ii) and (iii);
[0128] b) a polynucleotide encoding a desired polypeptide or a
nucleic acid of interest, operably linked to the nucleic acid
defined in (a) above;
[0129] c) Optionally, a nucleic acid comprising a 3'-regulatory
polynucleotide, preferably a 3'-regulatory polynucleotide of the
APM1 gene.
[0130] In a specific embodiment of the nucleic acid defined above,
said nucleic acid includes the 5'-untranslated region (5'-UTR) of
the APM1 cDNA, or a biologically active fragment or variant
thereof.
[0131] In a second specific embodiment of the nucleic acid defined
above, said nucleic acid includes the 3'-untranslated region
(3'-UTR) of the APM1 cDNA, or a biologically active fragment or
variant thereof.
[0132] The regulatory polynucleotide of SEQ ID No 2, and its
biologically active fragments or variants, is operably linked at
the 5'-end of the polynucleotide encoding the desired polypeptide
or polynucleotide.
[0133] The regulatory polynucleotide of SEQ ID No 3, or its
biologically active fragments or variants, is advantageously
operably linked at the 3'-end of the polynucleotide encoding the
desired polypeptide or polynucleotide.
[0134] The desired polypeptide encoded by the above-described
nucleic acid may be of various nature or origin, encompassing
proteins of prokaryotic or eukaryotic origin. Among the
polypeptides expressed under the control of a APM1 regulatory
region are included bacterial, fungal or viral antigens. Also
encompassed are eukaryotic proteins including, but not limited to,
intracellular proteins such as "house keeping" proteins,
membrane-bound proteins such as receptors, and secreted proteins
such as endogenous mediators, for example cytokines. The desired
polypeptide may be the APM1 protein, especially the protein of the
amino acid sequence of SEQ ID No 6, or a fragment or a variant
thereof.
[0135] The nucleic acids encoded by the above-described
polynucleotide, usually an RNA molecule, may be complementary to a
coding polynucleotide, for example to the APM1 coding sequence, and
thus useful as antisense polynucleotides.
[0136] Such a polynucleotide may be included in a recombinant
expression vector in order to express the desired polypeptide or
the desired nucleic acid in host cell or in a host organism.
Suitable recombinant vectors that contain a polynucleotide such as
described hereinbefore are disclosed elsewhere in the
specification.
[0137] Coding Regions
[0138] The APM1 open reading frame is contained in the
corresponding mRNA of SEQ ID No 5. More precisely, the effective
APM1 coding sequence (CDS) includes the region between nucleotide
position 49 (first nucleotide of the ATG codon) and nucleotide
position 783 (end nucleotide of the TGA codon) of SEQ ID No 5. The
present invention also embodies isolated, purified, and recombinant
polynucleotides which encode polypeptides comprising a contiguous
span of at least 6 amino acids, preferably at least 8 or 10 amino
acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100
amino acids of SEQ ID NO: 6, wherein said contiguous span includes
a glutamic acid residue at amino acid position 56 in SEQ ID NO:
6.
[0139] The coding sequence of APM1 may be expressed in a desired
host cell or a desired host organism, when this polynucleotide is
placed under the control of suitable expression signals. The
expression signals may be either the expression signals contained
in the regulatory regions in the APM1 gene of the invention or may
be exogenous regulatory nucleic sequences. Such a polynucleotide,
when placed under suitable expression signals, may also be inserted
in a vector for its expression and/or amplification.
[0140] Polynucleotide Constructs
[0141] The terms "polynucleotide construct" and "recombinant
polynucleotide" are used interchangeably herein to refer to linear
or circular, purified or isolated polynucleotides that have been
artificially designed and which comprise at least two nucleotide
sequences that are not found as contiguous nucleotide sequences in
their initial natural environment.
[0142] DNA Construct That Enables Directing Temporal and Spatial
APM1 Gene Expression in Recombinant Cell Hosts and in Transgenic
Animals.
[0143] In order to study the physiological and phenotypic
consequences of a lack of synthesis of the APM1 protein, both at
the cell level and at the multi cellular organism level, the
invention also encompasses DNA constructs and recombinant vectors
enabling a conditional expression of a specific allele of the APM1
genomic sequence or cDNA and also of a copy of this genomic
sequence or cDNA harboring substitutions, deletions, or additions
of one or more bases as regards to the APM1 nucleotide sequence of
SEQ ID Nos 1 and 5, or a fragment thereof, these base
substitutions, deletions or additions being located either in an
exon, an intron or a regulatory sequence, but preferably in the
5'-regulatory sequence or in an exon of the APM1 genomic sequence
or within the APM1 cDNA of SEQ ID No 5. In a preferred embodiment,
the APM1 sequence comprises a biallelic marker of the present
invention.
[0144] The present invention also embodies recombinant vectors
comprised of isolated, purified, or recombinant polynucleotides
which encode a polypeptides comprising a contiguous span of at
least 6 amino acids, preferably at least 8 or 10 amino acids, more
preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids
of SEQ ID NO: 6, wherein said contiguous span includes a glutamic
acid residue at amino acid position 56 in SEQ ID NO: 6.
Particularly preferred embodiments of the invention include
recombinant vectors comprised of isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 5 or the complements thereof,
wherein said contiguous span comprises positions selected from the
group consisting of a nucleotide T at the position 93, positions
1154-1157, a nucleotide G at the position 1997, positions
2083-2086, a nucleotide C at the position 2367, 2456, 2467, 2475,
or 2631, an nucleotide A at the position 2778, positions 2785-2788,
positions 2797-2801, a nucleotide T at the position 3594, a
nucleotide G at the position 3684, positions 3697-3701, positions
4026-4027, a nucleotide T at the position 4053, 4078, 4533 or 4536
of SEQ ID No 5. Alternative preferred embodiments of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 5 or the complements thereof, wherein said contiguous
span comprises positions selected from the group consisting of a
nucleotide G at the position 93, a nucleotide G at the position
1815, a nucleotide A at the position 1997, and a nucleotide T at
position 2475 of SEQ ID NO: 5. Other particularly preferred
embodiments of the invention include recombinant vectors comprised
of isolated, purified, or recombinant polynucleotides comprising a
contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No: 5
or the complements thereof, wherein said contiguous span comprises
at least 1, 2, 3, 5, or 10 of nucleotide positions 1 to 22 of SEQ
ID No 5. Such embodiments are particularly useful in expression
vectors, and when stably transfected into host cells and animals.
Particularly preferred recombinant vectors of the invention are
comprised of isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 1 or the complements thereof, wherein said contiguous
span comprises at least 1, 2, 3, 5, or 10 of the following
nucleotide positions of SEQ ID No 1: 1 to 3528, 4852 to 15143,
15366 to 16276, and 20560 to 20966. Other preferred recombinant
vectors of the invention are comprised of isolated, purified, or
recombinant polynucleotides comprising a contiguous span of at
least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
200, 500, or 1000 nucleotides of SEQ ID No: 1 or the complements
thereof, wherein said contiguous span comprises positions 4150 to
4154, or 17169 to 17170 of SEQ ID No: 1. Additional preferred
recombinant vectors of the invention are comprised of isolated,
purified, or recombinant polynucleotides comprising a contiguous
span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80,
90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No: 1 or the
complements thereof, wherein said contiguous span comprises a G at
position 3787, a G at position 3809, a T at position 4311, an A at
position 4328, an A at position 4683, or an A at position 15319 of
SEQ ID No: 1. Other particularly preferred recombinant vectors of
the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises at least 1, 2, 3, 5, or 10
of nucleotide positions I to 4833 of SEQ ID No 1.
[0145] A first preferred DNA construct is based on the tetracycline
resistance operon tet from E. coli transposon Tn110 for controlling
the APM1 gene expression, such as described by Gossen et al. (1992,
1995) and Furth et al. (1994). Such a DNA construct contains seven
tet operator sequences from Tn10 (tetop) that are fused to either a
minimal promoter or a 5'-regulatory sequence of the APM1 gene, said
minimal promoter or said APM1 regulatory sequence being operably
linked to a polynucleotide of interest that codes either for a
sense or an antisense oligonucleotide or for a polypeptide,
including a APM1 polypeptide or a peptide fragment thereof. This
DNA construct is functional as a conditional expression system for
the nucleotide sequence of interest when the same cell also
comprises a nucleotide sequence coding for either the wild type
(tTA) or the mutant (rTA) repressor fused to the activating domain
of viral protein VP16 of herpes simplex virus, placed under the
control of a promoter, such as the HCMVIE1 enhancer/promoter or the
MMTV-LTR. Indeed, a preferred DNA construct of the invention
comprises both the polynucleotide containing the tet operator
sequences and the polynucleotide containing a sequence coding for
the tTA or the rTA repressor.
[0146] In a specific embodiment, the conditional expression DNA
construct contains the sequence encoding the mutant tetracycline
repressor rTA, where the expression of the polynucleotide of
interest is silent in the absence of tetracycline and induced in
its presence.
[0147] DNA Constructs Allowing Homologous Recombination:
Replacement Vectors
[0148] A second preferred DNA construct will comprise, from 5'-end
to 3'-end: (a) a first nucleotide sequence that is comprised in the
APM1 genomic sequence; (b) a nucleotide sequence comprising a
positive selection marker, such as the marker for neomycine
resistance (neo); and (c) a second nucleotide sequence that is
comprised in the APM1 genomic sequence, and is located downstream
from the first APM1 nucleotide sequence (a).
[0149] In a preferred embodiment, this DNA construct also comprises
a negative selection marker located upstream from the nucleotide
sequence (a) or downstream from the nucleotide sequence (c).
Preferably, the negative selection marker consists of the thymidine
kinase (tk) gene (Thomas et al., 1986), the hygromycin beta gene
(Te Riele et al., 1990), the hprt gene ( Van der Lugt et al., 1991;
Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene
(Nada et al., 1993; Yagi et al.1990). Preferably, the positive
selection marker is located within a APM1 exon sequence so as to
interrupt the sequence encoding a APM1 protein. These replacement
vectors are described, for example, by Thomas et al. (1986; 1987),
Mansour et al. (1988) and Koller et al. (1992).
[0150] The first and second nucleotide sequences (a) and (c) may be
located within a APM1 regulatory sequence, an intronic sequence, an
exon sequence or a sequence containing both regulatory and/or
intronic and/or exon sequences. The size of the nucleotide
sequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to
10 kb, more preferably from 2 to 6 kb and most preferably from 2 to
4 kb.
[0151] DNA Constructs Allowing Homologous Recombination
[0152] The present invention also encompasses primary, secondary,
and immortalized homologously recombinant host cells of vertebrate
origin, preferably mammalian origin and particularly human origin,
that have been engineered to: a) insert exogenous (heterologous)
polynucleotides into the endogenous chromosomal DNA of a targeted
gene, b) delete endogenous chromosomal DNA, and/or c) replace
endogenous chromosomal DNA with exogenous polynucleotides.
Insertions, deletions, and/or replacements of polynucleotide
sequences may be to the coding sequences of the targeted gene
and/or to regulatory regions, such as promoter and enhancer
sequences, operably associated with the targeted gene.
[0153] The present invention further relates to a method of making
a homologously recombinant host cell in vitro or in vivo, wherein
the expression of a targeted gene not normally expressed in the
cell is altered. Preferably the alteration causes expression of the
targeted gene under normal growth conditions or under conditions
suitable for producing the polypeptide encoded by the targeted
gene. The method comprises the steps of: (a) transfecting the cell
in vitro or in vivo with a polynucleotide construct, the a
polynucleotide construct comprising; (i) a targeting sequence; (ii)
a regulatory sequence and/or a coding sequence; and (iii) an
unpaired splice donor site, if necessary, thereby producing a
transfected cell; and (b) maintaining the transfected cell in vitro
or in vivo under conditions appropriate for homologous
recombination.
[0154] The present invention further relates to a method of
altering the expression of a targeted gene in a cell in vitro or in
vivo wherein the gene is not normally expressed in the cell,
comprising the steps of: (a) transfecting the cell in vitro or in
vivo with a a polynucleotide construct, the a polynucleotide
construct comprising: (i) a targeting sequence; (ii) a regulatory
sequence and/or a coding sequence; and (iii) an unpaired splice
donor site, if necessary, thereby producing a transfected cell; and
(b) maintaining the transfected cell in vitro or in vivo under
conditions appropriate for homologous recombination, thereby
producing a homologously recombinant cell; and (c) maintaining the
homologously recombinant cell in vitro or in vivo under conditions
appropriate for expression of the gene.
[0155] The present invention further relates to a method of making
a polypeptide of the present invention by altering the expression
of a targeted endogenous gene in a cell in vitro or in vivo wherein
the gene is not normally expressed in the cell, comprising the
steps of: a) transfecting the cell in vitro with a a polynucleotide
construct, the a polynucleotide construct comprising: (i) a
targeting sequence; (ii) a regulatory sequence and/or a coding
sequence; and (iii) an unpaired splice donor site, if necessary,
thereby producing a transfected cell; (b) maintaining the
transfected cell in vitro or in vivo under conditions appropriate
for homologous recombination, thereby producing a homologously
recombinant cell; and c) maintaining the homologously recombinant
cell in vitro or in vivo under conditions appropriate for
expression of the gene thereby making the polypeptide.
[0156] The present invention further relates to a a polynucleotide
construct which alters the expression of a targeted gene in a cell
type in which the gene is not normally expressed. This occurs when
a polynucleotide construct is inserted into the chromosomal DNA of
the target cell, wherein the a polynucleotide construct comprises:
a) a targeting sequence; b) a regulatory sequence and/or coding
sequence; and c) an unpaired splice-donor site, if necessary.
Further included are a polynucleotide constructs, as described
above, wherein the construct further comprises a polynucleotide
which encodes a polypeptide and is in-frame with the targeted
endogenous gene after homologous recombination with chromosomal
DNA.
[0157] The compositions may be produced, and methods performed, by
techniques known in the art, such as those described in U.S. Pat.
Nos: 6,054,288; 6,048,729; 6,048,724; 6,048,524; 5,994,127;
5,968,502; 5,965,125; 5,869,239; 5,817,789; 5,783,385; 5,733,761;
5,641,670; 5,580,734 ; International Publication Nos:WO96/2941 1,
WO 94/12650; and scientific articles including 1994; Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989) (the disclosures of
each of which are incorporated by reference in their
entireties).
[0158] DNA Constructs Allowing Homologous Recombination: Cre-LoxP
System.
[0159] These new DNA constructs make use of the site specific
recombination system of the P1 phage. The P1 phage possesses a
recombinase called Cre which interacts specifically with a 34 base
pairs loxP site. The loxP site is composed of two palindromic
sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et
al., 1986). The recombination by the Cre enzyme between two loxP
sites having an identical orientation leads to the deletion of the
DNA fragment.
[0160] The Cre-loxP system used in combination with a homologous
recombination technique has been described by Gu et al. (1993,
1994). Briefly, a nucleotide sequence of interest to be inserted in
a targeted location of the genome harbors at least two loxP sites
in the same orientation and located at the respective ends of a
nucleotide sequence to be excised from the recombinant genome. The
excision event requires the presence of the recombinase (Cre)
enzyme within the nucleus of the recombinant cell host. The
recombinase enzyme may be provided by (a) incubating the
recombinant cell hosts in a culture medium containing this enzyme,
by injecting the Cre enzyme directly into the desired cell, such as
described by Araki et al. (1995), or by lipofection of the enzyme
into the cells, such as described by Baubonis et al. (1993); (b)
transfecting the cell host with a vector comprising the Cre coding
sequence operably linked to a promoter functional in the
recombinant cell host, which promoter being optionally inducible,
said vector being introduced in the recombinant cell host, such as
described by Gu et al. (1993) and Sauer et al. (1988); (c)
introducing in the genome of the cell host a polynucleotide
comprising the Cre coding sequence operably linked to a promoter
functional in the recombinant cell host, which promoter is
optionally inducible, and said polynucleotide being inserted in the
genome of the cell host either by a random insertion event or an
homologous recombination event, such as described by Gu et al.
(1994).
[0161] In a specific embodiment, the vector containing the sequence
to be inserted in the APM1 gene by homologous recombination is
constructed in such a way that selectable markers are flanked by
loxP sites in the same orientation. The selectable markers can be
removed while leaving the APM1 sequences of interest that have been
inserted by an homologous recombination event using the Cre enzyme.
Again, two selectable markers are needed: a positive selection
marker to select for the recombination event and a negative
selection marker to select for the homologous recombination event.
Vectors and methods using the Cre-loxP system are described by Zou
et al. (1 994).
[0162] Thus, a third preferred DNA construct of the invention
comprises, from 5'-end to 3'-end : (a) a first nucleotide sequence
that is comprised in the APM1 genomic sequence; (b) a nucleotide
sequence comprising a polynucleotide encoding a positive selection
marker, said nucleotide sequence comprising additionally two
sequences defining a site recognized by a recombinase, such as a
loxP site, the two sites being placed in the same orientation; and
(c) a second nucleotide sequence that is comprised in the APM1
genomic sequence, and is located on the genome downstream of the
first APM1 nucleotide sequence (a).
[0163] The sequences defining a site recognized by a recombinase,
such as a loxP site, are preferably located within the nucleotide
sequence (b) at suitable locations bordering the nucleotide
sequence for which the conditional excision is sought. In one
specific embodiment, two loxP sites are located at each side of the
positive selection marker sequence in order to allow its excision
at a desired time after the occurrence of the homologous
recombination event.
[0164] In a preferred embodiment of a method using the third DNA
construct described above, the excision of the polynucleotide
fragment bordered by the two sites recognized by a recombinase,
preferably two loxP sites, is performed at a desired time, due to
the presence within the genome of the recombinant host cell of a
sequence encoding the Cre enzyme operably linked to a promoter
sequence, preferably an inducible promoter, more preferably a
tissue-specific promoter sequence, and most preferably a promoter
sequence which is both inducible and tissue-specific, such as
described by Gu et al. (1994).
[0165] The presence of the Cre enzyme within the genome of the
recombinant cell host may be the result of the breeding of two
transgenic animals, the first transgenic animal bearing the
APM1-derived sequence of interest containing the loxP sites as
described above and the second transgenic animal bearing the Cre
coding sequence operably linked to a suitable promoter sequence,
such as described by Gu et al. (1994).
[0166] Spatio-temporal control of the Cre enzyme expression may
also be achieved with an adenovirus based vector that contains the
Cre gene thus allowing infection of cells, or in vivo infection of
organs, for delivery of the Cre enzyme, such as described by Anton
and Graham (1995) and Kanegae et al. (1995).
[0167] The DNA constructs described above may be used to introduce
a desired nucleotide sequence of the invention, preferably a APM1
genomic sequence or a APM1 cDNA sequence, and most preferably an
altered copy of a APM1 genomic or cDNA sequence, within a
predetermined location of the targeted genome, leading either to
the generation of an altered copy of a targeted gene (knock-out
homologous recombination) or to the replacement of a copy of the
targeted gene by another copy sufficiently homologous to allow an
homologous recombination event to occur (knock-in homologous
recombination). In a specific embodiment, the DNA constructs
described above may be used to introduce a APM1 genomic sequence or
a APM1 cDNA sequence comprising at least one biallelic marker of
the present invention, preferably at least one biallelic marker
selected from the group consisting of A1 to A26.
[0168] Nuclear Antisense DNA Constructs
[0169] Other compositions contain a vector of the invention
comprising an oligonucleotide fragment of the nucleic sequence SEQ
ID No 5, preferably a fragment including the start codon of the
APM1 gene, as an antisense tool that inhibits the expression of the
corresponding APM1 gene. Preferred methods using antisense
polynucleotide according to the present invention are described by
Sczakiel et al. (1995) or PCT Application No WO 95/24223, hereby
encorporated by reference herein in their entirety including any
drawings, figures, or tables.
[0170] Preferably, the antisense tools are chosen among the
polynucleotides (15-200 bp long) that are complementary to the
5'end of the APM1 mRNA. In one embodiment, a combination of
different antisense polynucleotides complementary to different
parts of the desired targeted gene are used.
[0171] Preferred antisense polynucleotides of the invention are
complementary to mRNA sequences of APM1 containing either the
translation initiation codon ATG or a splice site. Further
preferred antisense polynucleotides are complementary to a splice
site of APM1 mRNA.
[0172] Preferably, the antisense polynucleotides of the invention
have a 3' polyadenylation signal that has been replaced with a
self-cleaving ribozyme sequence, such that RNA polymerase II
transcripts are produced without poly(A) at their 3' ends. These
antisense polynucleotides are incapable of being exported from the
nucleus (Liu et al. (1994)). In a preferred embodiment, these APM1
antisense polynucleotides also comprise, within the ribozyme
cassette, a histone stem-loop structure to stabilize cleaved
transcripts against 3'-5' exonucleolytic degradation, such as the
structure described by Eckner et al. (1991).
[0173] Oligonucleotide Probes And Primers
[0174] Polynucleotides derived from the APM1 gene are useful in
order to detect the presence of at least a copy of a nucleotide
sequence of SEQ ID No 1, or a fragment, complement, or variant
thereof in a test sample.
[0175] Primers and probes according to the invention consist of
nucleic acids comprising at least 12, 15, 18, 20, 25, 30, 40, 50,
or 100 consecutive nucleotides of a nucleic acid selected from the
group consisting of:
[0176] a) the nucleotide sequence beginning at the nucleotide in
position 1 and ending at the nucleotide in position 4811 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; more particularly, the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 3528 of the nucleotide sequence of SEQ
ID No 1 or a variant thereof or a sequence complementary
thereto;
[0177] b) the nucleotide sequence beginning at the nucleotide in
position 4853 and ending at the nucleotide in position 15143 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0178] c) the nucleotide sequence beginning at the nucleotide in
position 15366 and ending at the nucleotide in position 16276 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0179] d) the nucleotide sequence beginning at the nucleotide in
position 20560 and ending at the nucleotide in position 20966 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0180] e) the nucleotide sequence beginning at the nucleotide in
position 1 and ending at the nucleotide in position 22 of the
nucleotide sequence of SEQ ID No 5 or a variant thereof or a
sequence complementary thereto.
[0181] Thus, the invention also relates to nucleic acid probes
characterized in that they hybridize specifically, under the
stringent hybridization conditions defined above, with a nucleic
acid selected from the group consisting of:
[0182] a) the nucleotide sequence beginning at the nucleotide in
position 1 and ending at the nucleotide in position 4811 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; more particularly, the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 3528 of the nucleotide sequence of SEQ
ID No 1 or a variant thereof or a sequence complementary
thereto;
[0183] b) the nucleotide sequence beginning at the nucleotide in
position 4853 and ending at the nucleotide in position 15143 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0184] c) the nucleotide sequence beginning at the nucleotide in
position 15366 and ending at the nucleotide in position 16276 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0185] d) the nucleotide sequence beginning at the nucleotide in
position 20560 and ending at the nucleotide in position 20966 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0186] e) the nucleotide sequence beginning at the nucleotide in
position 1 and ending at the nucleotide in position 22 of the
nucleotide sequence of SEQ ID No 5 or a variant thereof or a
sequence complementary thereto.
[0187] The formation of stable hybrids depends on the melting
temperature (Tm) of the DNA. The Tm depends on the length of the
primer or probe, the ionic strength of the solution and the G+C
content. The higher the G+C content of the primer or probe, the
higher is the melting temperature because G:C pairs are held by
three H bonds whereas A:T pairs have only two. The GC content in
the probes of the invention usually ranges between 10 and 75%,
preferably between 35 and 60%, and more preferably between 40 and
55%.
[0188] A probe or a primer according to the invention has between 8
and 1000 nucleotides in length, and ranges preferably at least 8,
10, 12, 15, 18 or 20 to 25, 35, 40, 50, 60, 70, 80, 100, 250, 500
or 1000 contiguous nucleotides of the nucleotide sequence of SEQ ID
Nos 1-3, or a variant thereof or a complementary sequence thereto,
or is specified to be at least 12, 15, 18, 20, 25, 35, 40, 50, 60,
70, 80, 100, 250, 500 or 1000 contiguous nucleotides of the
nucleotide sequence of SEQ ID Nos 1-3 or a variant thereof or a
complementary sequence thereto. More particularly, the length of
these probes can range from 8, 10, 15, 20, or 30 to 100
nucleotides, preferably from 10 to 50, more preferably from 15 to
30 nucleotides. Shorter probes tend to lack specificity for a
target nucleic acid sequence and generally require cooler
temperatures to form sufficiently stable hybrid complexes with the
template. Longer probes are expensive to produce and can sometimes
self-hybridize to form hairpin structures. The appropriate length
for primers and probes under a particular set of assay conditions
may be empirically determined by one of skill in the art. A
preferred probe or primer consists of a nucleic acid comprising a
polynucleotide selected from the group of nucleotide sequences
consisting of B1 to B23, C1 to C24, D1 to D26 and E1 to E26.
[0189] Additionally, another preferred embodiment of a probe
according to the invention consists of a nucleic acid comprising a
biallelic marker selected from the group consisting of A1 to A26 or
the complements thereto. Exemplary probes are given in Table 4 in
the Examples.
[0190] Particularly preferred probes and primers of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 1 or the complements thereof, wherein said contiguous
span comprises at least 1, 2, 3, 5, or 10 of the following
nucleotide positions of SEQ ID No 1: 1 to 3528, 4852 to 15143,
15366 to 16276, and 20560 to 20966. Other preferred primers and
probes of the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises positions 4150 to 4154, or
17169 to 17170 of SEQ ID No: 1. Additional preferred primers and
probes of the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 1 or the complements thereof,
wherein said contiguous span comprises a G at position 3787, a G at
position 3809, a T at position 4311, an A at position 4328, an A at
position 4683, or an A at position 15319 of SEQ ID No: 1.
Additional preferred primers and probes of the invention include
isolated, purified, or recombinant polynucleotides comprising a
contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No: 1
or the complements thereof, wherein said contiguous span comprises
a G at position 15196, a deletion of an A at position 17170, a G at
position 17829, an A at position 18011, and a T at position 18489.
Other particularly preferred primers and probes of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 1 or the complements thereof, wherein said contiguous
span comprises at least 1, 2, 3, 5, or 10 of nucleotide positions 1
to 4833 of SEQ ID No 1.
[0191] Another object of the invention is a purified, isolated, or
recombinant primers and probes comprising the nucleotide sequence
of SEQ ID No 5, complementary sequences thereto, as well as allelic
variants, and fragments thereof. Moreover, preferred primers and
probes of the invention include purified, isolated, or recombinant
APM1 cDNAs consisting of, consisting essentially of, or comprising
the sequence of SEQ ID No: 5. Particularly preferred embodiments of
the invention include isolated, purified, or recombinant
polynucleotides comprising a contiguous span of at least 12, 15,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or
1000 nucleotides of SEQ ID No: 5 or the complements thereof,
wherein said contiguous span comprises positions selected from the
group consisting of a nucleotide T at the position 93, positions
1154-1157, a nucleotide G at the position 1997, positions
2083-2086, a nucleotide C at the position 2367, 2456, 2467, 2475,
or 2631, an nucleotide A at the position 2778, positions 2785-2788,
positions 2797-2801, a nucleotide T at the position 3594, a
nucleotide G at the position 3684, positions 3697-3701, positions
4026-4027, a nucleotide T at the position 4053, 4078, 4533 or 4536
of SEQ ID No 5. Alternative preferred embodiments of the invention
include isolated, purified, or recombinant polynucleotides
comprising a contiguous span of at least 12, 15, 18, 20, 25, 30,
35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides
of SEQ ID No: 5 or the complements thereof, wherein said contiguous
span comprises positions selected from the group consisting of a
nucleotide G at the position 93, a nucleotide G at the position
1815, a nucleotide A at the position 1997, and a nucleotide T at
position 2475 of SEQ ID NO: 5. Other particularly preferred
embodiments of the invention include isolated, purified, or
recombinant polynucleotides comprising a contiguous span of at
least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,
200, 500, or 1000 nucleotides of SEQ ID No: 5 or the complements
thereof, wherein said contiguous span comprises at least 1, 2, 3,
5, or 10 of nucleotide positions 1 to 22 of SEQ ID No 5.
[0192] In one embodiment the invention encompasses isolated,
purified, and recombinant polynucleotides consisting of, or
consisting essentially of a contiguous span of 8 to 50 nucleotides
of SEQ ID 1 and the complement thereof, wherein said span includes
an APM1-related biallelic marker in said sequence; optionally,
wherein said APM1-related biallelic marker is selected from the
group consisting of A1, A2, A3, A5, A6, A7, A9, A10, A11, A12, A13,
A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, and A23;
optionally, wherein said APM1-related biallelic marker is selected
from the group consisting of A4 A8, A24, A25 and A26; optionally,
wherein said APM1-related biallelic marker is selected from the
group consisting of A1, A2, and A7 or the group consisting of A4
and A8; optionally, wherein said contiguous span is 18 to 35
nucleotides in length and said biallelic marker is within 4
nucleotides of the center of said polynucleotide; optionally,
wherein said polynucleotide consists of said contiguous span and
said contiguous span is 25 nucleotides in length and said biallelic
marker is at the center of said polynucleotide; optionally, wherein
the 3' end of said contiguous span is present at the 3' end of said
polynucleotide; and optionally, wherein the 3' end of said
contiguous span is located at the 3' end of said polynucleotide and
said biallelic marker is present at the 3' end of said
polynucleotide.
[0193] In another embodiment the invention encompasses isolated,
purified and recombinant polynucleotides consisting of, or
consisting essentially of a contiguous span of 8 to 50 nucleotides
of SEQ ID No: 1 or the complement thereof, wherein the 3' end of
said contiguous span is located at the 3' end of said
polynucleotide, and wherein the 3' end of said polynucleotide is
located within 20 nucleotides upstream of an APM1-related biallelic
marker in said sequence; optionally, wherein said APM1-related
biallelic marker is selected from the group consisting of A1, A2,
A3, A5, A6, A7, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18,
A19, A20, A21, A22, and A23, or wherein said APM1-related biallelic
marker is selected from the group consisting of A4, A8, A24, A25
and A26; optionally, wherein said APM1-related biallelic marker is
selected from the group consisting of A1, A2, and A7 or the group
consisting of A4 and A8; optionally, wherein the 3' end of said
polynucleotide is located 1 nucleotide upstream of said
APM1-related biallelic marker in said sequence; and optionally,
wherein said polynucleotide consists essentially of a sequence
selected from the following sequences: D1, D2, D3, D4, D5, D6, D7,
D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21,
D22, D23, D24, D25, D26, E1, E2, E3, E4, E5, E6, E7, E8, E9, E10,
E11, E12, E13, E14, E15, E16, E17, E18, E19, E20,21, E22, E23, E24,
E25, and E26.
[0194] In a further embodiment, the invention encompasses isolated,
purified, or recombinant polynucleotides consisting of, or
consisting essentially of a sequence selected from the following
sequences: B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13,
B14, B15, B16, B17, B18, B19, B20, B21, B22, B23, C1, C2, C3, C4,
C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18,
C19, C20, C21, C22, C23, and C24.
[0195] In an additional embodiment, the invention encompasses
polynucleotides for use in hybridization assays, sequencing assays,
and allele-specific amplification assays for determining the
identity of the nucleotide at an APM1-related biallelic marker in
SEQ ID No: 1 or the complement thereof, as well as polynucleotides
for use in amplifying segments of nucleotides comprising an
APM1-related biallelic marker in SEQ ID No: 1 or the complement
thereof; optionally, wherein said APM1-related biallelic marker is
selected from the group consisting of A1, A2, A3, A5, A6, A7, A9,
A10, A11, A12, A13, A14, A15, A16, A17A18, A19, A20, A21, A22, and
A23, or wherein said APM1-related biallelic marker is selected from
the group consisting of A4, A8, A24, A25 and A26; optionally,
wherein said APM1-related biallelic marker is selected from the
group consisting of A1, A2, and A7 or the group consisting of A4
and A8.
[0196] The primers and probes can be prepared by any suitable
method, including, for example, cloning and restriction of
appropriate sequences and direct chemical synthesis by a method
such as the phosphodiester method of Narang et al. (1979), the
phosphodiester method of Brown et al. (1979), the
diethylphosphoramidite method of Beaucage et al. (1981) and the
solid support method described in EP 0 707 592. The disclosures of
all these documents are incorporated herein by reference.
[0197] Detection probes are generally nucleic acid sequences or
uncharged nucleic acid analogs such as, for example peptide nucleic
acids which are disclosed in International Patent Application WO
92/20702, and morpholino analogs which are described in U.S. Pat.
Nos. 5,185,444, 5,034,506 and 5,142,047. The probe may have to be
rendered "non-extendable" in that additional dNTPs cannot be added
to the probe. In and of themselves analogs usually are
non-extendable and nucleic acid probes can be rendered
non-extendable by modifying the 3' end of the probe such that the
hydroxyl group is no longer capable of participating in elongation.
For example, the 3' end of the probe can be functionalized with the
capture or detection label to thereby consume or otherwise block
the hydroxyl group. Alternatively, the 3' hydroxyl group can be
cleaved, replaced or modified. U.S. patent application Ser. No.
07/049,061, filed Apr. 19, 1993, describes modifications, which can
be used to render a probe non-extendable.
[0198] Any of the polynucleotides of the present invention can be
labeled, if desired, by incorporating a label detectable by
spectroscopic, photochemical, biochemical, immunochemical, or
chemical means. For example, useful labels include radioactive
substances (.sup.32P, .sup.35S, .sup.3H, .sup.125I), fluorescent
dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene,
digoxigenin) and biotin. Preferably, polynucleotides are labeled at
their 3' and/or 5' ends. Examples of non-radioactive labeling of
nucleic acid fragments are described in French patent No.
FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al
(1988). In addition, the probes according to the present invention
may have structural characteristics such that they allow the signal
amplification, such structural characteristics being, for example,
branched DNA probes as those described by Urdea et al. in 1991 or
in European patent No. EP 0 225 807 (Chiron).
[0199] A label can also be used to capture the primer, so as to
facilitate the immobilization of either the primer or a primer
extension product, such as amplified DNA, on a solid support. A
capture label is attached to the primers or probes and can be a
specific binding member which forms a binding pair with the solid
phase reagent's specific binding member (e.g. biotin and
streptavidin). Therefore, depending upon the type of label carried
by a polynucleotide or a probe, it may be employed to capture or to
detect the target DNA. Further, it will be understood that the
polynucleotides, primers or probes provided herein, may,
themselves, serve as the capture label. For example, in the case
where a solid phase reagent's binding member is a nucleic acid
sequence, it may be selected such that it binds a complementary
portion of a primer or probe to thereby immobilize the primer or
probe to the solid phase. In cases where a polynucleotide probe
itself serves as the binding member, those skilled in the art will
recognize that the probe will contain a sequence or "tail" that is
not complementary to the target. In the case where a polynucleotide
primer itself serves as the capture label, at least a portion of
the primer will be free to hybridize with a nucleic acid on a solid
phase. DNA Labeling techniques are well known to the skilled
technician.
[0200] The probes of the present invention are useful for a number
of purposes. They can be notably used in Southern hybridization to
genomic DNA. The probes can also be used to detect PCR
amplification products. They may also be used to detect mismatches
in the APM1 gene or mRNA using other techniques well-known in the
art.
[0201] Any of the polynucleotides, primers and probes of the
present invention can be conveniently immobilized on a solid
support. Solid supports are known to those skilled in the art and
include the walls of wells of a reaction tray, test tubes,
polystyrene beads, magnetic beads, nitrocellulose strips,
membranes, microparticles such as latex particles, sheep (or other
animal) red blood cells, duracytes and others. The solid support is
not critical and can be selected by one skilled in the art. Thus,
latex particles, microparticles, magnetic or non-magnetic beads,
membranes, plastic tubes, walls of microtiter wells, glass or
silicon chips, sheep (or other suitable animal's) red blood cells
and duracytes are all suitable examples. Suitable methods for
immobilizing nucleic acids on solid phases include ionic,
hydrophobic, covalent interactions and the like. A solid support,
as used herein, refers to any material which is insoluble, or can
be made insoluble by a subsequent reaction. The solid support can
be chosen for its intrinsic ability to attract and immobilize the
capture reagent. Alternatively, the solid phase can retain an
additional receptor which has the ability to attract and immobilize
the capture reagent. The additional receptor can include a charged
substance that is oppositely charged with respect to the capture
reagent itself or to a charged substance conjugated to the capture
reagent. As yet another alternative, the receptor molecule can be
any specific binding member which is immobilized upon (attached to)
the solid support and which has the ability to immobilize the
capture reagent through a specific binding reaction. The receptor
molecule enables the indirect binding of the capture reagent to a
solid support material before the performance of the assay or
during the performance of the assay. The solid phase thus can be a
plastic, derivatized plastic, magnetic or non-magnetic metal, glass
or silicon surface of a test tube, microtiter well, sheet, bead,
microparticle, chip, sheep (or other suitable animal's) red blood
cells, duracytes.RTM. and other configurations known to those of
ordinary skill in the art. The polynucleotides of the invention can
be attached to or immobilized on a solid support individually or in
groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct
polynucleotides of the invention to a single solid support. In
addition, polynucleotides other than those of the invention may be
attached to the solid support that one or more polynucleotides of
the invention are attached to.
[0202] Consequently, the invention also deals with a method for
detecting the presence of a nucleic acid comprising a nucleotide
sequence selected from a group consisting of SEQ ID Nos 1-5, a
fragment or a variant thereof or a complementary sequence thereto
in a sample, said method comprising the following steps of:
[0203] a) bringing into contact a nucleic acid probe or a plurality
of nucleic acid probes that can hybridize with a nucleotide
sequence included in a nucleic acid selected from the group
consisting of the nucleotide sequences of SEQ ID Nos 1-5, a
fragment or a variant thereof or a complementary sequence thereto
and the sample to be assayed.
[0204] b) detecting the hybrid complex formed between the probe and
a nucleic acid in the sample.
[0205] In a first preferred embodiment of this detection method,
said nucleic acid probe or the plurality of nucleic acid probes are
labeled with a detectable molecule. In a second preferred
embodiment of said method, said nucleic acid probe or the plurality
of nucleic acid probes has been immobilized on a substrate. In a
third preferred embodiment, the nucleic acid probe or the plurality
of nucleic acid probes comprise either a sequence which is selected
from the group consisting of the nucleotide sequences of SEQ ID Nos
B1 to B23, C1 to C24, D1 to 26 and E1 to E26 or a biallelic marker
selected from the group consisting of A1 to A26 or the complements
thereto.
[0206] The invention further concerns a kit for detecting the
presence of a nucleic acid comprising a nucleotide sequence
selected from a group consisting of SEQ ID Nos 1-5, a fragment or a
variant thereof or a complementary sequence thereto in a sample,
said kit comprising:
[0207] a) a nucleic acid probe or a plurality of nucleic acid
probes that can hybridize with a nucleotide sequence included in a
nucleic acid selected form the group consisting of the nucleotide
sequences of SEQ ID Nos 1-5, a fragment or a variant thereof or a
complementary sequence thereto;
[0208] b) optionally, the reagents necessary for performing the
hybridization reaction.
[0209] In a first preferred embodiment of the detection kit, the
nucleic acid probe or the plurality of nucleic acid probes are
labeled with a detectable molecule. In a second preferred
embodiment of the detection kit, the nucleic acid probe or the
plurality of nucleic acid probes has been immobilized on a
substrate. In a third preferred embodiment of the detection kit,
the nucleic acid probe or the plurality of nucleic acid probes
comprise either a sequence which is selected from the group
consisting of the nucleotide sequences of SEQ ID Nos B1 to B23, C1
to C24, D1 to D26 and E1 to E26 or a biallelic marker selected from
the group consisting of A1 to A26 or the complements thereto.
[0210] Oligonucleotide Arrays
[0211] A substrate comprising a plurality of oligonucleotide
primers or probes of the invention may be used either for detecting
or amplifying targeted sequences in the APM1 gene and may also be
used for detecting mutations in the coding or in the non-coding
sequences of the APM1 gene.
[0212] Any polynucleotide provided herein may be attached in
overlapping areas or at random locations on the solid support.
Alternatively the polynucleotides of the invention may be attached
in an ordered array wherein each polynucleotide is attached to a
distinct region of the solid support which does not overlap with
the attachment site of any other polynucleotide. Preferably, such
an ordered array of polynucleotides is designed to be "addressable"
where the distinct locations are recorded and can be accessed as
part of an assay procedure. Addressable polynucleotide arrays
typically comprise a plurality of different oligonucleotide probes
that are coupled to a surface of a substrate in different known
locations. The knowledge of the precise location of each
polynucleotide makes these "addressable" arrays particularly useful
in hybridization assays. Any addressable array technology known in
the art can be employed with the polynucleotides of the invention.
One particular embodiment of these polynucleotide arrays, known as
Genechips.TM., has been generally described in U.S. Pat. No.
5,143,854 and PCT publications WO 90/15070 and 92/10092. These
arrays may generally be produced using mechanical synthesis methods
or light directed synthesis methods which incorporate a combination
of photolithographic methods and solid phase oligonucleotide
synthesis (Fodor et al., 1991). The immobilization of arrays of
oligonucleotides on solid supports has been rendered possible by
the development of a technology generally identified as "Very Large
Scale Immobilized Polymer Synthesis" (VLSIPS.TM.) in which,
typically, probes are immobilized in a high density array on a
solid surface of a chip. Examples of VLSIPS.TM. technologies are
provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT
Publications WO 90/15070, WO 92/10092 and WO 95/11995, which
describe methods for forming oligonucleotide arrays through
techniques such as light-directed synthesis techniques. In
designing strategies aimed at providing arrays of nucleotides
immobilized on solid supports, further presentation strategies were
developed to order and display the oligonucleotide arrays on the
chips in an attempt to maximize hybridization patterns and sequence
information. Examples of such presentation strategies are disclosed
in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO
97/31256.
[0213] In another embodiment of the oligonucleotide arrays of the
invention, an oligonucleotide probe matrix may advantageously be
used to detect mutations occurring in the APM1 gene and preferably
in its regulatory region. For this particular purpose, probes are
specifically designed to have a nucleotide sequence allowing their
hybridization to the genes that carry known mutations (either by
deletion, insertion or substitution of one or several nucleotides).
By known mutations is meant mutations on the APM1 gene that have
been identified according, for example, to the technique used by
Huang et al. (1996) or Samson et al. (1996).
[0214] Another technique that is used to detect mutations in the
APM1 gene is a high-density DNA array. Each oligonucleotide probe
constituting a unit element of the high density DNA array is
designed to match a specific subsequence of the APM1 genomic DNA or
cDNA. Thus, an array of wild-type Apm1 oligonucleotides
complementary to subsequences of the target gene sequence is used
to determine the identity of the target sequence, measure its
amount, and detect differences between the target sequence and the
reference sequence. In one such design (4L tiled array) uses a set
of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In
each set of four probes, the perfect complement will hybridize more
strongly than mismatched probes. Consequently, a nucleic acid
target of length L is scanned for mutations with a tiled array
containing 4L probes, the whole probe set containing all the
possible mutations in the known wild-type reference sequence. The
hybridization signals of the 15-mer probe set tiled array are
perturbed by a single base change in the target sequence. As a
consequence, there is a characteristic loss of signal or a
"footprint" for the probes flanking a mutation position. This
technique was described by Chee et al. in 1996, which is herein
incorporated by reference.
[0215] Consequently, the invention concerns an array of nucleic
acid molecules comprising at least one polynucleotide described
above as probes and primers. Preferably, the invention concerns an
array of nucleic acid sequencescomprising at least two
polynucleotides described above as probes and primers.
[0216] A further object of the invention consists of an array of
nucleic acid sequences comprising either at least one of the
sequences selected from the group consisting of SEQ ID Nos B1 to
B23, C1 to C24, D1 to D26 and E1 to E26 or the sequences
complementary thereto or a fragment thereof of, or at least 8, 10,
12, 15, 18, 20, 25, 30, or 40 consecutive nucleotides thereof, or
at least one sequence comprising a biallelic marker selected from
the group consisting of A1 to A26 or the complements thereto.
[0217] The invention also pertains to an array of nucleic acid
sequences comprising either at least two of the sequences selected
from the group consisting of SEQ ID Nos B1 to B23, C1 to C24, D1 to
D26 and E1 to E26 or the sequences complementary thereto or a
fragment thereof, or at least 8 consecutive nucleotides thereof, or
at least two sequences comprising a biallelic marker selected from
the group consisting of A1 to A26 or the complements thereto.
[0218] APM1 Proteins and Polypeptide Fragments:
[0219] The term "APM1 polypeptides" is used herein to embrace all
of the proteins and polypeptides of the present invention. Also
forming part of the invention are polypeptides encoded by the
polynucleotides of the invention, as well as fusion polypeptides
comprising such polypeptides. The invention embodies APM1 proteins
from humans, including isolated or purified APM1 proteins
consisting of, consisting essentially of, or comprising the
sequence of SEQ ID NO: 6. It should be noted the APM1 proteins of
the invention are based on the naturally-occurring variant of the
amino acid sequence of human APM1, wherein the aspartic acid
residue of amino acid position 56 has been replaced with a glutamic
acid residue. This variant protein and the fragments thereof which
contain amino acid position 56 of SEQ ID NO: 6 are collectively
referred to herein as "56-Glu variants."
[0220] The present invention embodies isolated, purified, and
recombinant polypeptides comprising a contiguous span of at least 6
amino acids, preferably at least 8 to 10 amino acids, more
preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids
of SEQ ID NO: 6, wherein said contiguous span includes a glutamic
acid residue at amino acid position 56 in SEQ ID NO: 6. In other
preferred embodiments the contiguous stretch of amino acids
comprises the site of a mutation or functional mutation, including
a deletion, addition, swap or truncation of the amino acids in the
APM1 protein sequence.
[0221] APM1 proteins are preferably isolated from human or
mammalian tissue samples or expressed from human or mammalian
genes. The APM1 polypeptides of the invention can be made using
routine expression methods known in the art. The polynucleotide
encoding the desired polypeptide, is ligated into an expression
vector suitable for any convenient host. Both eukaryotic and
prokaryotic host systems are used in forming recombinant
polypeptides, and a summary of some of the more common systems is
provided herein. The polypeptide is then isolated from lysed cells
or from the culture medium and purified to the extent needed for
its intended use. Purification is by any technique known in the
art, for example, differential extraction, salt fractionation,
chromatography, centrifugation, and the like. See, for example,
Methods in Enzymology for a variety of methods for purifying
proteins.
[0222] In addition, shorter protein fragments can be produced by
chemical synthesis. Alternatively the proteins of the invention are
extracted from cells or tissues of humans or non-human animals.
Methods for purifying proteins are known in the art, and include
the use of detergents or chaotropic agents to disrupt particles
followed by differential extraction and separation of the
polypeptides by ion exchange chromatography, affinity
chromatography, sedimentation according to density, and gel
electrophoresis, for example.
[0223] Any APM1 cDNA, including SEQ ID NO: 5, can be used to
express APM1 proteins and polypeptides. The nucleic acid encoding
the APM1 protein or polypeptide to be expressed is operably linked
to a promoter in an expression vector using conventional cloning
technology. The APM1 insert in the expression vector may comprise
the full coding sequence for the APM1 protein or a portion thereof.
For example, the APM1 derived insert may encode a polypeptide
comprising at least 10 consecutive amino acids of the APM1 protein
of SEQ ID NO: 6, where in said consecutive amino acids comprise a
glutamic acid residue in amino acid position 56.
[0224] The expression vector is any of the mammalian, yeast, insect
or bacterial expression systems known in the art. Commercially
available vectors and expression systems are available from a
variety of suppliers including Genetics Institute (Cambridge,
Mass.), Stratagene (La Jolla, Calif.), Promega (Madison, Wis.), and
Invitrogen (San Diego, Calif.). If desired, to enhance expression
and facilitate proper protein folding, the codon context and codon
pairing of the sequence can be optimized for expression in the
organism in which the expression vector is introduced, as explained
by Hatfield, et al., U.S. Pat. No. 5,082,767.
[0225] In one embodiment, the entire coding sequence of the APM1
cDNA through the poly A signal of the cDNA are operably linked to a
promoter in the expression vector. Alternatively, if the nucleic
acid encoding a portion of the APM1 protein lacks a methionine to
serve as the initiation site, an initiating methionine can be
introduced next to the first codon of the nucleic acid using
conventional techniques. Similarly, if the insert from the APM1
cDNA lacks a poly A signal, this sequence can be added to the
construct by, for example, splicing out the Poly A signal from pSG5
(Stratagene) using BglI and SalI restriction endonuclease enzymes
and incorporating it into the mammalian expression vector pXT1
(Stratagene). pXT1 contains the LTRs and a portion of the gag gene
from Moloney Murine Leukemia Virus. The position of the LTRs in the
construct allow efficient stable transfection. The vector includes
the Herpes Simplex Thymidine Kinase promoter and the selectable
neomycin gene. The nucleic acid encoding the APM1 protein or a
portion thereof is obtained by PCR from a bacterial vector
containing the APM1 cDNA of SEQ ID NO: 5. The oligonucleotide
primers used are complementary to the APM1 cDNA, or a portion
thereof, and contain restriction endonuclease sequences for Pst I
incorporated into the 5'primer and BglII at the 5' end of the
corresponding cDNA 3' primer, taking care to ensure that the
sequence encoding the APM1 protein, or portion thereof, is
positioned properly with respect to the poly A signal. The purified
fragment obtained from the resulting PCR reaction is digested with
PstI, blunt ended with an exonuclease, digested with Bgl II,
purified, and ligated to pXT1.
[0226] The ligated product is transfected into mouse NIH 3T3 cells
using Lipofectin (Life Technologies, Inc., Grand Island, N.Y.)
under conditions outlined in the product specification. Positive
transfectants are selected after growing the transfected cells in
600 .mu.g/mL G418 (Sigma, St. Louis, Mo.).
[0227] Alternatively, the nucleic acids encoding the APM1 protein
or a portion thereof are cloned into pED6dpc2 (Genetics Institute,
Cambridge, Mass.). The resulting pED6dpc2 constructs
is.backslash..backslash.are transfected into a suitable host cell,
such as COS 1 cells. Methotrexate resistant cells are selected and
expanded.
[0228] The above procedures may also be used to express a mutant
APM1 protein responsible for a detectable phenotype or a portion
thereof.
[0229] The expressed proteins are purified using conventional
purification techniques such as ammonium sulfate precipitation or
chromatographic separation based on size or charge. The protein
encoded by the nucleic acid insert may also be purified using
standard immunochromatography techniques. In such procedures, a
solution containing the expressed APM1 protein or portion thereof,
such as a cell extract, is applied to a column having antibodies
against the APM1 protein or portion thereof attached to the
chromatography matrix. The expressed protein is allowed to bind to
the immunochromatography column. Thereafter, the column is washed
to remove non-specifically bound proteins. The specifically bound
expressed protein is then released from the column and recovered
using standard techniques.
[0230] To confirm expression of the APM1 protein or a portion
thereof, the proteins expressed in host cells containing an
expression vector containing an insert encoding the APM1 protein or
a portion thereof can be compared to the proteins expressed in host
cells containing the expression vector without an insert. The
presence of a band in samples from cells containing the expression
vector with an insert which is absent in samples from cells
containing the expression vector without an insert indicates that
the APM1 protein or a portion thereof is being expressed.
Generally, the band will have the mobility expected for the APM1
protein or portion thereof. However, the band may have a mobility
different than that expected as a result of modifications such as
glycosylation, ubiquitination, or enzymatic cleavage.
[0231] Antibodies capable of specifically recognizing the expressed
APM1 protein or a portion thereof are described below.
[0232] If antibody production is not possible, the nucleic acids
encoding the APM1 protein or a portion thereof can be incorporated
into expression vectors designed for use in purification schemes
employing chimeric polypeptides. In such strategies, the nucleic
acid encoding the APM1 protein or a portion thereof is inserted in
frame with a gene encoding the other half of the chimera. The other
half of the chimera can be .beta.-globin or a nickel binding
polypeptide encoding sequence, for example. A chromatography matrix
having antibody to .beta.-globin or nickel attached thereto can
then be used to purify the chimeric protein. Protease cleavage
sites are engineered between the .beta.-globin gene or the nickel
binding polypeptide and the APM1 protein or portion thereof. Thus,
the two polypeptides of the chimera are separated from one another
by protease digestion.
[0233] One useful expression vector for generating .beta.-globin
chimerics is pSG5 (Stratagene), which encodes rabbit .beta.-globin.
Intron II of the rabbit .beta.-globin gene facilitates splicing of
the expressed transcript, and the polyadenylation signal
incorporated into the construct increases the level of expression.
These techniques are well known to those skilled in the art of
molecular biology. Standard methods are published in methods texts
such as Davis et al., (Basic Methods in Molecular Biology, L. G.
Davis, M. D. Dibner, and J. F. Battey, ed., Elsevier Press, N.Y.,
1986) and many of the methods are available from Stratagene, Life
Technologies, Inc., or Promega. Polypeptides may additionally be
produced from the construct using in vitro translation systems such
as the In vitro Express.TM. Translation Kit (Stratagene).
[0234] Antibodies That Bind APM1 Polypeptides of the Invention
[0235] Any APM1 polypeptide or whole protein may be used to
generate antibodies capable of specifically binding to expressed
APM1 protein or fragments thereof as described. The antibody
compositions of the invention are capable of specifically binding
or specifically bind to the 56-Glu variant of the APM1 protein. For
an antibody composition to specifically bind to the 56-Glu variant
of APM1 it must demonstrate at least a 5%, 10%, 15%, 20%, 25%, 50%,
or 100% greater binding affinity for the 56-Glu variant of APM1
than for the 56-Asp variant of APM1 in an ELISA, RIA, or other
antibody-based binding assay.
[0236] In a preferred embodiment of the invention antibody
compositions are capable of selectively binding, or selectively
bind to an epitope-containing fragment of a polypeptide comprising
a contiguous span of at least 6 amino acids, preferably at least 8
to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40,
50, or 100 amino acids of SEQ ID NO: 6, wherein said epitope
comprises a glutamic acid residue at amino acid position 56 in SEQ
ID NO: 6, wherein said antibody composition is optionally either
polyclonal or monoclonal.
[0237] The present invention also contemplates the use of
polypeptides comprising a contiguous span of at least 6 amino
acids, preferably at least 8 to 10 amino acids, more preferably at
least 12, 15, 20, 25, 50, or 100 amino acids of a APM1 polypeptide
in the manufacture of antibodies, wherein said contiguous span
comprises a glutamic acid residue at amino acid position 56 of SEQ
ID NO: 6. In a preferred embodiment such polypeptides are useful in
the manufacture of antibodies to detect the presence and absence of
the 56-Glu variant.
[0238] Non-human animals or mammals, whether wild-type or
transgenic, which express a different species of APM1 than the one
to which antibody binding is desired, and animals which do not
express APM1 (i.e. an APM1 knock out animal as described in herein)
are particularly useful for preparing antibodies. APM1 knock out
animals will recognize all or most of the exposed regions of APM1
as foreign antigens, and therefore produce antibodies with a wider
array of APM1 epitopes. Moreover, smaller polypeptides with only 10
to 30 amino acids may be useful in obtaining specific binding to
the 56-Glu variant. In addition, the humoral immune system of
animals which produce a species of APM1 that resembles the
antigenic sequence will preferentially recognize the differences
between the animal's native APM1 species and the antigen sequence,
and produce antibodies to these unique sites in the antigen
sequence. Such a technique will be particularly useful in obtaining
antibodies that specifically bind to the 56-Glu variant.
[0239] Amplification of the APM1 Gene.
[0240] 1. DNA Extraction
[0241] As for the source of the genomic DNA to be subjected to
analysis, almost any test sample can be used without any particular
limitation. These test samples include biological samples which can
be tested by the methods of the present invention described herein
and include human and animal body fluids such as whole blood,
serum, plasma, cerebrospinal fluid, urine, lymph fluids, and
various external secretions of the respiratory, intestinal and
genitourinary tracts, tears, saliva, milk, white blood cells,
myelomas and the like; biological fluids such as cell culture
supernatants; fixed tissue specimens including tumor and non-tumor
tissue and lymph node tissues; bone marrow aspirates and fixed cell
specimens. The preferred source of genomic DNA used in the context
of the present invention is from peripheral venous blood of each
donor.
[0242] The techniques of DNA extraction are well-known to the
technician of ordinary skill in the art. Such techniques are
described notably by Lin et al. (1998) and by Mackey et al.
(1998).
[0243] 2. DNA Amplification
[0244] DNA amplification techniques are well-known to those skilled
in the art. Amplification techniques that can be used in the
context of the present invention include, but are not limited to,
the ligase chain reaction (LCR) described in EP-A-320 308, WO
9320227 and EP-A-439 182, the disclosures of which are incorporated
herein by reference, the polymerase chain reaction (PCR, RT-PCR)
and techniques such as the nucleic acid sequence based
amplification (NASBA) described in Guatelli J. C., et al. (1990)
and in Compton J. (1991), Q-beta amplification as described in
European Patent Application No 4544610, strand displacement
amplification as described in Walker et al. (1996) and EP A 684 315
and, target mediated amplification as described in PCT Publication
WO 9322461, the disclosures of which are incorporated herein by
reference. For amplification of mRNAs, it is within the scope of
the present invention to reverse transcribe mRNA into cDNA followed
by polymerase chain reaction (RT-PCR); or, to use a single enzyme
for both steps as described in U.S. Pat. No. 5,322,770 or, to use
Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al.
(1994). AGLCR is a modification of GLCR that allows the
amplification of RNA.
[0245] The PCR technology is the preferred amplification technique
used in the present invention. A variety of PCR techniques are
familiar to those skilled in the art. For a review of PCR
technology, see White (1997) and the publication entitled "PCR
Methods and Applications" (1991, Cold Spring Harbor Laboratory
Press). In each of these PCR procedures, PCR primers on either side
of the nucleic acid sequences to be amplified are added to a
suitably prepared nucleic acid sample along with dNTPs and a
thermostable polymerase such as Taq polymerase, Pfu polymerase, or
Vent polymerase. The nucleic acid in the sample is denatured and
the PCR primers are specifically hybridized to complementary
nucleic acid sequences in the sample. The hybridized primers are
extended. Thereafter, another cycle of denaturation, hybridization,
and extension is initiated. The cycles are repeated multiple times
to produce an amplified fragment containing the nucleic acid
sequence between the primer sites. PCR has further been described
in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202
and 4,965,188. Each of these publications is incorporated herein by
reference.
[0246] One of the aspects of the present invention is a method for
the amplification of the human APM1 gene, particularly of the
genomic sequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID
No 5, or a fragment or a variant thereof in a test sample,
preferably using the PCR technology. This method comprises the
steps of contacting a test sample suspected of containing the
target APM1 encoding sequence or portion thereof with amplification
reaction reagents comprising a pair of amplification primers, and
eventually in some instances a detection probe that can hybridize
with an internal region of amplicon sequences to confirm that the
desired amplification reaction has taken place.
[0247] Thus, the present invention also relates to a method for the
amplification of a human APM1 gene sequence, particularly of a
portion of the genomic sequences of SEQ ID No 1 or of the cDNA
sequence of SEQ ID No 5, or a variant thereof in a test sample,
said method comprising the steps of:
[0248] a) contacting a test sample suspected of containing the
targeted APM1 gene sequence comprised in a nucleotide sequence
selected from a group consisting of SEQ ID Nos 1 and 5, or
fragments or variants thereof with amplification reaction reagents
comprising a pair of amplification primers as described above and
located on either side of the polynucleotide region to be
amplified, and
[0249] b) optionally, detecting the amplification products.
[0250] In a first preferred embodiment of the above amplification
method, the amplification product is detected by hybridization with
a labeled probe having a sequence which is complementary to the
amplified region. In a second preferred embodiment, the nucleic
acid primers comprise a sequence which is selected from the group
consisting of B1 to B23, C1 to C24, D1 to D26 and E1 to E26. The
primers are more particularly characterized in that they have
sufficient complementarity with any sequence of a strand of the
genomic sequence close to the region to be amplified, for example
with a non-coding sequence adjacent to the exons to be
amplified.
[0251] The invention also concerns a kit for the amplification of a
human APM1 gene sequence, particularly of a portion of the genomic
sequence of SEQ ID No 1 or of the cDNA sequence of SEQ ID No 5, or
a variant thereof in a test sample, wherein said kit comprises
[0252] a) a pair of oligonucleotide primers located on either side
of the APM1 region to be amplified;
[0253] b) optionally, the reagents necessary for performing the
amplification reaction.
[0254] In one embodiment of the above amplification kit, the
amplification product is detected by hybridization with a labeled
probe having a sequence which is complementary to the amplified
region.
[0255] In another embodiment of the above amplification kit,
primers comprise a sequence which is selected from the group
consisting of B1 to B23, C1 to C24, D1 to D26 and E1 to E26.
[0256] APM1-Related Biallelic Markers
[0257] Biallelic markers generally consist of a polymorphism at one
single base position. Each biallelic marker therefore corresponds
to two forms of a polynucleotide sequence which, when compared with
one another, present a nucleotide modification at one position.
Usually, the nucleotide modification involves the substitution of
one nucleotide for another (for example C instead of T).
[0258] Advantages of the Biallelic Markers of the Present
Invention
[0259] The APM1-related biallelic markers of the present invention
offer a number of important advantages over other genetic markers
such as RFLP (Restriction fragment length polymorphism) and VNTR
(Variable Number of Tandem Repeats) markers.
[0260] The first generation of markers, were RFLPs, which are
variations that modify the length of a restriction fragment. But
methods used to identify and to type RFLPs are relatively wasteful
of materials, effort, and time. The second generation of genetic
markers were VNTRs, which can be categorized as either
minisatellites or microsatellites. Minisatellites are tandemly
repeated DNA sequences present in units of 5-50 repeats which are
distributed along regions of the human chromosomes ranging from 0.1
to 20 kilobases in length. Since they present many possible
alleles, their informative content is very high. Minisatellites are
scored by performing Southern blots to identify the number of
tandem repeats present in a nucleic acid sample from the individual
being tested. However, there are only 10.sup.4 potential VNTRs that
can be typed by Southern blotting. Moreover, both RFLP and VNTR
markers are costly and time-consuming to develop and assay in large
numbers.
[0261] Single nucleotide polymorphism or biallelic markers can be
used in the same manner as RFLPs and VNTRs but offer several
advantages. SNP are densely spaced in the human genome and
represent the most frequent type of variation. An estimated number
of more than 10.sup.7 sites are scattered along the
3.times.10.sup.9 base pairs of the human genome. Therefore, SNP
occur at a greater frequency and with greater uniformity than RFLP
or VNTR markers which means that there is a greater probability
that such a marker will be found in close proximity to a genetic
locus of interest. SNP are less variable than VNTR markers but are
mutationally more stable.
[0262] Also, the different forms of a characterized single
nucleotide polymorphism, such as the biallelic markers of the
present invention, are often easier to distinguish and can
therefore be typed easily on a routine basis. Biallelic markers
have single nucleotide based alleles and they have only two common
alleles, which allows highly parallel detection and automated
scoring. The biallelic markers of the present invention offer the
possibility of rapid, high throughput genotyping of a large number
of individuals.
[0263] Biallelic markers are densely spaced in the genome,
sufficiently informative and can be assayed in large numbers. The
combined effects of these advantages make biallelic markers
extremely valuable in genetic studies. Biallelic markers can be
used in linkage studies in families, in allele sharing methods, in
linkage disequilibrium studies in populations, in association
studies of case-control populations or of trait positive and trait
negative populations. An important aspect of the present invention
is that biallelic markers allow association studies to be performed
to identify genes involved in complex traits. Association studies
examine the frequency of marker alleles in unrelated case- and
control-populations and are generally employed in the detection of
polygenic or sporadic traits. Association studies may be conducted
within the general population and are not limited to studies
performed on related individuals in affected families (linkage
studies). Biallelic markers in different genes can be screened in
parallel for direct association with disease or response to a
treatment. This multiple gene approach is a powerful tool for a
variety of human genetic studies as it provides the necessary
statistical power to examine the synergistic effect of multiple
genetic factors on a particular phenotype, drug response, sporadic
trait, or disease state with a complex genetic etiology.
[0264] Candidate Gene of the Present Invention
[0265] Different approaches can be employed to perform association
studies: genome-wide association studies, candidate region
association studies and candidate gene association studies.
Genome-wide association studies rely on the screening of genetic
markers evenly spaced and covering the entire genome. The candidate
gene approach is based on the study of genetic markers specifically
located in genes potentially involved in a biological pathway
related to the trait of interest. In the present invention, APM1 is
the candidate gene. Indeed, the APM1 gene seems to be involved in
obesity and in others disorders linked to obesity. The candidate
gene analysis clearly provides a short-cut approach to the
identification of genes and gene polymorphisms related to a
particular trait when some information concerning the biology of
the trait is available. However, it should be noted that all of the
biallelic markers disclosed in the instant application can be
employed as part of genome-wide association studies or as part of
candidate region association studies and such uses are specifically
contemplated in the present invention and claims.
[0266] APM1-Related Biallelic Markers and Polynucleotides Related
Thereto
[0267] The invention also concerns APM1-related biallelic markers.
As used herein the term "APM1-related biallelic marker" relates to
a set of biallelic markers in linkage disequilibrium with the APM1
gene. The term APM1-related biallelic marker includes the biallelic
markers designated A1 to A26.
[0268] A portion of the biallelic markers of the present invention
are disclosed in Tables A and B. Their location on the APM1 gene is
indicated in Tables A and B and also as a single base polymorphism
in the features of SEQ ID No 1. The pairs of primers allowing the
amplification of a nucleic acid containing the polymorphic base of
one APM1 biallelic marker are listed in Table 1 of Example 2.
2TABLE A List of biallelic markers surrounded by sequence that has
never been previously suggested in the art. Marker Biallelic
Localization in Frequency position in marker Marker Name APM1 gene
Polymorphism Of Allele 2 SEQ ID No 1 A1 9-27/261 5' regulatory
Allele 1: G 3787 region Allele 2: C A2 99-14387/129 Intron 1 Allele
1: A 11118 Allele 2: C A3 9-12/48 Intron 1 Allele 1: T 1.5% 15120
Allele 2: C A5 9-12/355 or Intron 2 Allele 1: G 26% 15427 9-13/297
Allele 2: T A6 9-12/428 or Intron 2 Allele 1: A 11% 15500 9-13/370
Allele 2: G A7 99-14405/105 Intron 2 Allele 1: G 37% 15863 Allele
2: A A9 17-30-216 5' regulatory Allele 1: G 945 region Allele 2: A
A10 9-27-211 5' regulatory Allele 1: A 3738 region Allele 2: G A11
9-27-246 5' regulatory Allele 1: G 3773 region Allele 2: A A12
17-31-298 Intron 1 Allele 1: A 5095 Allele 2: G A13 17-31-413
Intron 1 Allele 1: T 5210 Allele 2: C A14 17-32-24 Intron 1 Allele
1: T 10637 Allele 2: C A15 99-14387-50 Intron 1 Allele 1: C 11039
Allele 2: A A16 99-14387-199 Intron 1 Allele 1: A 11188 Allele 2: G
A17 17-33- Intron 1 Allele 1: no 13973 TGAGACT insert Allele 2:
TGAGACT insert A18 17-34-860 Intron 1 Allele 1: G 14702 Allele 2: A
A19 17-34-915 Intron 1 Allele 1: G 14757 Allele 2: A A20 17-35-71
Intron 1 Allele 1: C 14815 Allele 2: T A21 17-35-306 Intron 1
Allele 1: G 15050 Allele 2: T A22 17-36-47 Intron 2 Allele 1: G
15680 Allele 2: C A23 17-36-120 Intron 2 Allele 1: C 15790 Allele
2: T
[0269]
3TABLE B List of biallelic markers surrounded by previously
suggested sequence, where one allele, allele 2, has never been
previously suggested in the art. The identity of the nucleotide of
the original allele has been previously suggested in the art.
Marker Biallelic Localization in Frequency position in marker
Marker Name APM1 gene Polymorphism Of Allele 2 SEQ ID No 1 A4
9-12/124 or Exon 2 Allele 1: T 11.5% 15196 9-13/66 Allele 2: G A8
9-16/189 Exon 3 Allele 1: A 40% 17170 Allele 2: Del A24 17-37-629
Exon 3 Allele 1: A 17829 Allele 2: G A25 17-37-811 Exon 3 Allele 1:
G 18011 Allele 2: A A26 17-38-349 Exon 3 Allele 1: C 18489 Allele
2: T
[0270] The invention also relates to a purified and/or isolated
nucleotide sequence comprising a polymorphic base of a biallelic
marker located in the sequence of the APM1 gene, preferably of a
biallelic marker selected from the group consisting of A1, A2, A3,
A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17,
A18, A19, A20, A21, A22, A23, A24, A25 and A26, and the complements
thereof; optionally, wherein said APM1-related biallelic marker is
selected from the group consisting of A1, A2, and A7 or the group
consisting of A4 and A8. The sequence has between 8 and 1000
nucleotides in length, and preferably comprises at least 8, 10, 12,
15, 18, 20, 25, 35, 40, 50, 60, 70, 80, 100, 250, 500 or 1000
contiguous nucleotides of a nucleotide sequence selected from the
group consisting of SEQ ID Nos 1 and 5 or a variant thereof or a
complementary sequence thereto. These nucleotide sequences comprise
the polymorphic base of either allele 1 or allele 2 of the
considered biallelic marker. Optionally, said biallelic marker may
be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said
polynucleotide or at the center of said polynucleotide. Optionally,
the 3' end of said contiguous span may be present at the 3' end of
said polynucleotide. Optionally, biallelic marker may be present at
the 3' end of said polynucleotide. Optionally, the 3' end of said
polynucleotide may be located within or at least 2, 4, 6, 8, 10,
12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream
of a biallelic marker of the APM1 gene in said sequence.
Optionally, the 3' end of said polynucleotide may be located 1
nucleotide upstream of a biallelic marker of the APM1 gene in said
sequence. Optionally, said polynucleotide may further comprise a
label. Optionally, said polynucleotide can be attached to solid
support. In a further embodiment, the polynucleotides defined above
can be used alone or in any combination.
[0271] In a preferred embodiment, the sequences comprising a
polymorphic base of one of the biallelic markers listed in Tables A
and B are selected from the group consisting of the nucleotide
sequences that have a contiguous span of, that consist of, that are
comprised in, or that comprise a polynucleotide selected from the
group consisting of the nucleic acid sequences set forth as Nos
9-27, 99-14387, 9-12, 9-13, 99-14405, and 9-16 (listed in Table 1)
or a variant thereof or a complementary sequence thereto.
[0272] The invention further concerns a nucleic acid encoding the
APM1 protein, wherein said nucleic acid comprises a polymorphic
base of a biallelic marker selected from the group consisting of
A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15,
A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and A26 and the
complements thereof; optionally, wherein said APM1-related
biallelic marker is selected from the group consisting of A1, A2,
and A7 or the group consisting of A4 and A8.
[0273] The invention also encompasses the use of any polynucleotide
for, or any polynucleotide for use in, determining the identity of
one or more nucleotides at a APM1-related biallelic marker. In
addition, the polynucleotides of the invention for use in
determining the identity of one or more nucleotides at a
APM1-related biallelic marker encompass polynucleotides with any
further limitation described in this disclosure, or those
following, specified alone or in any combination. Optionally, said
APM1-related biallelic marker may be selected from the group
consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12,
A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and
A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8. Optionally,
said polynucleotide may comprise a sequence disclosed in the
present specification. Optionally, said polynucleotide may consist
of, or consist essentially of any polynucleotide described in the
present specification. Optionally, said determining may be
performed in a hybridization assay, sequencing assay,
microsequencing assay, or allele-specific amplification assay.
Optionally, said polynucleotide may be attached to a solid support,
array, or addressable array. Optionally, said polynucleotide may be
labeled. A preferred polynucleotide may be used in a hybridization
assay for determining the identity of the nucleotide at a biallelic
marker of the APM1 gene. Another preferred polynucleotide may be
used in a sequencing or microsequencing assay for determining the
identity of the nucleotide at a biallelic marker of the APM1 gene.
A third preferred polynucleotide may be used in an allele-specific
amplification assay for determining the identity of the nucleotide
at a biallelic marker of the APM1 gene. A fourth preferred
polynucleotide may be used in amplifying a segment of
polynucleotides comprising a biallelic marker of the APM1 gene.
Optionally, any of the polynucleotides described above may be
attached to a solid support, array, or addressable array.
Optionally, said polynucleotide may be labeled.
[0274] Additionally, the invention encompasses the use of any
polynucleotide for, or any polynucleotide for use in, amplifying a
segment of nucleotides comprising a APM1-related biallelic marker.
In addition, the polynucleotides of the invention for use in
amplifying a segment of nucleotides comprising a APM1-related
biallelic marker encompass polynucleotides with any further
limitation described in this disclosure, or those following,
specified alone or in any combination. Optionally, said
APM1-related biallelic marker may be selected from the group
consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12,
A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and
A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8. Optionally,
said polynucleotide may comprise a sequence disclosed in the
present specification. Optionally, said polynucleotide may consist
of, or consist essentially of any polynucleotide described in the
present specification. Optionally, said amplifying may be performed
by a PCR or LCR. Optionally, said polynucleotide may be attached to
a solid support, array, or addressable array. Optionally, said
polynucleotide may be labeled.
[0275] The primers for amplification or sequencing reaction of a
polynucleotide comprising a biallelic marker of the invention may
be designed from the disclosed sequences for any method known in
the art. A preferred set of primers are fashioned such that the 3'
end of the contiguous span of identity with a sequence selected
from the group consisting of SEQ ID Nos 1 or 5 or a sequence
complementary thereto or a variant thereof is present at the 3' end
of the primer. Such a configuration allows the 3' end of the primer
to hybridize to a selected nucleic acid sequence and dramatically
increases the efficiency of the primer for amplification or
sequencing reactions. Allele specific primers may be designed such
that a polymorphic base of a biallelic marker is at the 3' end of
the contiguous span and the contiguous span is present at the 3'
end of the primer. Such allele specific primers tend to selectively
prime an amplification or sequencing reaction so long as they are
used with a nucleic acid sample that contains one of the two
alleles present at a biallelic marker. The 3' end of the primer of
the invention may be located within or at least 2, 4, 6, 8, 10, 12,
15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of
a biallelic marker of APM1 in said sequence or at any other
location which is appropriate for their intended use in sequencing,
amplification or the location of novel sequences or markers. Thus,
another set of preferred amplification primers comprise an isolated
polynucleotide consisting essentially of a contiguous span of 8 to
50 nucleotides in a sequence selected from the group consisting of
SEQ ID Nos 1 and 5 or a sequence complementary thereto or a variant
thereof, wherein the 3' end of said contiguous span is located at
the 3'end of said polynucleotide, and wherein the 3'end of said
polynucleotide is located upstream of a biallelic marker of the
APM1 gene in said sequence. Preferably, those amplification primers
comprise a sequence selected from the group consisting of the
sequences B1 to B23 and C1 to C24. Primers with their 3' ends
located 1 nucleotide upstream of a biallelic marker of APM1 have a
special utility as microsequencing assays. Preferred
microsequencing primers are described in Table 3. Optionally, the
biallelic marker of the APM1 gene is selected from the group
consisting of A1, A2, A3, A5, A6, A7, A9, A10, A11, A12, A13, A14,
A15, A16, A17, A18, A19, A20, A21, A22, and A23 and the complements
thereof. Optionally, the biallelic marker of the APM1 gene is
selected from the group consisting of A4, A8, A24, A25 and A26 and
the complements thereof, optionally, wherein said APM1-related
biallelic marker is selected from the group consisting of A1, A2,
and A7 or the group consisting of A4 and A8. Optionally,
microsequencing primers are selected from the group consisting of
the nucleotide sequences D1 to D26 and E1 to E26. Alternatively
preferred microsequencing primers are selected from the group
consisting of the nucleotide sequences D3, E4, E5, E6, D7 and
D8.
[0276] The probes of the present invention may be designed from the
disclosed sequences for any method known in the art, particularly
methods which allow for testing if a marker disclosed herein is
present. A preferred set of probes may be designed for use in the
hybridization assays of the invention in any manner known in the
art such that they selectively bind to one allele of a biallelic
marker, but not the other under any particular set of assay
conditions. Preferred hybridization probes comprise the polymorphic
base of either allele 1 or allele 2 of the considered biallelic
marker. Optionally, said biallelic marker may be within 6, 5, 4, 3,
2, or 1 nucleotides of the center of the hybridization probe or at
the center of said probe. Exemplary probes are provided in Table 4
in the Examples.
[0277] The polynucleotides of the present invention are not limited
to having the exact flanking sequences surrounding the polymorphic
bases which are enumerated in the Sequence Listing. The flanking
sequences surrounding the biallelic markers may be lengthened or
shortened to any extent compatible with their intended use and the
present invention specifically contemplates such sequences. The
flanking regions outside of the contiguous span need not be
homologous to native flanking sequences that are known to occur in
human subjects. The addition of any nucleotide sequence that is
compatible with the nucleotides intended use is specifically
contemplated.
[0278] Primers and probes may be labeled or immobilized on a solid
support as described in "Oligonucleotide probes and primers".
[0279] The polynucleotides of the invention which are attached to a
solid support encompass polynucleotides with any further limitation
described in this disclosure, or those following, specified alone
or in any combination. Optionally, said polynucleotides may be
specified as attached individually or in groups of at least 2, 5,
8, 10, 12, 15, 20, or 25 distinct polynucleotides of the invention
to a single solid support. Optionally, polynucleotides other than
those of the invention may attached to the same solid support as
polynucleotides of the invention. Optionally, when multiple
polynucleotides are attached to a solid support they may be
attached at random locations, or in an ordered array. Optionally,
said ordered array may be addressable.
[0280] The invention also pertains to a method of genotyping
comprising determining the identity of a nucleotide at a biallelic
marker of the APM1 gene selected from the group consisting of A1,
A2, A3, A5, A6, A7, A9, A10, A11, A12, A13, A14, A15, A16, A17,
A18, A19, A20, A21, A22, and A23 and the complements thereof in a
biological sample; optionally, wherein said APM1-related biallelic
marker is selected from the group consisting of A1, A2, and A7 or
the group consisting of A4 and A8.
[0281] The invention further deals with a method of genotyping
comprising determining the identity of a nucleotide at an
APM1-related biallelic marker, preferably a biallelic marker of the
APM1 gene selected from the group consisting of A4, A8, A24, A25
and A26 and the complements thereof in a biological sample.
[0282] Optionally, the biological sample is derived from a single
subject. Optionally, the identity of the nucleotides at said
biallelic marker is determined for both copies of said biallelic
marker present in said individual's genome. Optionally, the
biological sample is derived from multiple subjects. Optionally,
the method of genotyping described above further comprises
amplifying a portion of said sequence comprising the biallelic
marker prior to said determining step, for example by a PCR
amplification.
[0283] The determining step of the above genotyping method may be
performed using a hybridization assay, a sequencing assay, an
allele-specific amplification assay or a microsequencing assay.
Thus, the invention also encompasses methods of genotyping a
biological sample comprising determining the identity of a
nucleotide at a APM1-related biallelic marker. In addition, the
genotyping methods of the invention encompass methods with any
further limitation described in this disclosure, or those
following, specified alone or in any combination. Optionally, said
APM1-related biallelic marker may be selected from the group
consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12,
A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and
A26, and the complements thereof, optionally, wherein said APM1
-related biallelic marker is selected from the group consisting of
A1, A2, and A7 or the group consisting of A4 and A8. Optionally,
said biological sample is derived from a single individual or
subject. Optionally, said method is performed in vitro. Optionally,
said biallelic marker is determined for both copies of said
biallelic marker present in said individual's genome. Optionally,
said biological sample is derived from multiple subjects or
individuals. Optionally, said method further comprises amplifying a
portion of said sequence comprising the biallelic marker prior to
said determining step. Optionally, wherein said amplifying is
performed by PCR, LCR, or replication of a recombinant vector
comprising an origin of replication and said portion in a host
cell. Optionally, wherein said determining is performed by a
hybridization assay, sequencing assay, microsequencing assay, or
allele-specific amplification assay.
[0284] The present invention also encompasses diagnostic kits
comprising one or more polynucleotides of the invention with a
portion or all of the necessary reagents and instructions for
genotyping a test subject by determining the identity of a
nucleotide at a biallelic marker of APM1. The polynucleotides of a
kit may optionally be attached to a solid support, or be part of an
array or addressable array of polynucleotides. The kit may provide
for the determination of the identity of the nucleotide at a marker
position by any method known in the art including, but not limited
to, a sequencing assay method, a microsequencing assay method, a
hybridization assay method, or an allele specific amplification
method. Optionally such a kit may include instructions for scoring
the results of the determination with respect to the test subjects'
risk of suffering of obesity or disorders linked to obesity.
[0285] Methods For De Novo Identification Of Biallelic Markers
[0286] Any of a variety of methods can be used to screen a genomic
fragment for single nucleotide polymorphisms such as differential
hybridization with oligonucleotide probes, detection of changes in
the mobility measured by gel electrophoresis or direct sequencing
of the amplified nucleic acid. A preferred method for identifying
biallelic markers involves comparative sequencing of genomic DNA
fragments from an appropriate number of unrelated individuals.
[0287] In a first embodiment, DNA samples from unrelated
individuals are pooled together, following which the genomic DNA of
interest is amplified and sequenced. The nucleotide sequences thus
obtained are then analyzed to identify significant polymorphisms.
One of the major advantages of this method resides in the fact that
the pooling of the DNA samples substantially reduces the number of
DNA amplification reactions and sequencing reactions, which must be
carried out. Moreover, this method is sufficiently sensitive so
that a biallelic marker obtained thereby usually demonstrates a
sufficient frequency of its less common allele to be useful in
conducting association studies.
[0288] In a second embodiment, the DNA samples are not pooled and
are therefore amplified and sequenced individually. This method is
usually preferred when biallelic markers need to be identified in
order to perform association studies within candidate genes.
Preferably, highly relevant gene regions such as promoter regions
or exon regions may be screened for biallelic markers. A biallelic
marker obtained using this method may show a lower degree of
informativeness for conducting association studies, e.g. if the
frequency of its less frequent allele may be less than about 10%.
Such a biallelic marker will, however, be sufficiently informative
to conduct association studies and it will further be appreciated
that including less informative biallelic markers in the genetic
analysis studies of the present invention, may allow in some cases
the direct identification of causal mutations, which may, depending
on their penetrance, be rare mutations.
[0289] The following is a description of the various parameters of
a preferred method used by the inventors for the identification of
the biallelic markers of the present invention.
[0290] Genomic DNA Samples
[0291] The genomic DNA samples from which the biallelic markers of
the present invention are generated are preferably obtained from
unrelated individuals corresponding to a heterogeneous population
of known ethnic background. The number of individuals from whom DNA
samples are obtained can vary substantially, preferably from about
10 to about 1000, preferably from about 50 to about 200
individuals. It is usually preferred to collect DNA samples from at
least about 100 individuals in order to have sufficient polymorphic
diversity in a given population to identify as many markers as
possible and to generate statistically significant results.
[0292] As for the source of the genomic DNA to be subjected to
analysis, any test sample can be foreseen without any particular
limitation. These test samples include biological samples, which
can be tested by the methods of the present invention described
herein, and include human and animal body fluids such as whole
blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and
various external secretions of the respiratory, intestinal and
genitourinary tracts, tears, saliva, milk, white blood cells,
myelomas and the like; biological fluids such as cell culture
supernatants; fixed tissue specimens including tumor and non-tumor
tissue and lymph node tissues; bone marrow aspirates and fixed cell
specimens. The preferred source of genomic DNA used in the present
invention is from peripheral venous blood of each donor. Techniques
to prepare genomic DNA from biological samples are well known to
the skilled technician. Details of a preferred embodiment are
provided in Example 1. The person skilled in the art can choose to
amplify pooled or unpooled DNA samples.
[0293] DNA Amplification
[0294] The identification of biallelic markers in a sample of
genomic DNA may be facilitated through the use of DNA amplification
methods. DNA samples can be pooled or unpooled for the
amplification step. DNA amplification techniques are well known to
those skilled in the art. Various methods to amplify DNA fragments
carrying biallelic markers are further described hereinbefore in
"Amplification of the APM1 gene". The PCR technology is the
preferred amplification technique used to identify new biallelic
markers. A typical example of a PCR reaction suitable for the
purposes of the present invention is provided in Example 2.
[0295] In a first embodiment of the present invention, biallelic
markers are identified using genomic sequence information generated
by the inventors. Sequenced genomic DNA fragments are used to
design primers for the amplification of 500 bp fragments. These 500
bp fragments are amplified from genomic DNA and are scanned for
biallelic markers. Primers may be designed using the OSP software
(Hillier L. and Green P., 1991). All primers may contain, upstream
of the specific target bases, a common oligonucleotide tail that
serves as a sequencing primer. Those skilled in the art are
familiar with primer extensions, which can be used for these
purposes.
[0296] Preferred primers, useful for the amplification of genomic
sequences encoding the candidate genes, focus on promoters, exons
and splice sites of the genes. A biallelic marker presents a higher
probability to be an eventual causal mutation if it is located in
these functional regions of the gene. Preferred amplification
primers of the invention include the nucleotide sequences Nos B1 to
B23 and the nucleotide sequences Nos C1 to C24 disclosed in Example
2.
[0297] Sequencing of Amplified Genomic DNA and Identification of
Single Nucleotide Polymorphisms
[0298] The amplification products generated as described above, are
then sequenced using any method known and available to the skilled
technician. Methods for sequencing DNA using either the
dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method
are widely known to those of ordinary skill in the art. Such
methods are for example disclosed in Sambrook et al. (1989).
Alternative approaches include hybridization to high-density DNA
probe arrays as described in Chee et al. (1996).
[0299] Preferably, the amplified DNA is subjected to automated
dideoxy terminator sequencing reactions using a dye-primer cycle
sequencing protocol. The products of the sequencing reactions are
run on sequencing gels and the sequences are determined using gel
image analysis. The polymorphism search is based on the presence of
superimposed peaks in the electrophoresis pattern resulting from
different bases occurring at the same position. Because each
dideoxy terminator is labeled with a different fluorescent
molecule, the two peaks corresponding to a biallelic site present
distinct colors corresponding to two different nucleotides at the
same position on the sequence. However, the presence of two peaks
can be an artifact due to background noise. To exclude such an
artifact, the two DNA strands are sequenced and a comparison
between the peaks is carried out. In order to be registered as a
polymorphic sequence, the polymorphism has to be detected on both
strands.
[0300] The above procedure permits those amplification products,
which contain biallelic markers to be identified. The detection
limit for the frequency of biallelic polymorphisms detected by
sequencing pools of 100 individuals is approximately 0.1 for the
minor allele, as verified by sequencing pools of known allelic
frequencies. However, more than 90% of the biallelic polymorphisms
detected by the pooling method have a frequency for the minor
allele higher than 0.25. Therefore, the biallelic markers selected
by this method have a frequency of at least 0.1 for the minor
allele and less than 0.9 for the major allele. Preferably at least
0.2 for the minor allele and less than 0.8 for the major allele,
more preferably at least 0.3 for the minor allele and less than 0.7
for the major allele, thus a heterozygosity rate higher than 0.18,
preferably higher than 0.32, more preferably higher than 0.42.
[0301] In another embodiment, biallelic markers are detected by
sequencing individual DNA samples, the frequency of the minor
allele of such a biallelic marker may be less than 0.1.
[0302] Validation of the Biallelic Markers of the Present
Invention
[0303] The polymorphisms are evaluated for their usefulness as
genetic markers by validating that both alleles are present in a
population. Validation of the biallelic markers is accomplished by
genotyping a group of individuals by a method of the invention and
demonstrating that both alleles are present. Microsequencing is a
preferred method of genotyping alleles. The validation by
genotyping step may be performed on individual samples derived from
each individual in the group or by genotyping a pooled sample
derived from more than one individual. The group can be as small as
one individual if that individual is heterozygous for the allele in
question. Preferably the group contains at least three individuals,
more preferably the group contains five or six individuals, so that
a single validation test will be more likely to result in the
validation of more of the biallelic markers that are being tested.
It should be noted, however, that when the validation test is
performed on a small group it may result in a false negative result
if as a result of sampling error none of the individuals tested
carries one of the two alleles. Thus, the validation process is
less useful in demonstrating that a particular initial result is an
artifact, than it is at demonstrating that there is a bona fide
biallelic marker at a particular position in a sequence. All of the
genotyping, haplotyping, association, and interaction study methods
of the invention may optionally be performed solely with validated
biallelic markers.
[0304] Evaluation of the Frequency of the Biallelic Markers of the
Present Invention
[0305] The validated biallelic markers are further evaluated for
their usefulness as genetic markers by determining the frequency of
the least common allele at the biallelic marker site. The higher
the frequency of the less common allele the greater the usefulness
of the biallelic marker is association and interaction studies. The
determination of the least common allele is accomplished by
genotyping a group of individuals by a method of the invention and
demonstrating that both alleles are present. This determination of
frequency by genotyping step may be performed on individual samples
derived from each individual in the group or by genotyping a pooled
sample derived from more than one individual. The group must be
large enough to be representative of the population as a whole.
Preferably the group contains at least 20 individuals, more
preferably the group contains at least 50 individuals, most
preferably the group contains at least 100 individuals. Of course
the larger the group the greater the accuracy of the frequency
determination because of reduced sampling error. For an indication
of the frequency for the less common allele of a particular
biallelic marker of the invention see Table A and B. A biallelic
marker wherein the frequency of the less common allele is 30% or
more is termed a "high quality biallelic marker." All of the
genotyping, haplotyping, association, and interaction study methods
of the invention may optionally be performed solely with high
quality biallelic markers.
[0306] The invention also relates to methods of estimating the
frequency of an allele in a population comprising determining the
proportional representation of a nucleotide at a APM1-related
biallelic marker in said population. In addition, the methods of
estimating the frequency of an allele in a population of the
invention encompass methods with any further limitation described
in this disclosure, or those following, specified alone or in any
combination. Optionally, said APM1-related biallelic marker may be
selected from the group consisting of A1, A2, A3, A4, A5, A6, A7,
A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21,
A22, A23, A24, A25 and A26, and the complements thereof,
optionally, wherein said APM1-related biallelic marker is selected
from the group consisting of A1, A2, and A7 or the group consisting
of A4 and A8. Optionally, determining the proportional
representation of a nucleotide at a APM1-related biallelic marker
may be accomplished by determining the identity of the nucleotides
for both copies of said biallelic marker present in the genome of
each individual in said population and calculating the proportional
representation of said nucleotide at said APM1-related biallelic
marker for the population. Optionally, determining the proportional
representation may be accomplished by performing a genotyping
method of the invention on a pooled biological sample derived from
a representative number of individuals, or each individual, in said
population, and calculating the proportional amount of said
nucleotide compared with the total.
[0307] Methods for Genotyping an Individual for Biallelic
Markers
[0308] Methods are provided to genotype a biological sample for one
or more biallelic markers of the present invention, all of which
may be performed in vitro. Such methods of genotyping comprise
determining the identity of a nucleotide at an APM1 biallelic
marker site by any method known in the art. These methods find use
in genotyping case-control populations in association studies as
well as individuals in the context of detection of alleles of
biallelic markers which are known to be associated with a given
trait, in which case both copies of the biallelic marker present in
individual's genome are determined so that an individual may be
classified as homozygous or heterozygous for a particular
allele.
[0309] These genotyping methods can be performed on nucleic acid
samples derived from a single individual or pooled DNA samples.
[0310] Genotyping can be performed using similar methods as those
described above for the identification of the biallelic markers, or
using other genotyping methods such as those further described
below. In preferred embodiments, the comparison of sequences of
amplified genomic fragments from different individuals is used to
identify new biallelic markers whereas microsequencing is used for
genotyping known biallelic markers in diagnostic and association
study applications.
[0311] In one embodiment the invention encompasses methods of
genotyping comprising determining the identity of a nucleotide at
an APM1-related biallelic marker of SEQ ID No: 1 or the complement
thereof in a biological sample. Optionally, the biological sample
is derived from a single subject. Optionally, the identity of the
nucleotides at said biallelic marker is determined for both copies
of said biallelic marker present in said individual's genome.
Optionally, the biological sample is derived from multiple
subjects. Optionally, the method further comprises amplifying a
portion of said sequence comprising the biallelic marker prior to
said determining step. Optionally, the amplifying step is performed
by PCR. Optionally, the determining step is performed by a
hybridization assay, a sequencing assay, a microsequencing assay,
or an allele-specific amplification assay.
[0312] Source of DNA for Genotyping
[0313] Any source of nucleic acids, in purified or non-purified
form, can be utilized as the starting nucleic acid, provided it
contains or is suspected of containing the specific nucleic acid
sequence desired. DNA or RNA may be extracted from cells, tissues,
body fluids and the like as described above. While nucleic acids
for use in the genotyping methods of the invention can be derived
from any mammalian source, the test subjects and individuals from
which nucleic acid samples are taken are generally understood to be
human.
[0314] Amplification of DNA Fragments Comprising Biallelic
Markers
[0315] Methods and polynucleotides are provided to amplify a
segment of nucleotides comprising one or more biallelic marker of
the present invention. It will be appreciated that amplification of
DNA fragments comprising biallelic markers may be used in various
methods and for various purposes and is not restricted to
genotyping. Nevertheless, many genotyping methods, although not
all, require the previous amplification of the DNA region carrying
the biallelic marker of interest. Such methods specifically
increase the concentration or total number of sequences that span
the biallelic marker or include that site and sequences located
either distal or proximal to it. Diagnostic assays may also rely on
amplification of DNA segments carrying a biallelic marker of the
present invention. Amplification of DNA may be achieved by any
method known in the art. Amplification techniques are described
above in the section entitled, Amplification of the APM1 Gene.
[0316] Some of these amplification methods are particularly suited
for the detection of single nucleotide polymorphisms and allow the
simultaneous amplification of a target sequence and the
identification of the polymorphic nucleotide as it is further
described below.
[0317] The identification of biallelic markers as described above
allows the design of appropriate oligonucleotides, which can be
used as primers to amplify DNA fragments comprising the biallelic
markers of the present invention. Amplification can be performed
using the primers initially used to discover new biallelic markers
which are described herein or any set of primers allowing the
amplification of a DNA fragment comprising a biallelic marker of
the present invention.
[0318] In some embodiments the present invention provides primers
for amplifying a DNA fragment containing one or more biallelic
markers of the present invention. Preferred amplification primers
are listed in Example 2. It will be appreciated that the primers
listed are merely exemplary and that any other set of primers which
produce amplification products containing one or more biallelic
markers of the present invention.
[0319] The spacing of the primers determines the length of the
segment to be amplified. In the context of the present invention,
amplified segments carrying biallelic markers can range in size
from at least about 25 bp to 35 kbp. Amplification fragments from
25-3000 bp are typical, fragments from 50-1000 bp are preferred and
fragments from 100-600 bp are highly preferred. It will be
appreciated that amplification primers for the biallelic markers
may be any sequence which allow the specific amplification of any
DNA fragment carrying the markers. Amplification primers may be
labeled or immobilized on a solid support as described in
"Oligonucleotide probes and primers".
[0320] Methods of Genotyping DNA Samples for Biallelic Markers
[0321] Any method known in the art can be used to identify the
nucleotide present at a biallelic marker site. Since the biallelic
marker allele to be detected has been identified and specified in
the present invention, detection will prove simple for one of
ordinary skill in the art by employing any of a number of
techniques. Many genotyping methods require the previous
amplification of the DNA region carrying the biallelic marker of
interest. While the amplification of target or signal is often
preferred at present, ultrasensitive detection methods which do not
require amplification are also encompassed by the present
genotyping methods. Methods well-known to those skilled in the art
that can be used to detect biallelic polymorphisms include methods
such as, conventional dot blot analyzes, single strand
conformational polymorphism analysis (SSCP) described by Orita et
al. (1989), denaturing gradient gel electrophoresis (DGGE),
heteroduplex analysis, mismatch cleavage detection, and other
conventional techniques as described in Sheffield et al. (1991),
White et al. (1992), Grompe et al. (1989 and 1993). Another method
for determining the identity of the nucleotide present at a
particular polymorphic site employs a specialized
exonuclease-resistant nucleotide derivative as described in U.S.
Pat. No. 4,656,127.
[0322] Preferred methods involve directly determining the identity
of the nucleotide present at a biallelic marker site by sequencing
assay, allele-specific amplification assay, or hybridization assay.
The following is a description of some preferred methods. A highly
preferred method is the microsequencing technique. The term
"sequencing" is used herein to refer to polymerase extension of
duplex primer/template complexes and includes both traditional
sequencing and microsequencing.
[0323] 1) Sequencing Assays
[0324] The nucleotide present at a polymorphic site can be
determined by sequencing methods. In a preferred embodiment, DNA
samples are subjected to PCR amplification before sequencing as
described above. DNA sequencing methods are described in
"Sequencing Of Amplified Genomic DNA And Identification Of Single
Nucleotide Polymorphisms".
[0325] Preferably, the amplified DNA is subjected to automated
dideoxy terminator sequencing reactions using a dye-primer cycle
sequencing protocol. Sequence analysis allows the identification of
the base present at the biallelic marker site.
[0326] 2) Microsequencing Assays
[0327] In microsequencing methods, the nucleotide at a polymorphic
site in a target DNA is detected by a single nucleotide primer
extension reaction. This method involves appropriate
microsequencing primers which, hybridize just upstream of the
polymorphic base of interest in the target nucleic acid. A
polymerase is used to specifically extend the 3' end of the primer
with one single ddNTP (chain terminator) complementary to the
nucleotide at the polymorphic site. Next the identity of the
incorporated nucleotide is determined in any suitable way.
[0328] Typically, microsequencing reactions are carried out using
fluorescent ddNTPs and the extended microsequencing primers are
analyzed by electrophoresis on ABI 377 sequencing machines to
determine the identity of the incorporated nucleotide as described
in EP 412 883. Alternatively capillary electrophoresis can be used
in order to process a higher number of assays simultaneously. An
example of a typical microsequencing procedure that can be used in
the context of the present invention is provided in Example 4.
[0329] Different approaches can be used for the labeling and
detection of ddNTPs. A homogeneous phase detection method based on
fluorescence resonance energy transfer has been described by Chen
and Kwok (1997) and Chen et al. (1997). In this method, amplified
genomic DNA fragments containing polymorphic sites are incubated
with a 5'-fluorescein-labeled primer in the presence of allelic
dye-labeled dideoxyribonucleoside triphosphates and a modified Taq
polymerase. The dye-labeled primer is extended one base by the
dye-terminator specific for the allele present on the template. At
the end of the genotyping reaction, the fluorescence intensities of
the two dyes in the reaction mixture are analyzed directly without
separation or purification. All these steps can be performed in the
same tube and the fluorescence changes can be monitored in real
time. Alternatively, the extended primer may be analyzed by
MALDI-TOF Mass Spectrometry. The base at the polymorphic site is
identified by the mass added onto the microsequencing primer (see
Haff and Smirnov, 1997).
[0330] Microsequencing may be achieved by the established
microsequencing method or by developments or derivatives thereof.
Alternative methods include several solid-phase microsequencing
techniques. The basic microsequencing protocol is the same as
described previously, except that the method is conducted as a
heterogeneous phase assay, in which the primer or the target
molecule is immobilized or captured onto a solid support. To
simplify the primer separation and the terminal nucleotide addition
analysis, oligonucleotides are attached to solid supports or are
modified in such ways that permit affinity separation as well as
polymerase extension. The 5' ends and internal nucleotides of
synthetic oligonucleotides can be modified in a number of different
ways to permit different affinity separation approaches, e.g.,
biotinylation. If a single affinity group is used on the
oligonucleotides, the oligonucleotides can be separated from the
incorporated terminator regent. This eliminates the need of
physical or size separation. More than one oligonucleotide can be
separated from the terminator reagent and analyzed simultaneously
if more than one affinity group is used. This permits the analysis
of several nucleic acid species or more nucleic acid sequence
information per extension reaction. The affinity group need not be
on the priming oligonucleotide but could alternatively be present
on the template. For example, immobilization can be carried out via
an interaction between biotinylated DNA and streptavidin-coated
microtitration wells or avidin-coated polystyrene particles. In the
same manner, oligonucleotides or templates may be attached to a
solid support in a high-density format. In such solid phase
microsequencing reactions, incorporated ddNTPs can be radiolabeled
(Syvnen, 1994) or linked to fluorescein (Livak and Hainer, 1994).
The detection of radiolabeled ddNTPs can be achieved through
scintillation-based techniques. The detection of fluorescein-linked
ddNTPs can be based on the binding of antifluorescein antibody
conjugated with alkaline phosphatase, followed by incubation with a
chromogenic substrate (such as p-nitrophenyl phosphate). Other
possible reporter-detection pairs include: ddNTP linked to
dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate
(Harju et al., 1993) or biotinylated ddNTP and horseradish
peroxidase-conjugated streptavidin with o-phenylenediamine as a
substrate (WO 92/15712). As yet another alternative solid-phase
microsequencing procedure, Nyren et al. (1993) described a method
relying on the detection of DNA polymerase activity by an enzymatic
luminometric inorganic pyrophosphate detection assay (ELIDA).
[0331] Pastinen et al. (1997) describe a method for multiplex
detection of single nucleotide polymorphism in which the solid
phase minisequencing principle is applied to an oligonucleotide
array format. High-density arrays of DNA probes attached to a solid
support (DNA chips) are further described below.
[0332] In one aspect the present invention provides polynucleotides
and methods to genotype one or more biallelic markers of the
present invention by performing a microsequencing assay. Preferred
microsequencing primers include the nucleotide sequences Nos D1 to
D26 and E1 to E26. More preferred microsequencing primers are
selected from the group consisting of the nucleotide sequences Nos
D3, E4, E5, E6, D7, and D8. It will be appreciated that the
microsequencing primers listed in Example 4 are merely exemplary
and that, any primer having a 3' end immediately adjacent to the
polymorphic nucleotide may be used. Similarly, it will be
appreciated that microsequencing analysis may be performed for any
biallelic marker or any combination of biallelic markers of the
present invention. One aspect of the present invention is a solid
support which includes one or more microsequencing primers listed
in Example 4, or fragments comprising at least 8, 12, 15, 20, 25,
30, 40, or 50 consecutive nucleotides thereof and having a 3'
terminus immediately upstream of the corresponding biallelic
marker, for determining the identity of a nucleotide at a biallelic
marker site.
[0333] 3) Allele-Specific Amplification Assay Methods
[0334] In one aspect the present invention provides polynucleotides
and methods to determine the allele of one or more biallelic
markers of the present invention in a biological sample, by
allele-specific amplification assays. Methods, primers and various
parameters to amplify DNA fragments comprising biallelic markers of
the present invention are further described above in "Amplification
Of DNA Fragments Comprising Biallelic Markers".
[0335] Allele Specific Amplification Primers
[0336] Discrimination between the two alleles of a biallelic marker
can also be achieved by allele specific amplification, a selective
strategy, whereby one of the alleles is amplified without
amplification of the other allele. This is accomplished by placing
the polymorphic base at the 3' end of one of the amplification
primers. Because the extension forms from the 3'end of the primer,
a mismatch at or near this position has an inhibitory effect on
amplification. Therefore, under appropriate amplification
conditions, these primers only direct amplification on their
complementary allele. Determining the precise location of the
mismatch and the corresponding assay conditions are well with the
ordinary skill in the art.
[0337] Ligation/Amplification Based Methods
[0338] The "Oligonucleotide Ligation Assay" (OLA) uses two
oligonucleotides which are designed to be capable of hybridizing to
abutting sequences of a single strand of a target molecules. One of
the oligonucleotides is biotinylated, and the other is detectably
labeled. If the precise complementary sequence is found in a target
molecule, the oligonucleotides will hybridize such that their
termini abut, and create a ligation substrate that can be captured
and detected. OLA is capable of detecting single nucleotide
polymorphisms and may be advantageously combined with PCR as
described by Nickerson et al. (1990). In this method, PCR is used
to achieve the exponential amplification of target DNA, which is
then detected using OLA.
[0339] Other amplification methods which are particularly suited
for the detection of single nucleotide polymorphism include LCR
(ligase chain reaction), Gap LCR (GLCR) which are described above
in "Amplification of the APM1 gene". LCR uses two pairs of probes
to exponentially amplify a specific target. The sequences of each
pair of oligonucleotides, is selected to permit the pair to
hybridize to abutting sequences of the same strand of the target.
Such hybridization forms a substrate for a template-dependant
ligase. In accordance with the present invention, LCR can be
performed with oligonucleotides having the proximal and distal
sequences of the same strand of a biallelic marker site. In one
embodiment, either oligonucleotide will be designed to include the
biallelic marker site. In such an embodiment, the reaction
conditions are selected such that the oligonucleotides can be
ligated together only if the target molecule either contains or
lacks the specific nucleotide that is complementary to the
biallelic marker on the oligonucleotide. In an alternative
embodiment, the oligonucleotides will not include the biallelic
marker, such that when they hybridize to the target molecule, a
"gap" is created as described in WO 90/01069. This gap is then
"filled" with complementary dNTPs (as mediated by DNA polymerase),
or by an additional pair of oligonucleotides. Thus at the end of
each cycle, each single strand has a complement capable of serving
as a target during the next cycle and exponential allele-specific
amplification of the desired sequence is obtained.
[0340] Ligase/Polymerase-mediated Genetic Bit Analysis.TM. is
another method for determining the identity of a nucleotide at a
preselected site in a nucleic acid molecule (WO 95/21271). This
method involves the incorporation of a nucleoside triphosphate that
is complementary to the nucleotide present at the preselected site
onto the terminus of a primer molecule, and their subsequent
ligation to a second oligonucleotide. The reaction is monitored by
detecting a specific label attached to the reaction's solid phase
or by detection in solution.
[0341] 4) Hybridization Assay Methods
[0342] A preferred method of determining the identity of the
nucleotide present at a biallelic marker site involves nucleic acid
hybridization. The hybridization probes, which can be conveniently
used in such reactions, preferably include the probes defined
herein. Any hybridization assay may be used including Southern
hybridization, Northern hybridization, dot blot hybridization and
solid-phase hybridization (see Sambrook et al., 1989).
[0343] Hybridization refers to the formation of a duplex structure
by two single stranded nucleic acids due to complementary base
pairing. Hybridization can occur between exactly complementary
nucleic acid strands or between nucleic acid strands that contain
minor regions of mismatch. Specific probes can be designed that
hybridize to one form of a biallelic marker and not to the other
and therefore are able to discriminate between different allelic
forms. Allele-specific probes are often used in pairs, one member
of a pair showing perfect match to a target sequence containing the
original allele and the other showing a perfect match to the target
sequence containing the alternative allele. Hybridization
conditions should be sufficiently stringent that there is a
significant difference in hybridization intensity between alleles,
and preferably an essentially binary response, whereby a probe
hybridizes to only one of the alleles. Stringent, sequence specific
hybridization conditions, under which a probe will hybridize only
to the exactly complementary target sequence are well known in the
art (Sambrook et al., 1989). Stringent conditions are sequence
dependent and will be different in different circumstances.
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. Although such
hybridizations can be performed in solution, it is preferred to
employ a solid-phase hybridization assay. The target DNA comprising
a biallelic marker of the present invention may be amplified prior
to the hybridization reaction. The presence of a specific allele in
the sample is determined by detecting the presence or the absence
of stable hybrid duplexes formed between the probe and the target
DNA. The detection of hybrid duplexes can be carried out by a
number of methods. Various detection assay formats are well known
which utilize detectable labels bound to either the target or the
probe to enable detection of the hybrid duplexes. Typically,
hybridization duplexes are separated from unhybridized nucleic
acids and the labels bound to the duplexes are then detected. Those
skilled in the art will recognize that wash steps may be employed
to wash away excess target DNA or probe as well as unbound
conjugate. Further, standard heterogeneous assay formats are
suitable for detecting the hybrids using the labels present on the
primers and probes.
[0344] Two recently developed assays allow hybridization-based
allele discrimination with no need for separations or washes (see
Landegren U. et al., 1998). The TaqMan assay takes advantage of the
5' nuclease activity of Taq DNA polymerase to digest a DNA probe
annealed specifically to the accumulating amplification product.
TaqMan probes are labeled with a donor-acceptor dye pair that
interacts via fluorescence energy transfer. Cleavage of the TaqMan
probe by the advancing polymerase during amplification dissociates
the donor dye from the quenching acceptor dye, greatly increasing
the donor fluorescence. All reagents necessary to detect two
allelic variants can be assembled at the beginning of the reaction
and the results are monitored in real time (see Livak et al.,
1995). In an alternative homogeneous hybridization based procedure,
molecular beacons are used for allele discriminations. Molecular
beacons are hairpin-shaped oligonucleotide probes that report the
presence of specific nucleic acids in homogeneous solutions. When
they bind to their targets they undergo a conformational
reorganization that restores the fluorescence of an internally
quenched fluorophore (Tyagi et al., 1998).
[0345] The polynucleotides provided herein can be used to produce
probes which can be used in hybridization assays for the detection
of biallelic marker alleles in biological samples. These probes are
characterized in that they preferably comprise between 8 and 50
nucleotides, and in that they are sufficiently complementary to a
sequence comprising a biallelic marker of the present invention to
hybridize thereto and preferably sufficiently specific to be able
to discriminate the targeted sequence for only one nucleotide
variation. A particularly preferred probe is 25 nucleotides in
length. Preferably the biallelic marker is within 4 nucleotides of
the center of the polynucleotide probe. In particularly preferred
probes, the biallelic marker is at the center of said
polynucleotide. Preferred probes comprise a nucleotide sequence
selected from the group consisting of Nos 9-27, 99-14387, 9-12,
9-13, 99-14405, and 9-16 and the sequences complementary thereto,
or a fragment thereof, said fragment comprising at least about 8
consecutive nucleotides, preferably 10, 15, 20, more preferably 25,
30, 40, 47, or 50 consecutive nucleotides and containing a
polymorphic base. In preferred embodiments the polymorphic base is
within 5, 4, 3, 2, 1, nucleotides of the center of the said
polynucleotide, more preferably at the center of said
polynucleotide.
[0346] Preferably the probes of the present invention are labeled
or immobilized on a solid support. Labels and solid supports are
further described in "Oligonucleotide Probes and Primers". The
probes can be non-extendable as described in "Oligonucleotide
Probes and Primers".
[0347] By assaying the hybridization to an allele specific probe,
one can detect the presence or absence of a biallelic marker allele
in a given sample. High-Throughput parallel hybridizations in array
format are specifically encompassed within "hybridization assays"
and are described below.
[0348] 5) Hybridization to Addressable Arrays of
Oligonucleotides
[0349] Hybridization assays based on oligonucleotide arrays rely on
the differences in hybridization stability of short
oligonucleotides to perfectly matched and mismatched target
sequence variants. Efficient access to polymorphism information is
obtained through a basic structure comprising high-density arrays
of oligonucleotide probes attached to a solid support (e.g., the
chip) at selected positions. Each DNA chip can contain thousands to
millions of individual synthetic DNA probes arranged in a grid-like
pattern and miniaturized to the size of a dime.
[0350] The chip technology has already been applied with success in
numerous cases. For example, the screening of mutations has been
undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and
in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker
et al., 1996 ; Kozal et al., 1996). Chips of various formats for
use in detecting biallelic polymorphisms can be produced on a
customized basis by Affymetrix (GeneChip.TM.), Hyseq (HyChip and
HyGnostics), and Protogene Laboratories.
[0351] In general, these methods employ arrays of oligonucleotide
probes that are complementary to target nucleic acid sequence
segments from an individual which, target sequences include a
polymorphic marker. EP 785280 describes a tiling strategy for the
detection of single nucleotide polymorphisms. Briefly, arrays may
generally be "tiled" for a large number of specific polymorphisms.
By "tiling" is generally meant the synthesis of a defined set of
oligonucleotide probes which is made up of a sequence complementary
to the target sequence of interest, as well as preselected
variations of that sequence, e.g., substitution of one or more
given positions with one or more members of the basis set of
monomers, i.e. nucleotides. Tiling strategies are further described
in PCT application No. WO 95/11995. In a particular aspect, arrays
are tiled for a number of specific, identified biallelic marker
sequences. In particular, the array is tiled to include a number of
detection blocks, each detection block being specific for a
specific biallelic marker or a set of biallelic markers. For
example, a detection block may be tiled to include a number of
probes, which span the sequence segment that includes a specific
polymorphism. To ensure probes that are complementary to each
allele, the probes are synthesized in pairs differing at the
biallelic marker. In addition to the probes differing at the
polymorphic base, monosubstituted probes are also generally tiled
within the detection block. These monosubstituted probes have bases
at and up to a certain number of bases in either direction from the
polymorphism, substituted with the remaining nucleotides (selected
from A, T, G, C and U). Typically the probes in a tiled detection
block will include substitutions of the sequence positions up to
and including those that are 5 bases away from the biallelic
marker. The monosubstituted probes provide internal controls for
the tiled array, to distinguish actual hybridization from
artefactual cross-hybridization. Upon completion of hybridization
with the target sequence and washing of the array, the array is
scanned to determine the position on the array to which the target
sequence hybridizes. The hybridization data from the scanned array
is then analyzed to identify which allele or alleles of the
biallelic marker are present in the sample. Hybridization and
scanning may be carried out as described in PCT application No. WO
92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186.
[0352] Thus, in some embodiments, the chips may comprise an array
of nucleic acid sequences of fragments of about 15 nucleotides in
length. In further embodiments, the chip may comprise an array
including at least one of the sequences selected from the group
consisting of 9-27, 99-14387, 9-12, 9-13, 99-14405, and 9-16 and
the sequences complementary thereto, or a fragment thereof, said
fragment comprising at least about 8 consecutive nucleotides,
preferably 10, 15, 20, more preferably 25, 30, 40, 47, or 50
consecutive nucleotides and containing a polymorphic base. In
preferred embodiments the polymorphic base is within 5, 4, 3, 2, 1,
nucleotides of the center of the said polynucleotide, more
preferably at the center of said polynucleotide. In some
embodiments, the chip may comprise an array of at least 2, 3, 4, 5,
6, 7, 8 or more of these polynucleotides of the invention. Solid
supports and polynucleotides of the present invention attached to
solid supports are further described in "oligonucleotide probes and
primers".
[0353] 6) Integrated Systems
[0354] Another technique, which may be used to analyze
polymorphisms, includes multicomponent integrated systems, which
miniaturize and compartmentalize processes such as PCR and
capillary electrophoresis reactions in a single functional device.
An example of such technique is disclosed in U.S. Pat. No.
5,589,136, which describes the integration of PCR amplification and
capillary electrophoresis in chips.
[0355] Integrated systems can be envisaged mainly when microfluidic
systems are used. These systems comprise a pattern of microchannels
designed onto a glass, silicon, quartz, or plastic wafer included
on a microchip. The movements of the samples are controlled by
electric, electroosmotic or hydrostatic forces applied across
different areas of the microchip to create functional microscopic
valves and pumps with no moving parts. Varying the voltage controls
the liquid flow at intersections between the micro-machined
channels and changes the liquid flow rate for pumping across
different sections of the microchip.
[0356] For genotyping biallelic markers, the microfluidic system
may integrate nucleic acid amplification, microsequencing,
capillary electrophoresis and a detection method such as
laser-induced fluorescence detection.
[0357] In a first step, the DNA samples are amplified, preferably
by PCR. Then, the amplification products are subjected to automated
microsequencing reactions using ddNTPs (specific fluorescence for
each ddNTP) and the appropriate oligonucleotide microsequencing
primers which hybridize just upstream of the targeted polymorphic
base. Once the extension at the 3' end is completed, the primers
are separated from the unincorporated fluorescent ddNTPs by
capillary electrophoresis. The separation medium used in capillary
electrophoresis can for example be polyacrylamide,
polyethyleneglycol or dextran. The incorporated ddNTPs in the
single-nucleotide primer extension products are identified by
fluorescence detection. This microchip can be used to process at
least 96 to 384 samples in parallel. It can use the usual four
color laser induced fluorescence detection of the ddNTPs.
[0358] Methods of Genetic Analysis Using the Biallelic Markers of
the Present Invention
[0359] Different methods are available for the genetic analysis of
complex traits (see Lander and Schork, 1994). The search for
disease-susceptibility genes is conducted using two main methods:
the linkage approach in which evidence is sought for cosegregation
between a locus and a putative trait locus using family studies,
and the association approach in which evidence is sought for a
statistically significant association between an allele and a trait
or a trait causing allele (Khoury et al., 1993). In general, the
biallelic markers of the present invention find use in any method
known in the art to demonstrate a statistically significant
correlation between a genotype and a phenotype. The biallelic
markers may be used in parametric and non-parametric linkage
analysis methods. Preferably, the biallelic markers of the present
invention are used to identify genes associated with detectable
traits using association studies, an approach which does not
require the use of affected families and which permits the
identification of genes associated with complex and sporadic
traits.
[0360] The genetic analysis using the biallelic markers of the
present invention may be conducted on any scale. The whole set of
biallelic markers of the present invention or any subset of
biallelic markers of the present invention corresponding to the
candidate gene may be used. Further, any set of genetic markers
including a biallelic marker of the present invention may be used.
A set of biallelic polymorphisms that could be used as genetic
markers in combination with the biallelic markers of the present
invention has been described in WO 98/20165. As mentioned above, it
should be noted that the biallelic markers of the present invention
may be included in any complete or partial genetic map of the human
genome. These different uses are specifically contemplated in the
present invention and claims.
[0361] The invention also comprises methods of detecting an
association between a genotype and a phenotype, comprising the
steps of a) genotyping at least one APM1-related biallelic marker
in a trait positive population according to a genotyping method of
the invention; b) genotyping said APM1-related biallelic marker in
a control population according to a genotyping method of the
invention; and c) determining whether a statistically significant
association exists between said genotype and said phenotype. In
addition, the methods of detecting an association between a
genotype and a phenotype of the invention encompass methods with
any further limitation described in this disclosure, or those
following, specified alone or in any combination. Optionally, said
APM1-related biallelic marker may be selected from the group
consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12,
A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and
A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8. Optionally,
said control population may be a trait negative population, or a
random population. Optionally, each of said genotyping steps a) and
b) may be performed on a pooled biological sample derived from each
of said populations. Optionally, each of said genotyping of steps
a) and b) is performed separately on biological samples derived
from each individual in said population or a subsample thereof.
Optionally, said phenotype is obesity or disorders related to
obesity. Optionally, wherein said disorder related to obesity is
selected from the group consisting of atherosclerosis, insulin
resistance, hypertension, hyperlipidemia, hypertriglyceridemia,
cardiovascular disease, microangiopathic in obese individuals with
Type II diabetes, ocular lesions associated with microangiopathy in
obese individuals with Type II diabetes, renal lesions associated
with microangiopathy in obese individuals with Type II diabetes,
and Syndrome X.
[0362] The invention also encompasses methods of estimating the
frequency of a haplotype for a set of biallelic markers in a
population, comprising the steps of: a) genotyping at least two
APM1-related biallelic marker for each individual in said
population or a subsample thereof, according to a genotyping method
of the invention; and b) applying a haplotype determination method
to the identities of the nucleotides determined in steps a) to
obtain an estimate of said frequency. In addition, the methods of
estimating the frequency of a haplotype of the invention encompass
methods with any further limitation described in this disclosure,
or those following, specified alone or in any combination:
Optionally, said biallelic marker may be selected from the group
consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12,
A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and
A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8. Optionally,
said haplotype determination method is performed by asymmetric PCR
amplification, double PCR amplification of specific alleles, the
Clark algorithm, or an expectation-maximization algorithm.
[0363] An additional embodiment of the present invention
encompasses methods of detecting an association between a haplotype
and a phenotype, comprising the steps of: a) estimating the
frequency of at least one haplotype in a trait positive population,
according to a method of the invention for estimating the frequency
of a haplotype; b) estimating the frequency of said haplotype in a
control population, according to a method of the invention for
estimating the frequency of a haplotype; and c) determining whether
a statistically significant association exists between said
haplotype and said phenotype. In addition, the methods of detecting
an association between a haplotype and a phenotype of the invention
encompass methods with any further limitation described in this
disclosure, or those following. Optionally, said biallelic marker
may be selected from the group consisting of A1, A2, A3, A4, A5,
A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19,
A20, A21, A22, A23, A24, A25 and A26, and the complements thereof;
optionally, wherein said APM1-related biallelic marker is selected
from the group consisting of A1, A2, and A7 or the group consisting
of A4 and A8. Optionally, said control population is a trait
negative population, or a random population. Optionally, said
phenotype is obesity or a disorder related to obesity. Optionally,
said method comprises the additional steps of determining the
phenotype in said trait positive and said control populations prior
to step c). Optionally, wherein said disorder related to obesity is
selected from the group consisting of atherosclerosis, insulin
resistance, hypertension, hyperlipidemia, hypertriglyceridemia,
cardiovascular disease, microangiopathic in obese individuals with
Type II diabetes, ocular lesions associated with microangiopathy in
obese individuals with Type II diabetes, renal lesions associated
with microangiopathy in obese individuals with Type II diabetes,
and Syndrome X.
[0364] Linkage Analysis
[0365] Linkage analysis is based upon establishing a correlation
between the transmission of genetic markers and that of a specific
trait throughout generations within a family. Thus, the aim of
linkage analysis is to detect marker loci that show cosegregation
with a trait of interest in pedigrees.
[0366] Parametric Methods
[0367] When data are available from successive generations there is
the opportunity to study the degree of linkage between pairs of
loci. Estimates of the recombination fraction enable loci to be
ordered and placed onto a genetic map. With loci that are genetic
markers, a genetic map can be established, and then the strength of
linkage between markers and traits can be calculated and used to
indicate the relative positions of markers and genes affecting
those traits (Weir, 1996). The classical method for linkage
analysis is the logarithm of odds (lod) score method (see Morton,
1955; Ott, 1991). Calculation of lod scores requires specification
of the mode of inheritance for the disease (parametric method).
Generally, the length of the candidate region identified using
linkage analysis is between 2 and 20 Mb. Once a candidate region is
identified as described above, analysis of recombinant individuals
using additional markers allows further delineation of the
candidate region. Linkage analysis studies have generally relied on
the use of a maximum of 5,000 microsatellite markers, thus limiting
the maximum theoretical attainable resolution of linkage analysis
to about 600 kb on average.
[0368] Linkage analysis has been successfully applied to map simple
genetic traits that show clear Mendelian inheritance patterns and
which have a high penetrance (i.e., the ratio between the number of
trait positive carriers of allele a and the total number of a
carriers in the population). However, parametric linkage analysis
suffers from a variety of drawbacks. First, it is limited by its
reliance on the choice of a genetic model suitable for each studied
trait. Furthermore, as already mentioned, the resolution attainable
using linkage analysis is limited, and complementary studies are
required to refine the analysis of the typical 2 Mb to 20 Mb
regions initially identified through linkage analysis. In addition,
parametric linkage analysis approaches have proven difficult when
applied to complex genetic traits, such as those due to the
combined action of multiple genes and/or environmental factors. It
is very difficult to model these factors adequately in a lod score
analysis. In such cases, too large an effort and cost are needed to
recruit the adequate number of affected families required for
applying linkage analysis to these situations, as recently
discussed by Risch, N. and Merikangas, K. (1996).
[0369] Non-Parametric Methods
[0370] The advantage of the so-called non-parametric methods for
linkage analysis is that they do not require specification of the
mode of inheritance for the disease, they tend to be more useful
for the analysis of complex traits. In non-parametric methods, one
tries to prove that the inheritance pattern of a chromosomal region
is not consistent with random Mendelian segregation by showing that
affected relatives inherit identical copies of the region more
often than expected by chance. Affected relatives should show
excess "allele sharing" even in the presence of incomplete
penetrance and polygenic inheritance. In non-parametric linkage
analysis the degree of agreement at a marker locus in two
individuals can be measured either by the number of alleles
identical by state (IBS) or by the number of alleles identical by
descent (IBD). Affected sib pair analysis is a well-known special
case and is the simplest form of these methods.
[0371] The biallelic markers of the present invention may be used
in both parametric and non-parametric linkage analysis. Preferably
biallelic markers may be used in non-parametric methods which allow
the mapping of genes involved in complex traits. The biallelic
markers of the present invention may be used in both IBD- and
IBS-methods to map genes affecting a complex trait. In such
studies, taking advantage of the high density of biallelic markers,
several adjacent biallelic marker loci may be pooled to achieve the
efficiency attained by multi-allelic markers (Zhao et al.,
1998).
[0372] Population Association Studies
[0373] The present invention comprises methods for identifying if
the APM1 gene is associated with a detectable trait using the
biallelic markers of the present invention. In one embodiment the
present invention comprises methods to detect an association
between a biallelic marker allele or a biallelic marker haplotype
and a trait. Further, the invention comprises methods to identify a
trait causing allele in linkage disequilibrium with any biallelic
marker allele of the present invention.
[0374] As described above, alternative approaches can be employed
to perform association studies: genome-wide association studies,
candidate region association studies and candidate gene association
studies. In a preferred embodiment, the biallelic markers of the
present invention are used to perform candidate gene association
studies. The candidate gene analysis clearly provides a short-cut
approach to the identification of genes and gene polymorphisms
related to a particular trait when some information concerning the
biology of the trait is available. Further, the biallelic markers
of the present invention may be incorporated in any map of genetic
markers of the human genome in order to perform genome-wide
association studies. Methods to generate a high-density map of
biallelic markers has been described in U.S. Provisional Patent
application serial No. 60/082,614. The biallelic markers of the
present invention may further be incorporated in any map of a
specific candidate region of the genome (a specific chromosome or a
specific chromosomal segment for example).
[0375] As mentioned above, association studies may be conducted
within the general population and are not limited to studies
performed on related individuals in affected families. Association
studies are extremely valuable as they permit the analysis of
sporadic or multifactor traits. Moreover, association studies
represent a powerful method for fine-scale mapping enabling much
finer mapping of trait causing alleles than linkage studies.
Studies based on pedigrees often only narrow the location of the
trait causing allele. Association studies using the biallelic
markers of the present invention can therefore be used to refine
the location of a trait causing allele in a candidate region
identified by Linkage Analysis methods. Moreover, once a chromosome
segment of interest has been identified, the presence of a
candidate gene such as a candidate gene of the present invention,
in the region of interest can provide a shortcut to the
identification of the trait causing allele. Biallelic markers of
the present invention can be used to demonstrate that a candidate
gene is associated with a trait. Such uses are specifically
contemplated in the present invention.
[0376] Determining the Frequency of a Biallelic Marker Allele or of
a Biallelic Marker Haplotype in a Population
[0377] Association studies explore the relationships among
frequencies for sets of alleles between loci.
[0378] Determining the Frequency of an Allele in a Population
[0379] Allelic frequencies of the biallelic markers in a
populations can be determined using one of the methods described
above under the heading "Methods for genotyping an individual for
biallelic markers", or any genotyping procedure suitable for this
intended purpose. Genotyping pooled samples or individual samples
can determine the frequency of a biallelic marker allele in a
population. One way to reduce the number of genotypings required is
to use pooled samples. A major obstacle in using pooled samples is
in terms of accuracy and reproducibility for determining accurate
DNA concentrations in setting up the pools. Genotyping individual
samples provides higher sensitivity, reproducibility and accuracy
and; is the preferred method used in the present invention.
Preferably, each individual is genotyped separately and simple gene
counting is applied to determine the frequency of an allele of a
biallelic marker or of a genotype in a given population.
[0380] Determining the Frequency of a Haplotype in a Population
[0381] The gametic phase of haplotypes is unknown when diploid
individuals are heterozygous at more than one locus. Using
genealogical information in families gametic phase can sometimes be
inferred (Perlin et al., 1994). When no genealogical information is
available different strategies may be used. One possibility is that
the multiple-site heterozygous diploids can be eliminated from the
analysis, keeping only the homozygotes and the single-site
heterozygote individuals, but this approach might lead to a
possible bias in the sample composition and the underestimation of
low-frequency haplotypes. Another possibility is that single
chromosomes can be studied independently, for example, by
asymmetric PCR amplification (see Newton et al, 1989; Wu et al.,
1989) or by isolation of single chromosome by limit dilution
followed by PCR amplification (see Ruano et al., 1990). Further, a
sample may be haplotyped for sufficiently close biallelic markers
by double PCR amplification of specific alleles (Sarkar, G. and
Sommer S. S., 1991). These approaches are not entirely satisfying
either because of their technical complexity, the additional cost
they entail, their lack of generalization at a large scale, or the
possible biases they introduce. To overcome these difficulties, an
algorithm to infer the phase of PCR-amplified DNA genotypes
introduced by Clark, A. G. (1990) may be used. Briefly, the
principle is to start filling a preliminary list of haplotypes
present in the sample by examining unambiguous individuals, that
is, the complete homozygotes and the single-site heterozygotes.
Then other individuals in the same sample are screened for the
possible occurrence of previously recognized haplotypes. For each
positive identification, the complementary haplotype is added to
the list of recognized haplotypes, until the phase information for
all individuals is either resolved or identified as unresolved.
This method assigns a single haplotype to each multiheterozygous
individual, whereas several haplotypes are possible when there are
more than one heterozygous site. Alternatively, one can use methods
estimating haplotype frequencies in a population without assigning
haplotypes to each individual. Preferably, a method based on an
expectation-maximization (EM) algorithm (Dempster et al., 1977)
leading to maximum-likelihood estimates of haplotype frequencies
under the assumption of Hardy-Weinberg proportions (random mating)
is used (see Excoffier L. and Slatkin M., 1995). The EM algorithm
is a generalized iterative maximum-likelihood approach to
estimation that is useful when data are ambiguous and/or
incomplete. The EM algorithm is used to resolve heterozygotes into
haplotypes. Haplotype estimations are further described below under
the heading "Statistical Methods." Any other method known in the
art to determine or to estimate the frequency of a haplotype in a
population may be used.
[0382] Linkage Disequilibrium Analysis
[0383] Linkage disequilibrium is the non-random association of
alleles at two or more loci and represents a powerful tool for
mapping genes involved in disease traits (see Ajioka R. S. et al.,
1997). Biallelic markers, because they are densely spaced in the
human genome and can be genotyped in greater numbers than other
types of genetic markers (such as RFLP or VNTR markers), are
particularly useful in genetic analysis based on linkage
disequilibrium.
[0384] When a disease mutation is first introduced into a
population (by a new mutation or the immigration of a mutation
carrier), it necessarily resides on a single chromosome and thus on
a single "background" or "ancestral" haplotype of linked markers.
Consequently, there is complete disequilibrium between these
markers and the disease mutation: one finds the disease mutation
only in the presence of a specific set of marker alleles. Through
subsequent generations recombination events occur between the
disease mutation and these marker polymorphisms, and the
disequilibrium gradually dissipates. The pace of this dissipation
is a function of the recombination frequency, so the markers
closest to the disease gene will manifest higher levels of
disequilibrium than those that are further away. When not broken up
by recombination, "ancestral" haplotypes and linkage disequilibrium
between marker alleles at different loci can be tracked not only
through pedigrees but also through populations. Linkage
disequilibrium is usually seen as an association between one
specific allele at one locus and another specific allele at a
second locus.
[0385] The pattern or curve of disequilibrium between disease and
marker loci is expected to exhibit a maximum that occurs at the
disease locus. Consequently, the amount of linkage disequilibrium
between a disease allele and closely linked genetic markers may
yield valuable information regarding the location of the disease
gene. For fine-scale mapping of a disease locus, it is useful to
have some knowledge of the patterns of linkage disequilibrium that
exist between markers in the studied region. As mentioned above the
mapping resolution achieved through the analysis of linkage
disequilibrium is much higher than that of linkage studies. The
high density of biallelic markers combined with linkage
disequilibrium analysis provides powerful tools for fine-scale
mapping. Different methods to calculate linkage disequilibrium are
described below under the heading "Statistical Methods".
[0386] Population-Based Case-Control Studies of Trait-Marker
Associations
[0387] As mentioned above, the occurrence of pairs of specific
alleles at different loci on the same chromosome is not random and
the deviation from random is called linkage disequilibrium.
Association studies focus on population frequencies and rely on the
phenomenon of linkage disequilibrium. If a specific allele in a
given gene is directly involved in causing a particular trait, its
frequency will be statistically increased in an affected (trait
positive) population, when compared to the frequency in a trait
negative population or in a random control population. As a
consequence of the existence of linkage disequilibrium, the
frequency of all other alleles present in the haplotype carrying
the trait-causing allele will also be increased in trait positive
individuals compared to trait negative individuals or random
controls. Therefore, association between the trait and any allele
(specifically a biallelic marker allele) in linkage disequilibrium
with the trait-causing allele will suffice to suggest the presence
of a trait-related gene in that particular region. Case-control
populations can be genotyped for biallelic markers to identify
associations that narrowly locate a trait causing allele. As any
marker in linkage disequilibrium with one given marker associated
with a trait will be associated with the trait. Linkage
disequilibrium allows the relative frequencies in case-control
populations of a limited number of genetic polymorphisms
(specifically biallelic markers) to be analyzed as an alternative
to screening all possible functional polymorphisms in order to find
trait-causing alleles. Association studies compare the frequency of
marker alleles in unrelated case-control populations, and represent
powerful tools for the dissection of complex traits.
[0388] Case-Control Populations (Inclusion Criteria)
[0389] Population-based association studies do not concern familial
inheritance but compare the prevalence of a particular genetic
marker, or a set of markers, in case-control populations. They are
case-control studies based on comparison of unrelated case
(affected or trait positive) individuals and unrelated control
(unaffected, trait negative or random) individuals. Preferably the
control group is composed of unaffected or trait negative
individuals. Further, the control group is ethnically matched to
the case population. Moreover, the control group is preferably
matched to the case-population for the main known confusion factor
for the trait under study (for example age-matched for an
age-dependent trait). Ideally, individuals in the two samples are
paired in such a way that they are expected to differ only in their
disease status. The terms "trait positive population", "case
population" and "affected population" are used interchangeably
herein.
[0390] An important step in the dissection of complex traits using
association studies is the choice of case-control populations (see
Lander and Schork, 1994). A major step in the choice of
case-control populations is the clinical definition of a given
trait or phenotype. Any genetic trait may be analyzed by the
association method proposed here by carefully selecting the
individuals to be included in the trait positive and trait negative
phenotypic groups. Four criteria are often useful: clinical
phenotype, age at onset, family history and severity. The selection
procedure for continuous or quantitative traits (such as blood
pressure for example) involves selecting individuals at opposite
ends of the phenotype distribution of the trait under study, so as
to include in these trait positive and trait negative populations
individuals with non-overlapping phenotypes. Preferably,
case-control populations consist of phenotypically homogeneous
populations. Trait positive and trait negative populations consist
of phenotypically uniform populations of individuals representing
each between 1 and 98%, preferably between 1 and 80%, more
preferably between 1 and 50%, and more preferably between 1 and
30%, most preferably between 1 and 20% of the total population
under study, and preferably selected among individuals exhibiting
non-overlapping phenotypes. The clearer the difference between the
two trait phenotypes, the greater the probability of detecting an
association with biallelic markers. The selection of those
drastically different but relatively uniform phenotypes enables
efficient comparisons in association studies and the possible
detection of marked differences at the genetic level, provided that
the sample sizes of the populations under study are significant
enough.
[0391] In preferred embodiments, a first group of between 50 and
300 trait positive individuals, preferably about 100 individuals,
are recruited according to their phenotypes. A similar number of
trait negative individuals are included in such studies.
[0392] In the present invention, typical examples of inclusion
criteria include obesity and disorders related to obesity.
[0393] Association Analysis
[0394] The general strategy to perform association studies using
biallelic markers derived from a region carrying a candidate gene
is to scan two groups of individuals (case-control populations) in
order to measure and statistically compare the allele frequencies
of the biallelic markers of the present invention in both
groups.
[0395] If a statistically significant association with a trait is
identified for at least one or more of the analyzed biallelic
markers, one can assume that: either the associated allele is
directly responsible for causing the trait (i.e. the associated
allele is the trait causing allele), or more likely the associated
allele is in linkage disequilibrium with the trait causing allele.
The specific characteristics of the associated allele with respect
to the candidate gene function usually give further insight into
the relationship between the associated allele and the trait
(causal or in linkage disequilibrium). If the evidence indicates
that the associated allele within the candidate gene is most
probably not the trait causing allele but is in linkage
disequilibrium with the real trait causing allele, then the trait
causing allele can be found by sequencing the vicinity of the
associated marker, and performing further association studies with
the polymorphisms that are revealed in an iterative manner.
[0396] Association studies are usually run in two successive steps.
In a first phase, the frequencies of a reduced number of biallelic
markers from the candidate gene are determined in the trait
positive and trait negative populations. In a second phase of the
analysis, the position of the genetic loci responsible for the
given trait is further refined using a higher density of markers
from the relevant region. However, if the candidate gene under
study is relatively small in length, as is the case for APM1, a
single phase may be sufficient to establish significant
associations.
[0397] Haplotype Analysis
[0398] As described above, when a chromosome carrying a disease
allele first appears in a population as a result of either mutation
or migration, the mutant allele necessarily resides on a chromosome
having a set of linked markers: the ancestral haplotype. This
haplotype can be tracked through populations and its statistical
association with a given trait can be analyzed. Complementing
single point (allelic) association studies with multi-point
association studies also called haplotype studies increases the
statistical power of association studies. Thus, a haplotype
association study allows one to define the frequency and the type
of the ancestral carrier haplotype. A haplotype analysis is
important in that it increases the statistical power of an analysis
involving individual markers.
[0399] In a first stage of a haplotype frequency analysis, the
frequency of the possible haplotypes based on various combinations
of the identified biallelic markers of the invention is determined.
The haplotype frequency is then compared for distinct populations
of trait positive and control individuals. The number of trait
positive individuals, which should be, subjected to this analysis
to obtain statistically significant results usually ranges between
30 and 300, with a preferred number of individuals ranging between
50 and 150. The same considerations apply to the number of
unaffected individuals (or random control) used in the study. The
results of this first analysis provide haplotype frequencies in
case-control populations, for each evaluated haplotype frequency a
p-value and an odd ratio are calculated. If a statistically
significant association is found the relative risk for an
individual carrying the given haplotype of being affected with the
trait under study can be approximated.
[0400] Interaction Analysis
[0401] The biallelic markers of the present invention may also be
used to identify patterns of biallelic markers associated with
detectable traits resulting from polygenic interactions. The
analysis of genetic interaction between alleles at unlinked loci
requires individual genotyping using the techniques described
herein. The analysis of allelic interaction among a selected set of
biallelic markers with appropriate level of statistical
significance can be considered as a haplotype analysis. Interaction
analysis consists in stratifying the case-control populations with
respect to a given haplotype for the first loci and performing a
haplotype analysis with the second loci with each
subpopulation.
[0402] Statistical Methods Used in Association Studies.
[0403] Testing for Linkage in the Presence of Association
[0404] The biallelic markers of the present invention may further
be used in TDT (transmission/disequilibrium test). TDT tests for
both linkage and association and is not affected by population
stratification. TDT requires data for affected individuals and
their parents or data from unaffected sibs instead of from parents
(see Spielmann S. et al., 1993; Schaid D. J. et al., 1996,
Spielmann S. and Ewens W. J., 1998). Such combined tests generally
reduce the false-positive errors produced by separate analyses.
[0405] Statistical Methods
[0406] In general, any method known in the art to test whether a
trait and a genotype show a statistically significant correlation
may be used.
[0407] 1) Methods in Linkage Analysis
[0408] Statistical methods and computer programs useful for linkage
analysis are well-known to those skilled in the art (see
Terwilliger J. D. and Ott J., 1994; Ott J., 1991).
[0409] 2) Methods to Estimate Haplotype Frequencies in a
Population
[0410] As described above, when genotypes are scored, it is often
not possible to distinguish heterozygotes so that haplotype
frequencies cannot be easily inferred. When the gametic phase is
not known, haplotype frequencies can be estimated from the
multilocus genotypic data. Any method known to person skilled in
the art can be used to estimate haplotype frequencies (see Lange
K., 1997; Weir, B. S., 1996) Preferably, maximum-likelihood
haplotype frequencies are computed using an
Expectation-Maximization (EM) algorithm (see Dempster et al., 1977;
Excoffier L. and Slatkin M., 1995). This procedure is an iterative
process aiming at obtaining maximum-likelihood estimates of
haplotype frequencies from multi-locus genotype data when the
gametic phase is unknown. Haplotype estimations are usually
performed by applying the EM algorithm using for example the
EM-HAPLO program (Hawley M. E. et al., 1994) or the Arlequin
program (Schneider et al., 1997). The EM algorithm is a generalized
iterative maximum likelihood approach to estimation and is briefly
described below.
[0411] Please note that in the present section, "Methods To
Estimate Haplotype Frequencies In A Population," of this text,
phenotypes will refer to multi-locus genotypes with unknown phase.
Genotypes will refer to known-phase multi-locus genotypes.
[0412] A sample of N unrelated individuals is typed for K markers.
The data observed are the unknown-phase K-locus phenotypes that can
categorized in F different phenotypes. Suppose that we have H
underlying possible haplotypes (in case of K biallelic markers,
H=2.sup.K).
[0413] For phenotype j, suppose that c.sub.j genotypes are
possible. We thus have the following equation 1 P j = i = 1 c j pr
( genotype i ) = i = 1 c j pr ( h k , h l ) Equation 1
[0414] where Pj is the probability of the phenotype j, h.sub.k and
h.sub.l are the two haplotypes constituent the genotype i. Under
the Hardy-Weinberg equilibrium, pr(h.sub.k,h.sub.l) becomes:
pr(h.sub.k, h.sub.l)=pr(h.sub.k).sup.2 if h.sub.k=h.sub.l,
pr(h.sub.k, h.sub.l)=2pr(h.sub.k). pr(h.sub.l) if
h.sub.k.noteq.h.sub.l. Equation 2
[0415] The successive steps of the E-M algorithm can be described
as follows:
[0416] Starting with initial values of the of haplotypes
frequencies, noted p.sub.1.sup.(0), p.sub.2.sup.(0), . . .
p.sub.H.sup.(0), these initial values serve to estimate the
genotype frequencies (Expectation step) and then estimate another
set of haplotype frequencies (Maximization step), noted
p.sub.1.sup.(1), p.sub.2.sup.(1), . . . p.sub.H.sup.(1), these two
steps are iterated until changes in the sets of haplotypes
frequency are very small.
[0417] A stop criterion can be that the maximum difference between
haplotype frequencies between two iterations is less than
10.sup.-7. These values can be adjusted according to the desired
precision of estimations.
[0418] At a given iteration s, the Expectation step consists in
calculating the genotypes frequencies by the following equation: 2
pr ( genotype i ) ( s ) = pr ( phenotype j ) pr ( genotype i
phenotype j ) ( s ) = n j N pr ( h k , h l ) ( s ) P j ( s )
Equation 3
[0419] where genotype i occurs in phenotype j, and where h.sub.k
and h.sub.l constitute genotype i. Each probability is derived
according to eq. 1, and eq. 2 described above.
[0420] Then the Maximization step simply estimates another set of
haplotype frequencies given the genotypes frequencies. This
approach is also known as the gene-counting method (Smith, 1957). 3
p t ( s + 1 ) = 1 2 j = 1 F i = 1 c j it pr ( genotype i ) ( s )
Equation 4
[0421] Where .delta..sub.it is an indicator variable which count
the number of time haplotype t in genotype i. It takes the values
of 0, 1 or 2.
[0422] To ensure that the estimation finally obtained is the
maximum-likelihood estimation several values of departures are
required. The estimations obtained are compared and if they are
different the estimations leading to the best likelihood are
kept.
[0423] 3) Methods to Calculate Linkage Disequilibrium Between
Markers
[0424] A number of methods can be used to calculate linkage
disequilibrium between any two genetic positions, in practice
linkage disequilibrium is measured by applying a statistical
association test to haplotype data taken from a population.
[0425] Linkage disequilibrium between any pair of biallelic markers
comprising at least one of the biallelic markers of the present
invention (M.sub.i, M.sub.j) having alleles (a.sub.i/b.sub.i) at
marker M.sub.i and alleles (a.sub.j/b.sub.j) at marker M.sub.j can
be calculated for every allele combination (a.sub.i,a.sub.j;
a.sub.i,b.sub.j; b.sub.i,a.sub.j and b.sub.i,b.sub.j), according to
the Piazza formula:
.DELTA..sub.aiaj={square root}.theta.4-{square
root}(.theta.4+.theta.3) (.theta.4+.theta.2), where:
[0426] .theta.4=--=frequency of genotypes not having allele a.sub.i
at M.sub.i and not having allele a.sub.j at M.sub.j
[0427] .theta.3=-+=frequency of genotypes not having allele a.sub.i
at M.sub.i and having allele a.sub.j at M.sub.j
[0428] .theta.2=+-=frequency of genotypes having allele a.sub.i at
M.sub.i and not having allele a.sub.j at M.sub.j
[0429] Linkage disequilibrium (LD) between pairs of biallelic
markers (M.sub.i, M.sub.j) can also be calculated for every allele
combination (ai,aj; ai,bj; b.sub.i,a.sub.j and b.sub.i,b.sub.j),
according to the maximum-likelihood estimate (MLE) for delta (the
composite genotypic disequilibrium coefficient), as described by
Weir (Weir B. S., 1996). The MLE for the composite linkage
disequilibrium is:
D.sub.aiaj=(2n.sub.1+n.sub.2+n.sub.3+n.sub.4/2)/N-2(pr(a.sub.i).
pr(a.sub.j))
[0430] Where n.sub.1=.SIGMA. phenotype (a.sub.i/a.sub.i,
a.sub.j/a.sub.j), n.sub.2=.SIGMA. phenotype (a.sub.i/a.sub.i,
a.sub.j/b.sub.j), n.sub.3=.SIGMA. phenotype (a.sub.i/b.sub.i,
a.sub.j/a.sub.j), n4=.SIGMA. phenotype (a.sub.i/b.sub.i,
a.sub.j/b.sub.j) and N is the number of individuals in the
sample.
[0431] This formula allows linkage disequilibrium between alleles
to be estimated when only genotype, and not haplotype, data are
available.
[0432] Another means of calculating the linkage disequilibrium
between markers is as follows. For a couple of biallelic markers,
M.sub.i(a.sub.i/b.sub.i) and M.sub.j(a.sub.j/b.sub.j), fitting the
Hardy-Weinberg equilibrium, one can estimate the four possible
haplotype frequencies in a given population according to the
approach described above.
[0433] The estimation of gametic disequilibrium between ai and aj
is simply:
D.sub.aiaj=pr(haplotype(a.sub.i,a.sub.j))-pr(a.sub.i).
pr(a.sub.j).
[0434] Where pr(a.sub.i) is the probability of allele a.sub.i and
pr(a.sub.j) is the probability of allele a.sub.j and where
pr(haplotype (a.sub.i, a.sub.j)) is estimated as in Equation 3
above.
[0435] For a couple of biallelic marker only one measure of
disequilibrium is necessary to describe the association between
M.sub.i and M.sub.j.
[0436] Then a normalized value of the above is calculated as
follows:
D'.sub.aiaj=D.sub.aiaj/max(-pr(a.sub.i). pr(a.sub.j), -pr(b.sub.i).
pr(b.sub.j)) with D.sub.aiaj<0
D'.sub.aiaj=D.sub.aiaj/max (pr(b.sub.i). pr(a.sub.j), pr(a.sub.i).
pr(b.sub.j)) with D.sub.aiaj>0
[0437] The skilled person will readily appreciate that other LD
calculation methods can be used.
[0438] Linkage disequilibrium among a set of biallelic markers
having an adequate heterozygosity rate can be determined by
genotyping between 50 and 1000 unrelated individuals, preferably
between 75 and 200, more preferably around 100.
[0439] 4) Testing for Association
[0440] Methods for determining the statistical significance of a
correlation between a phenotype and a genotype, in this case an
allele at a biallelic marker or a haplotype made up of such
alleles, may be determined by any statistical test known in the art
and with any accepted threshold of statistical significance being
required. The application of particular methods and thresholds of
significance are well with in the skill of the ordinary
practitioner of the art.
[0441] Testing for association is performed by determining the
frequency of a biallelic marker allele in case and control
populations and comparing these frequencies with a statistical test
to determine if their is a statistically significant difference in
frequency which would indicate a correlation between the trait and
the biallelic marker allele under study. Similarly, a haplotype
analysis is performed by estimating the frequencies of all possible
haplotypes for a given set of biallelic markers in case and control
populations, and comparing these frequencies with a statistical
test to determine if their is a statistically significant
correlation between the haplotype and the phenotype (trait) under
study. Any statistical tool useful to test for a statistically
significant association between a genotype and a phenotype may be
used. Preferably the statistical test employed is a chi-square test
with one degree of freedom. A P-value is calculated (the P-value is
the probability that a statistic as large or larger than the
observed one would occur by chance).
[0442] Statistical Significance
[0443] In preferred embodiments, significance for diagnosis
purposes, either as a positive basis for further diagnostic tests
or as a preliminary starting point for early preventive therapy,
the p value related to a biallelic marker association is preferably
about 1.times.10.sup.-2 or less, more preferably about
1.times.10.sup.-4 or less, for a single biallelic marker analysis
and about 1.times.10.sup.-3 or less, still more preferably
1.times.10.sup.-6 or less and most preferably of about
1.times.10.sup.-8 or less, for a haplotype analysis involving two
or more markers. These values are believed to be applicable to any
association studies involving single or multiple marker
combinations.
[0444] The skilled person can use the range of values set forth
above as a starting point in order to carry out association studies
with biallelic markers of the present invention. In doing so,
significant associations between the biallelic markers of the
present invention and obesity or disorders related to obesity can
be revealed and used for diagnosis and drug screening purposes.
[0445] Phenotypic Permutation
[0446] In order to confirm the statistical significance of the
first stage haplotype analysis described above, it might be
suitable to perform further analyses in which genotyping data from
case-control individuals are pooled and randomized with respect to
the trait phenotype. Each individual genotyping data is randomly
allocated to two groups, which contain the same number of
individuals as the case-control populations used to compile the
data obtained in the first stage. A second stage haplotype analysis
is preferably run on these artificial groups, preferably for the
markers included in the haplotype of the first stage analysis
showing the highest relative risk coefficient. This experiment is
reiterated preferably at least between 100 and 10000 times. The
repeated iterations allow the determination of the percentage of
obtained haplotypes with a significant p-value level below about
1.times.10.sup.-3.
[0447] Assessment of Statistical Association
[0448] To address the problem of false positives similar analysis
may be performed with the same case-control populations in random
genomic regions. Results in random regions and the candidate region
are compared as described in a co-pending U.S. Provisional Patent
Application entitled "Methods, Software And Apparati For
Identifying Genomic Regions Harboring A Gene Associated With A
Detectable Trait," U.S. Ser. No. 60/107,986, filed Nov. 10, 1998,
the contents of which are incorporated herein by reference.
[0449] 5) Evaluation of Risk Factors
[0450] The association between a risk factor (in genetic
epidemiology the risk factor is the presence or the absence of a
certain allele or haplotype at marker loci) and a disease is
measured by the odds ratio (OR) and by the relative risk (RR). If
P(R.sup.+) is the probability of developing the disease for
individuals with R and P(R.sup.-) is the probability for
individuals without the risk factor, then the relative risk is
simply the ratio of the two probabilities, that is:
RR=P(R.sup.+)/P(R.sup.-)
[0451] In case-control studies, direct measures of the relative
risk cannot be obtained because of the sampling design. However,
the odds ratio allows a good approximation of the relative risk for
low-incidence diseases and can be calculated:
OR=(F.sup.+/(1-F.sup.+))/(F.sup.-/(1-F.sup.-))
[0452] F.sup.+ is the frequency of the exposure to the risk factor
in cases and F.sup.- is the frequency of the exposure to the risk
factor in controls. F.sup.+ and F.sup.- are calculated using the
allelic or haplotype frequencies of the study and further depend on
the underlying genetic model (dominant, recessive, additive,
etc).
[0453] One can further estimate the attributable risk (AR) which
describes the proportion of individuals in a population exhibiting
a trait due to a given risk factor. This measure is important in
quantifying the role of a specific factor in disease etiology and
in terms of the public health impact of a risk factor. The public
health relevance of this measure lies in estimating the proportion
of cases of disease in the population that could be prevented if
the exposure of interest were absent. AR is determined as
follows:
AR=P.sub.E(RR-1)/(P.sub.E(RR-1)+1)
[0454] AR is the risk attributable to a biallelic marker allele or
a biallelic marker haplotype. P.sub.E is the frequency of exposure
to an allele or a haplotype within the population at large; and RR
is the relative risk which, is approximated with the odds ratio
when the trait under study has a relatively low incidence in the
general population.
[0455] Identification of Biallelic Markers in Linkage
Disequilibrium With the Biallelic Markers of the Invention
[0456] Once a first biallelic marker has been identified in a
genomic region of interest, the practitioner of ordinary skill in
the art, using the teachings of the present invention, can easily
identify additional biallelic markers in linkage disequilibrium
with this first marker. As mentioned before any marker in linkage
disequilibrium with a first marker associated with a trait will be
associated with the trait. Therefore, once an association has been
demonstrated between a given biallelic marker and a trait, the
discovery of additional biallelic markers associated with this
trait is of great interest in order to increase the density of
biallelic markers in this particular region. The causal gene or
mutation will be found in the vicinity of the marker or set of
markers showing the highest correlation with the trait.
[0457] Identification of additional markers in linkage
disequilibrium with a given marker involves: (a) amplifying a
genomic fragment comprising a first biallelic marker from a
plurality of individuals; (b) identifying of second biallelic
markers in the genomic region harboring said first biallelic
marker; (c) conducting a linkage disequilibrium analysis between
said first biallelic marker and second biallelic markers; and (d)
selecting said second biallelic markers as being in linkage
disequilibrium with said first marker. Subcombinations comprising
steps (b) and (c) are also contemplated.
[0458] Methods to identify biallelic markers and to conduct linkage
disequilibrium analysis are described herein and can be carried out
by the skilled person without undue experimentation. The present
invention then also concerns biallelic markers which are in linkage
disequilibrium with the specific biallelic markers A1, A2, A3, A4,
A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18,
A19, A20, A21, A22, A23, A24, A25 and A26 and which are expected to
present similar characteristics in terms of their respective
association with a given trait; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8.
[0459] Mapping Studies: Identification of Functional Mutations
[0460] Once a positive association is confirmed with a biallelic
marker of the present invention, gene in the associated candidate
region (within linkage disequillibrium of the APM1 gene) can be
scanned for mutations by comparing the sequences of a selected
number of trait positive and trait negative individuals. In a
preferred embodiment, functional regions such as exons and splice
sites, promoters and other regulatory regions of the APM1 gene are
scanned for mutations. Preferably, trait positive individuals carry
the haplotype shown to be associated with the trait, and trait
negative individuals do not carry the haplotype or allele
associated with the trait. The mutation detection procedure is
essentially similar to that used for biallelic site
identification.
[0461] The method used to detect such mutations generally comprises
the following steps: (a) amplification of a region of the candidate
gene comprising a biallelic marker or a group of biallelic markers
associated with the trait from DNA samples of trait positive
patients and trait negative controls; (b) sequencing of the
amplified region; (c) comparison of DNA sequences from
trait-positive patients and trait-negative controls; and (d)
determination of mutations specific to trait-positive patients.
Subcombinations which comprise steps (b) and (c) are specifically
contemplated.
[0462] It is preferred that candidate polymorphisms be then
verified by screening a larger population of cases and controls by
means of any genotyping procedure such as those described herein,
preferably using a microsequencing technique in an individual test
format. Polymorphisms are considered as candidate mutations when
present in cases and controls at frequencies compatible with the
expected association results.
[0463] Biallelic Markers of the Invention in Methods of Genetic
Diagnostics
[0464] The biallelic markers of the present invention can also be
used to develop diagnostic tests capable of identifying individuals
who express a detectable trait as the result of a specific genotype
or individuals whose genotype places them at risk of developing a
detectable trait at a subsequent time.
[0465] It will of course be understood by practitioners skilled in
the treatment or diagnosis of obesity and disorders related to
obesity that the present invention does not intend to provide an
absolute identification of individuals who could be at risk of
developing a particular disease involving obesity and disorders
related to obesity but rather to indicate a certain degree or
likelihood of developing a disease. However, this information is
extremely valuable as it can, in certain circumstances, be used to
initiate preventive treatments or to allow an individual carrying a
significant haplotype to foresee warning signs such as minor
symptoms. In diseases in which attacks may be extremely violent and
sometimes fatal if not treated on time, the knowledge of a
potential predisposition, even if this predisposition is not
absolute, might contribute in a very significant manner to
treatment efficacy.
[0466] The diagnostic techniques of the present invention may
employ a variety of methodologies to determine whether a test
subject has a biallelic marker pattern associated with an increased
risk of developing a detectable trait or whether the individual
suffers from a detectable trait as a result of a particular
mutation, including methods which enable the analysis of individual
chromosomes for haplotyping, such as family studies, single sperm
DNA analysis or somatic hybrids. The trait analyzed using the
present diagnostics may be any detectable trait, including obesity
and disorders related to obesity.
[0467] Another aspect of the present invention relates to a method
of determining whether an individual is at risk of developing a
trait or whether an individual expresses a trait as a consequence
of possessing a particular trait-causing allele. The present
invention also relates to a method of determining whether an
individual is at risk of developing a plurality of traits or
whether an individual expresses a plurality of traits as a result
of possessing a particular trait-causing allele. These methods
involve obtaining a nucleic acid sample from the individual and
determining whether the nucleic acid sample contains one or more
alleles of one or more biallelic markers indicative of a risk of
developing the trait or indicative that the individual expresses
the trait as a result of possessing a particular trait-causing
allele. These methods also involve obtaining a nucleic acid sample
from the individual and, determining, whether the nucleic acid
sample contains at least one allele or at least one biallelic
marker haplotype, indicative of a risk of developing the trait or
indicative that the individual expresses the trait as a result of
possessing a particular APM1 polymorphism or mutation
(trait-causing allele).
[0468] Preferably, in such diagnostic methods, a nucleic acid
sample is obtained from the individual and this sample is genotyped
using methods described above in "Methods Of Genotyping DNA Samples
For Biallelic markers. The diagnostics may be based on a single
biallelic marker or on a group of biallelic markers. In each of
these methods, a nucleic acid sample is obtained from the test
subject and the biallelic marker pattern of one or more of the
biallelic markers A1 to A26 is determined. Alternatively, the one
or more biallelic markers are selected from the group consisting of
A1, A2, A4, A7, and A8. Alternatively, one or more biallelic
markers are selected from the group consisting of A1, A2, and
A7.
[0469] In one embodiment, a PCR amplification is conducted on the
nucleic acid sample to amplify regions in which polymorphisms
associated with a detectable phenotype have been identified. The
amplification products are sequenced to determine whether the
individual possesses one or more APM1 polymorphisms associated with
a detectable phenotype. The primers used to generate amplification
products may comprise the primers listed in Table 1. Alternatively,
the nucleic acid sample is subjected to microsequencing reactions
as described above to determine whether the individual possesses
one or more APM1 polymorphisms associated with a detectable
phenotype resulting from a mutation or a polymorphism in the APM1
gene. The primers used in the microsequencing reactions may include
the primers listed in Table 4.
[0470] In another embodiment, the nucleic acid sample is contacted
with one or more allele specific oligonucleotide probes which
specifically hybridize to one or more APM1 alleles associated with
a detectable phenotype. The probes used in the hybridization assay
may include the probes listed in Table 3. In another embodiment,
the nucleic acid sample is contacted with a second APM1
oligonucleotide capable of producing an amplification product when
used with the allele specific oligonucleotide in an amplification
reaction. The presence of an amplification product in the
amplification reaction indicates that the individual possesses one
or more APM1 alleles associated with a detectable phenotype.
[0471] As described herein, the diagnostics may be based on a
single biallelic marker or a group of biallelic markers.
Preferably, the biallelic marker or combination of biallelic makers
is selected from the group consisting of A1 to A26 and the
complements thereof or any combination or subset thereof. More
preferably, the one or more biallelic markers are selected from the
group consisting of A1, A2, A4, A7, and A8, and the complements
thereof or any combination or subset thereof. Alternatively, the
one or more biallelic markers are selected from the group
consisting of A1, A2, and A7. Diagnostic kits comprise any of the
polynucleotides of the present invention.
[0472] These diagnostic methods are extremely valuable as they can,
in certain circumstances, be used to initiate preventive treatments
or to allow an individual carrying a significant genotype or
haplotype to foresee warning signs such as minor symptoms. For
example, in the study described in Example 6, the subjects were all
adolescent girls who did not yet have significant disease. However,
by identifying the girls as adolescents who are at risk for obesity
and obesity-related diseases and disorders later in their life,
they could be targeted now for more intensive treatment to prevent
the onset of later severe disease, such as diabetes, or
cardiovascular complications, or any of the other obesity-related
diseases discussed herein. An association has been shown between
APM1 markers and indicators of obesity and diabetes, specifically,
as well as indicating susceptibility to other related diseases
(Example 6).
[0473] Diagnostics, which analyze and predict response to a drug or
side effects to a drug, may be used to determine whether an
individual should be treated with a particular drug. For example,
if the diagnostic indicates a likelihood that an individual will
respond positively to treatment with a particular drug, the drug
may be administered to the individual. Conversely, if the
diagnostic indicates that an individual is likely to respond
negatively to treatment with a particular drug, an alternative
course of treatment may be prescribed. A negative response may be
defined as either the absence of an efficacious response or the
presence of toxic side effects. For example, in the study described
in Example 6, the identified APM1 markers would be useful for
genotyping a population of obese people to determine which people
are more likely to be susceptibile to drugs designed to lower
leptin levels or free fatty acid levels. Other associations between
APM1 markers and other traits associated with obesity can also be
determined using the methods of the invention without undue
experimentation and would indicate other markers useful to identify
sub-populations of people likely to be susceptible (or not) to a
drug targeting those traits. In addition, specific associations can
be performed looking at drug outcome (treatment/side effect) to
identify other useful markers for predicting risks/successful
treatment.
[0474] Clinical drug trials represent another application for the
markers of the present invention. One or more markers indicative of
response to an agent acting against an obesity-related disease or
to side effects to an agent acting against an obesity-related
disease may be identified using the methods described above.
Thereafter, potential participants in clinical trials of such an
agent may be screened to identify those individuals most likely to
respond favorably to the drug and/or exclude those likely to
experience side effects. In that way, the effectiveness of drug
treatment may be measured in individuals who have the potential to
respond positively to the drug, without lowering the measurement as
a result of the inclusion of individuals who are unlikely to
respond positively in the study and/or without risking undesirable
safety problems.
[0475] Based on Example 6, herein, subgroups for clinical trials
could be identified that had the rare allele of biallelic markers
Al and/or A2, any or all of the biallelic markers A4, A7 and A8, or
both sets of biallelic markers. The first set of markers was shown
to be associated with increased leptin levels, and the second set
was associated with higher free fatty acid levels in obese girls.
Having the rare allele from either of the sets of markers indicated
a higher risk of obesity in later life compared with a group of
individuals who remained thin throughout life with the same
ethnicity background (data not shown). Thus these markers can be
used to predict patients who might be susceptible to drugs designed
to target/ameliorate these symptoms.
[0476] Obviously, the methods of the invention can be used to
identify other markers and find other associations with traits
associated with obesity such as hypertriglyceridemia, or
hypertension, for example.
[0477] Recombinant Vectors
[0478] The term "vector" is used herein to designate either a
circular or a linear DNA or RNA molecule, which is either
double-stranded or single-stranded, and which comprise at least one
polynucleotide of interest that is sought to be transferred in a
cell host or in a unicellular or multicellular host organism.
[0479] The present invention encompasses a family of recombinant
vectors that comprise a regulatory polynucleotide derived from the
APM1 genomic sequence, or a coding polynucleotide from the APM1
genomic sequence. Consequently, the present invention further deals
with a recombinant vector comprising either a regulatory
polynucleotide comprised in the nucleic acids of SEQ ID Nos 2 and 3
or a polynucleotide comprising the APM1 coding sequence or
both.
[0480] In a first preferred embodiment, a recombinant vector of the
invention is used to amplify the inserted polynucleotide derived
from a APM1 genomic sequence selected from the group consisting of
the nucleic acids of SEQ ID No 1, 2 and 3 or a APM1 cDNA, for
example the cDNA of SEQ ID NO 5 in a suitable cell host, this
polynucleotide being amplified at every time that the recombinant
vector replicates.
[0481] Generally, a recombinant vector of the invention may
comprise any of the polynucleotides described herein, including
regulatory sequences and coding sequences, as well as any APM1
primer or probe as defined above.
[0482] A second preferred embodiment of the recombinant vectors
according to the invention consists of expression vectors
comprising either a regulatory polynucleotide or a coding nucleic
acid of the invention, or both. Within certain embodiments,
expression vectors are employed to express the APM1 polypeptide
which can be then purified and, for example be used in ligand
screening assays or as an immunogen in order to raise specific
antibodies directed against the APM1 protein. In other embodiments,
the expression vectors are used for constructing transgenic animals
and also for gene therapy. Expression requires that appropriate
signals are provided in the vectors, said signals including various
regulatory elements, such as enhancers/promoters from both viral
and mammalian sources that drive expression of the genes of
interest in host cells. Dominant drug selection markers for
establishing permanent, stable cell clones expressing the products
are generally included in the expression vectors of the invention,
as they are elements that link expression of the drug selection
markers to expression of the polypeptide.
[0483] More particularly, the present invention relates to
expression vectors which include nucleic acids encoding a APM1
protein, preferably the APM1 protein of the amino acid sequence of
SEQ ID No 6 or variants or fragments thereof, under the control of
a regulatory sequence selected among the APM1 regulatory
polynucleotides, or alternatively under the control of an exogenous
regulatory sequence.
[0484] Consequently, preferred expression vectors of the invention
are selected from the group consisting of: (a) the APM1 regulatory
sequence comprised therein drives the expression of a coding
polynucleotide operably linked thereto; (b) the APM1 coding
sequence is operably linked to regulation sequences allowing its
expression in a suitable cell host and/or host organism.
[0485] A recombinant expression vector comprising a nucleic acid
selected from the group consisting of SEQ ID No 2, or biologically
active fragments or variants thereof, is also part of the present
invention.
[0486] In a preferred embodiment, a recombinant expression vector
of the invention comprises a regulatory nucleotide sequence
selected from the group consisting of:
[0487] (i) a nucleotide sequence comprising a polynucleotide of SEQ
ID NO 2 or a complementary sequence thereto;
[0488] (ii) a nucleotide sequence comprising a polynucleotide
having at least 95% of nucleotide identity with the nucleotide
sequence of SEQ ID No 2 or a complementary sequence thereto;
[0489] (iii) a nucleotide sequence comprising a polynucleotide that
hybridizes under stringent hybridization conditions with the
nucleotide sequence of SEQ ID No 2 or a complementary sequence
thereto; and
[0490] (iv) a biologically active fragment or variant of the
polynucleotides in (i), (ii) and (iii).
[0491] The invention also encompasses a recombinant expression
vector comprising:
[0492] a) a nucleic acid comprising a regulatory nucleotide
sequence selected from the group consisting of:
[0493] (i) a nucleotide sequence comprising a polynucleotide of SEQ
ID NO 2 or a complementary sequence thereto;
[0494] (ii) a nucleotide sequence comprising a polynucleotide
having at least 95% of nucleotide identity with the nucleotide
sequence of SEQ ID No 2 or a complementary sequence thereto;
[0495] (iii) a nucleotide sequence comprising a polynucleotide that
hybridizes under stringent hybridization conditions with the
nucleotide sequence of SEQ ID No 2 or a complementary sequence
thereto; and
[0496] (iv) a biologically active fragment or variant of the
polynucleotides in (i), (ii) and (iii); and
[0497] b) a polynucleotide encoding a desired polypeptide or
nucleic acid of interest, operably linked to the nucleic acid
defined in (a) above.
[0498] Additionally, the recombinant expression vector described
above may also comprise a nucleic acid comprising a 3'-regulatory
polynucleotide, preferably a 3'-regulatory polynucleotide of the
APM1 gene. The APM1 3'-regulatory polynucleotide may also comprise
the 3'-UTR sequence contained in the nucleotide sequence of SEQ ID
NO 5.
[0499] The 5'-regulatory polynucleotide may also include the 5'-UTR
sequence of the APM1 cDNA, or a biologically active fragment or
variant thereof.
[0500] The invention also pertains to a recombinant expression
vector useful for the expression of the APM1 coding sequence,
wherein said vector comprises a nucleic acid of SEQ ID No 5.
[0501] Another preferred recombinant expression vector consists of
a vector for expressing a APM1 coding sequence, wherein said vector
comprises a nucleic acid of SEQ ID No 1 or a fragment thereof or a
nucleic acid having at least 95% nucleotide identity with a
polynucleotide of SEQ ID No 1 or a fragment thereof.
[0502] Recombinant vectors comprising a nucleic acid containing a
APM1-related biallelic marker is also part of the invention. In a
preferred embodiment, said biallelic marker is selected from the
group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11,
A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24,
A25 and A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8.
[0503] Some of the elements which can be found in the vectors of
the present invention are described in further detail in the
following sections.
[0504] 1. General Features of the Expression Vectors of the
Invention
[0505] A recombinant vector according to the invention comprises,
but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC
(Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a
plasmid or even a linear DNA molecule which may consist of a
chromosomal, non-chromosomal, semi-synthetic and synthetic DNA.
Such a recombinant vector can comprise a transcriptional unit
comprising an assembly of:
[0506] (1) a genetic element or elements having a regulatory role
in gene expression, for example promoters or enhancers. Enhancers
are cis-acting elements of DNA, usually from about 10 to 300 bp in
length that act on the promoter to increase the transcription.
[0507] (2) a structural or coding sequence which is transcribed
into mRNA and eventually translated into a polypeptide, said
structural or coding sequence being operably linked to the
regulatory elements described in (1); and
[0508] (3) appropriate transcription initiation and termination
sequences. Structural units intended for use in yeast or eukaryotic
expression systems preferably include a leader sequence enabling
extracellular secretion of translated protein by a host cell.
Alternatively, when a recombinant protein is expressed without a
leader or transport sequence, it may include a N-terminal residue.
This residue may or may not be subsequently cleaved from the
expressed recombinant protein to provide a final product.
[0509] Generally, recombinant expression vectors will include
origins of replication, selectable markers permitting
transformation of the host cell, and a promoter derived from a
highly expressed gene to direct transcription of a downstream
structural sequence. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably a leader sequence capable of
directing secretion of the translated protein into the periplasmic
space or the extracellular medium. In a specific embodiment wherein
the vector is adapted for transfecting and expressing desired
sequences in mammalian host cells, preferred vectors will comprise
an origin of replication in the desired host, a suitable promoter
and enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5'-flanking
non-transcribed sequences. DNA sequences derived from the SV40
viral genome, for example SV40 origin, early promoter, enhancer,
splice and polyadenylation sites may be used to provide the
required non-transcribed genetic elements.
[0510] The in vivo expression of a APM1 polypeptide of SEQ ID No 6
or fragments or variants thereof may be useful in order to correct
a genetic defect related to the expression of the native gene in a
host organism or to the production of a biologically inactive APM1
protein.
[0511] Consequently, the present invention also deals with
recombinant expression vectors mainly designed for the in vivo
production of the APM1 polypeptide of SEQ ID No 6 or fragments or
variants thereof by the introduction of the appropriate genetic
material in the organism of the patient to be treated. This genetic
material may be introduced in vitro in a cell that has been
previously extracted from the organism, the modified cell being
subsequently reintroduced in the said organism, directly in vivo
into the appropriate tissue.
[0512] 2. Regulatory Elements
[0513] Promoters
[0514] The suitable promoter regions used in the expression vectors
according to the present invention are chosen taking into account
the cell host in which the heterologous gene has to be expressed.
The particular promoter employed to control the expression of a
nucleic acid sequence of interest is not believed to be important,
so long as it is capable of directing the expression of the nucleic
acid in the targeted cell. Thus, where a human cell is targeted, it
is preferable to position the nucleic acid coding region adjacent
to and under the control of a promoter that is capable of being
expressed in a human cell, such as, for example, a human or a viral
promoter.
[0515] A suitable promoter may be heterologous with respect to the
nucleic acid for which it controls the expression or alternatively
can be endogenous to the native polynucleotide containing the
coding sequence to be expressed. Additionally, the promoter is
generally heterologous with respect to the recombinant vector
sequences within which the construct promotor/coding sequence has
been inserted.
[0516] Promoter regions can be selected from any desired gene
using, for example, CAT (chloramphenicol transferase) vectors and
more preferably pKK232-8 and pCM7 vectors.
[0517] Preferred bacterial promoters are the LacI, LacZ, the T3 or
T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL
and trp promoters (EP 0036776), the polyhedrin promoter, or the p10
protein promoter from baculovirus (Kit Novagen) (Smith et al.,
1983; O'Reilly et al., 1992), the lambda PR promoter or also the
trc promoter.
[0518] Eukaryotic promoters include CMV immediate early, HSV
thymidine kinase, early and late SV40, LTRs from retrovirus, and
mouse metallothionein-L. Selection of a convenient vector and
promoter is well within the level of ordinary skill in the art.
[0519] The choice of a promoter is well within the ability of a
person skilled in the field of genetic engineering (Sambrook et al.
(1989) And Fuller et al. (1996)).
[0520] Other Regulatory Elements
[0521] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed such as human growth hormone and SV40 polyadenylation
signals. Also contemplated as an element of the expression cassette
is a terminator. These elements can serve to enhance message levels
and to minimize read through from the cassette into other
sequences.
[0522] The vector containing the appropriate DNA sequence as
described above, more preferably APM1 gene regulatory
polynucleotide, a polynucleotide encoding the APM1 polypeptide
selected from the group consisting of SEQ ID No 1 or a fragment or
a variant thereof and SEQ ID No 5, or both of them, can be utilized
to transform an appropriate host to allow the expression of the
desired polypeptide or polynucleotide.
[0523] 3. Selectable Markers
[0524] Selectable markers confer an identifiable change to the cell
permitting easy identification of cells containing the expression
construct. The selectable marker genes for selection of transformed
host cells are preferably dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or
tetracycline, rifampicin or ampicillin resistance in E. coli, or
levan saccharase for mycobacteria, this latter marker being a
negative selection marker.
[0525] 3. Preferred Vectors.
[0526] Bacterial Vectors
[0527] As a representative but non-limiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and a bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of
pBR322 (ATCC 37017). Such commercial vectors include, for example,
pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec,
Madison, Wis., USA).
[0528] Large numbers of other suitable vectors are known to those
of skill in the art, and commercially available, such as the
following bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs,
pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A,
pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 (Pharmacia); pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene);
pSVK3, pBPV, pMSG, pSVL (Pharmacia); pQE-30 (QIAexpress).
[0529] Bacteriophage Vectors
[0530] The P1 bacteriophage vector may contain large inserts
ranging from about 80 to about 100 kb.
[0531] The construction of P1 bacteriophage vectors such as p158 or
p158/neo8 are notably described by Stemberg (1992, 1994).
Recombinant P1 clones comprising APM1 nucleotide sequences may be
designed for inserting large polynucleotides of more than 40 kb
(Linton et al., 1993). To generate P1 DNA for transgenic
experiments, a preferred protocol is the protocol described by
McCormick et al. (1994). Briefly, E. coli (preferably strain
NS3529) harboring the P1 plasmid are grown overnight in a suitable
broth medium containing 25 .mu.g/ml of kanamycin. The P1 DNA is
prepared from the E. coli by alkaline lysis using the Qiagen
Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to
the manufacturer's instructions. The P1 DNA is purified from the
bacterial lysate on two Qiagen-tip 500 columns, using the washing
and elution buffers contained in the kit. A phenol/chloroform
extraction is then performed before precipitating the DNA with 70%
ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4,
1 mM EDTA), the concentration of the DNA is assessed by
spectrophotometry.
[0532] To express a P1 clone comprising APM1 nucleotide sequences
in a transgenic animal, typically transgenic mice, it is desirable
to remove vector sequences from the P1 DNA fragment, for example by
cleaving the P1 DNA at rare-cutting sites within the P1 polylinker
(SfiI, NotI or SalI). The P1 insert is then purified from vector
sequences on a pulsed-field agarose gel, using methods similar
using methods similar to those originally reported for the
isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al.,
1993). At this stage, the resulting purified insert DNA can be
concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit
(Millipore, Bedford, Mass., USA--30,000 molecular weight limit) and
then dialyzed against microinjection buffer (10 mM Tris-HCl, pH
7.4; 250 .mu.M EDTA) containing 100 mM NaCl, 30 .mu.M spermine, 70
.mu.M spermidine on a microdyalisis membrane (type VS, 0. 025 .mu.M
from Millipore). The intactness of the purified P1 DNA insert is
assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC
Bio-products) pulse-field gel and staining with ethidium
bromide.
[0533] Baculovirus Vectors
[0534] A suitable vector for the expression of the APM1 polypeptide
of SEQ ID No 6 or fragments or variants thereof is a baculovirus
vector that can be propagated in insect cells and in insect cell
lines. A specific suitable host vector system is the pVL1392/1393
baculovirus transfer vector (Pharmingen) that is used to transfect
the SF9 cell line (ATCC N.degree.CRL 1711) which is derived from
Spodoptera frugiperda.
[0535] Other suitable vectors for the expression of the APM1
polypeptide of SEQ ID No 6 or fragments or variants thereof in a
baculovirus expression system include those described by Chai et
al. (1993), Vlasak et al. (1983) and Lenhard et al. (1996).
[0536] Viral Vectors
[0537] In one specific embodiment, the vector is derived from an
adenovirus. Preferred adenovirus vectors according to the invention
are those described by Feldman and Steg (1996) or Ohno et al.
(1994). Another preferred recombinant adenovirus according to this
specific embodiment of the present invention is the human
adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal
origin (French patent application N.degree. FR-93. 05954).
[0538] Retrovirus vectors and adeno-associated virus vectors are
generally understood to be the recombinant gene delivery systems of
choice for the transfer of exogenous polynucleotides in vivo,
particularly to mammals, including humans. These vectors provide
efficient delivery of genes into cells, and the transferred nucleic
acids are stably integrated into the chromosomal DNA of the
host.
[0539] Particularly preferred retroviruses for the preparation or
construction of retroviral in vitro or in vitro gene delivery
vehicles of the present invention include retroviruses selected
from the group consisting of Mink-Cell Focus Inducing Virus, Murine
Sarcoma Virus, Reticuloendotheliosis virus and Rous Sarcoma virus.
Particularly preferred Murine Leukemia Viruses include the 4070A
and the 1504A viruses, Abelson (ATCC No VR-999), Friend (ATCC No
VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and
Moloney Murine Leukemia Virus (ATCC No VR-190; PCT Application No
WO 94/24298). Particularly preferred Rous Sarcoma Viruses include
Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and
VR-728). Other preferred retroviral vectors are those described in
Roth et al. (1996), PCT Application No WO 93/25234, PCT Application
No WO 94/06920, Roux et al., 1989, Julan et al., 1992 and Neda et
al., 1991.
[0540] Yet another viral vector system that is contemplated by the
invention consists in the adeno-associated virus (AAV). The
adeno-associated virus is a naturally occurring defective virus
that requires another virus, such as an adenovirus or a herpes
virus, as a helper virus for efficient replication and a productive
life cycle (Muzyczka et al., 1992). It is also one of the few
viruses that may integrate its DNA into non-dividing cells, and
exhibits a high frequency of stable integration (Flotte et al.,
1992; Samulski et al., 1989; McLaughlin et al., 1989). One
advantageous feature of AAV derives from its reduced efficacy for
transducing primary cells relative to transformed cells.
[0541] BAC Vectors
[0542] The bacterial artificial chromosome (BAC) cloning system
(Shizuya et al., 1992) has been developed to stably maintain large
fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC
vector consists of pBeloBAC11 vector that has been described by Kim
et al. (l996). BAC libraries are prepared with this vector using
size-selected genomic DNA that has been partially digested using
enzymes that permit ligation into either the Bam HI or HindIII
sites in the vector. Flanking these cloning sites are T7 and SP6
RNA polymerase transcription initiation sites that can be used to
generate end probes by either RNA transcription or PCR methods.
After the construction of a BAC library in E. coli, BAC DNA is
purified from the host cell as a supercoiled circle. Converting
these circular molecules into a linear form precedes both size
determination and introduction of the BACs into recipient cells.
The cloning site is flanked by two Not I sites, permitting cloned
segments to be excised from the vector by Not I digestion.
Alternatively, the DNA insert contained in the pBeloBAC11 vector
may be linearized by treatment of the BAC vector with the
commercially available enzyme lambda terminase that leads to the
cleavage at the unique cosN site, but this cleavage method results
in a full length BAC clone containing both the insert DNA and the
BAC sequences.
[0543] 5. Delivery of the Recombinant Vectors
[0544] In order to effect expression of the polynucleotides and
polynucleotide constructs of the invention, these constructs must
be delivered into a cell. This delivery may be accomplished in
vitro, as in laboratory procedures for transforming cell lines, or
in vivo or ex vivo, as in the treatment of certain diseases
states.
[0545] One mechanism is viral infection where the expression
construct is encapsulated in an infectious viral particle.
[0546] Several non-viral methods for the transfer of
polynucleotides into cultured mammalian cells are also contemplated
by the present invention, and include, without being limited to,
calcium phosphate precipitation (Graham et al., 1973; Chen et al.,
1987;), DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland et
al., 1985), DNA-loaded liposomes (Nicolau et al., 1982; Fraley et
al., 1979), and receptor-mediate transfection (Wu and Wu, 1987;
1988). Some of these techniques may be successfully adapted for in
vivo or ex vivo use.
[0547] Once the expression polynucleotide has been delivered into
the cell, it may be stably integrated into the genome of the
recipient cell. This integration may be in the cognate location and
orientation via homologous recombination (gene replacement) or it
may be integrated in a random, non specific location (gene
augmentation). In yet further embodiments, the nucleic acid may be
stably maintained in the cell as a separate, episomal segment of
DNA. Such nucleic acid segments or "episomes" encode sequences
sufficient to permit maintenance and replication independent of or
in synchronization with the host cell cycle.
[0548] One specific embodiment for a method for delivering a
protein or peptide to the interior of a cell of a vertebrate in
vivo comprises the step of introducing a preparation comprising a
physiologically acceptable carrier and a naked polynucleotide
operatively coding for the polypeptide of interest into the
interstitial space of a tissue comprising the cell, whereby the
naked polynucleotide is taken up into the interior of the cell and
has a physiological effect. This is particularly applicable for
transfer in vitro but it may be applied to in vivo as well.
[0549] Compositions for use in vitro and in vivo comprising a
"naked" polynucleotide are described in PCT application N.degree.
WO 90/11092 (Vical Inc.) and also in PCT application No. WO
95/11307 (Institut Pasteur, INSERM, Universit d'Ottawa) as well as
in the articles of Tacson et al. (1996) and of Huygen et al.
(1996).
[0550] In still another embodiment of the invention, the transfer
of a naked polynucleotide of the invention, including a
polynucleotide construct of the invention, into cells may be
proceeded with a particle bombardment (biolistic), said particles
being DNA-coated microprojectiles accelerated to a high velocity
allowing them to pierce cell membranes and enter cells without
killing them, such as described by Klein et al. (1987).
[0551] In a further embodiment, the polynucleotide of the invention
may be entrapped in a liposome (Ghosh and Bacchawat, 1991; Wong et
al., 1980; Nicolau et al., 1987)
[0552] In a specific embodiment, the invention provides a
composition for the in vivo production of the APM1 protein or
polypeptide described herein. It comprises a naked polynucleotide
operatively coding for this polypeptide, in solution in a
physiologically acceptable carrier, and suitable for introduction
into a tissue to cause cells of the tissue to express the said
protein or polypeptide.
[0553] The amount of vector to be injected to the desired host
organism varies according to the site of injection. As an
indicative dose, it will be injected between 0,1 and 100 .mu.g of
the vector in an animal body, preferably a mammal body, for example
a mouse body.
[0554] In another embodiment of the vector according to the
invention, it may be introduced in vitro in a host cell, preferably
in a host cell previously harvested from the animal to be treated
and more preferably a somatic cell such as a muscle cell. In a
subsequent step, the cell that has been transformed with the vector
coding for the desired APM1 polypeptide or the desired fragment
thereof is reintroduced into the animal body in order to deliver
the recombinant protein within the body either locally or
systemically.
[0555] Cell Hosts
[0556] Another object of the invention consists of a host cell that
have been transformed or transfected with one of the
polynucleotides described therein, and more precisely a
polynucleotide either comprising a APM1 regulatory polynucleotide
or the coding sequence of the APM1 polypeptide selected from the
group consisting of SEQ ID No 1 or a fragment or a variant thereof
and SEQ ID No 5. Are included host cells that are transformed
(prokaryotic cells) or that are transfected (eukaryotic cells) with
a recombinant vector such as one of those described above.
[0557] Generally, a recombinant host cell of the invention
comprises any one of the polynucleotides or the recombinant vectors
described therein.
[0558] A preferred recombinant host cell according to the invention
comprises a polynucleotide selected from the following group of
polynucleotides:
[0559] a) a purified or isolated nucleic acid encoding a APM1
polypeptide, or a polypeptide fragment or variant thereof;
[0560] b) a purified or isolated nucleic comprising at least 8,
preferably at least 15, more preferably at least 25, consecutive
nucleotides of a nucleotide sequence selected from the group
consisting of:
[0561] 1) the nucleotide sequence beginning at the nucleotide in
position 1 and ending at the nucleotide in position 4811 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; more particularly, the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 3529 of the nucleotide sequence of SEQ
ID No 1 or a variant thereof or a sequence complementary
thereto;
[0562] 2) the nucleotide sequence beginning at the nucleotide in
position 4852 and ending at the nucleotide in position 15142 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0563] 3) the nucleotide sequence beginning at the nucleotide in
position 15366 and ending at the nucleotide in position 16276 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; and
[0564] 4) the nucleotide sequence beginning at the nucleotide in
position 20560 and ending at the nucleotide in position 20966 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0565] c) a purified or isolated nucleic acid comprising at least 8
consecutive nucleotides, preferably at least 15 of the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 22 of the nucleotide sequence of SEQ ID
No 5 or a variant thereof or a sequence complementary thereto;
[0566] d) a purified or isolated nucleic acid comprising an exon of
the APM1 gene, a sequence complementary thereto or a variant
thereof;
[0567] e) a purified or isolated nucleic acid comprising a
combination of at least two exons of the APM1 gene, or the
sequences complementary thereto wherein the polynucleotides are
arranged within the nucleic acid, from the 5' end to the 3'end of
said nucleic acid, in the same order than in SEQ ID No 1;
[0568] f) a purified or isolated nucleic acid comprising the
nucleotide sequence SEQ ID No 2 or the sequences complementary
thereto or a biologically active fragment or a variant thereof;
[0569] g) a purified or isolated nucleic acid comprising the
nucleotide sequence SEQ ID No 3, or the sequence complementary
thereto or a biologically active fragment or a variant thereof;
[0570] h) a polynucleotide consisting of:
[0571] (1) a nucleic acid comprising a regulatory polynucleotide of
SEQ ID No 2 or the sequences complementary thereto or a
biologically active fragment or variant thereof;
[0572] (2) a polynucleotide encoding a desired polypeptide or
nucleic acid; or
[0573] (3) optionally, a nucleic acid comprising a regulatory
polynucleotide of SEQ ID No 3, or the sequence complementary
thereto or a biologically active fragment or variant thereof;
and
[0574] i) a DNA construct as described previously in the present
specification.
[0575] Another preferred recombinant cell host according to the
present invention is characterized in that its genome or genetic
background (including chromosome, plasmids) is modified by the
nucleic acid coding for the APM1 polypeptide of SEQ ID No 5 or
fragments or variants thereof. 104291 A further recombinant cell
host according to the invention comprises a polynucleotide
containing a biallelic marker selected from the group consisting of
A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15,
A16, A17, A18, A19, A20, A21, A22, A23, A24, A25 and A26, and the
complements thereof; optionally, wherein said APM1-related
biallelic marker is selected from the group consisting of A1, A2,
and A7 or the group consisting of A4 and A8.
[0576] Preferred host cells used as recipients for the expression
vectors of the invention are the following:
[0577] a) Prokaryotic host cells: Escherichia coli strains
(I.E.DH5-.alpha. strain), Bacillus subtilis, Salmonella
typhimurium, and strains from species like Pseudomonas,
Streptomyces and Staphylococcus;
[0578] b) Eukaryotic host cells: HeLa cells (ATCC N.degree.CCL2;
N.degree.CCL2. 1; N.degree.CCL2. 2), Cv 1 cells (ATCC
N.degree.CCL70), COS cells (ATCC N.degree.CRL1650;
N.degree.CRL1651), Sf-9 cells (ATCC N.degree.CRL1711), C127 cells
(ATCC N.degree. CRL-1804), 3T3 (ATCC N.degree.CRL-6361), CHO (ATCC
N.degree. CCL-61), human kidney 293. (ATCC N.degree. 45504;
N.degree. CRL-1573) and BHK (ECACC N.degree. 84100501; N.degree.
84111301); and
[0579] c) other mammalian host cells.
[0580] The APM1 gene expression in mammalian, and typically human,
cells may be rendered defective, or alternatively it may be
proceeded with the insertion of a APM1 genomic or cDNA sequence
with the replacement of the APM1 gene counterpart in the genome of
an animal cell by a APM1 polynucleotide according to the invention.
These genetic alterations may be generated by homologous
recombination events using specific DNA constructs that have been
previously described.
[0581] Host cells that may be used include mammalian zygotes, such
as murine zygotes. For example, murine zygotes may undergo
microinjection with a purified DNA molecule of interest, for
example a purified DNA molecule that has previously been adjusted
to a concentration range from 1 ng/mL--for BAC inserts--3
ng/lL--for P1 bacteriophage inserts--in 10 mM Tris-HCl, pH 7.4, 250
.mu.M EDTA containing 100 mM NaCl, 30 .mu.M spermine, and 70 .mu.M
spermidine. When the DNA to be microinjected has a large size,
polyamines and high salt concentrations can be used in order to
avoid mechanical breakage of this DNA, as described by Schedl et al
(1993b).
[0582] Any of the polynucleotides of the invention, including the
DNA constructs described herein, may be introduced in an embryonic
stem (ES) cell line, preferably a mouse ES cell line. ES cell lines
are derived from pluripotent, uncommitted cells of the inner cell
mass of pre-implantation blastocysts. Preferred ES cell lines
include the following: ES-E14TG2a (ATCC n.degree. CRL-1821), ES-D3
(ATCC n.degree. CRL1934 and n.degree. CRL-11632), YS001 (ATCC
n.degree. CRL-11776), 36. 5 (ATCC n.degree. CRL-11116). To maintain
ES cells in an uncommitted state, they are cultured in the presence
of growth inhibited feeder cells that provide the appropriate
signals to preserve this embryonic phenotype and serve as a matrix
for ES cell adherence. Preferred feeder cells consist of primary
embryonic fibroblasts that are established from tissue of day
13-day 14 embryos of virtually any mouse strain, that are
maintained in culture, such as described by Abbondanzo et al.
(1993) and are inhibited in growth by irradiation, such as
described by Robertson (1987), or by the presence of an inhibitory
concentration of LIF, such as described by Pease and Williams
(1990).
[0583] The constructs in the host cells can be used in a
conventional manner to produce the gene product encoded by the
recombinant sequence.
[0584] Following transformation of a suitable host and growth of
the host to an appropriate cell density, the selected promoter is
induced by appropriate means, such as temperature shift or chemical
induction, and cells are cultivated for an additional period.
[0585] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0586] Microbial cells employed in the expression of proteins can
be disrupted by any convenient method, including freeze-thaw
cycling, sonication, mechanical disruption, or use of cell lysing
agents. Such methods are well known by the skill artisan.
[0587] Transgenic Animals
[0588] The terms "transgenic animals" or "host animals" are used
herein designate animals that have their genome genetically and
artificially manipulated so as to include one of the nucleic acids
according to the invention. Preferred animals are non-human mammals
and include those belonging to a genus selected from Mus (e.g.
mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have
their genome artificially and genetically altered by the insertion
of a nucleic acid according to the invention. In one embodiment,
the invention encompasses non-human host mammals and animals
comprising a recombinant vector of the invention or an APM1 gene
disrupted by homologous recombination with a knock out vector.
[0589] The transgenic animals of the invention all include within a
plurality of their cells a cloned recombinant or synthetic DNA
sequence, more specifically one of the purified or isolated nucleic
acids comprising a APM1 coding sequence, a APM1 regulatory
polynucleotide or a DNA sequence encoding an antisense
polynucleotide such as described in the present specification.
[0590] Preferred transgenic animals according to the invention
contain in their somatic cells and/or in their germ line cells a
polynucleotide selected from the following group of
polynucleotides:
[0591] a) a purified or isolated nucleic acid encoding a APM1
polypeptide, or a polypeptide fragment or variant thereof;
[0592] b) a purified or isolated nucleic comprising at least 8,
preferably at least 15, more preferably at least 25, consecutive
nucleotides of a nucleotide sequence selected from the group
consisting of:
[0593] 1) the nucleotide sequence beginning at the nucleotide in
position I and ending at the nucleotide in position 4811 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; more particularly, the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 3529 of the nucleotide sequence of SEQ
ID No 1 or a variant thereof or a sequence complementary
thereto;
[0594] 2) the nucleotide sequence beginning at the nucleotide in
position 4852 and ending at the nucleotide in position 15142 of the
nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0595] 3) the nucleotide sequence beginning at the nucleotide in
position 15366 and ending at the nucleotide in position 16276 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto; and
[0596] 4) the nucleotide sequence beginning at the nucleotide in
position 20560 and ending at the nucleotide in position 20966 of
the nucleotide sequence of SEQ ID No 1 or a variant thereof or a
sequence complementary thereto;
[0597] c) a purified or isolated nucleic acid comprising at least 8
consecutive nucleotides, preferably at least 15 of the nucleotide
sequence beginning at the nucleotide in position 1 and ending at
the nucleotide in position 22 of the nucleotide sequence of SEQ ID
No 5 or a variant thereof or a sequence complementary thereto;
[0598] d) a purified or isolated nucleic acid comprising an exon of
the APM1 gene, a sequence complementary thereto or a variant
thereof;
[0599] e) a purified or isolated nucleic acid comprising a
combination of at least two exons of the APM1 gene, or the
sequences complementary thereto wherein the polynucleotides are
arranged within the nucleic acid, from the 5' end to the 3'end of
said nucleic acid, in the same order than in SEQ ID No 1;
[0600] f) a purified or isolated nucleic acid comprising the
nucleotide sequence SEQ ID No 2 or the sequences complementary
thereto or a biologically active fragment or a variant thereof;
[0601] g) a purified or isolated nucleic acid comprising the
nucleotide sequence SEQ ID No 3, or the sequence complementary
thereto or a biologically active fragment or a variant thereof;
[0602] h) a polynucleotide consisting of:
[0603] (1) a nucleic acid comprising a regulatory polynucleotide of
SEQ ID No 2 or the sequences complementary thereto or a
biologically active fragment or variant thereof;
[0604] (2) a polynucleotide encoding a desired polypeptide or
nucleic acid; or
[0605] (3) optionally, a nucleic acid comprising a regulatory
polynucleotide of SEQ ID No 3, or the sequence complementary
thereto or a biologically active fragment or variant thereof;
and
[0606] i) a DNA construct as described previously in the present
specification.
[0607] The transgenic animals of the invention thus contain
specific sequences of exogenous genetic material such as the
nucleotide sequences described above in detail.
[0608] A further transgenic animals according to the invention
contains in their somatic cells and/or in their germ line cells a
polynucleotide comprising a biallelic marker selected from the
group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11,
A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22, A23, A24,
A25 and A26, and the complements thereof; optionally, wherein said
APM1-related biallelic marker is selected from the group consisting
of A1, A2, and A7 or the group consisting of A4 and A8.
[0609] In a first preferred embodiment, these transgenic animals
may be good experimental models in order to study the diverse
pathologies related to cell differentiation, in particular
concerning the transgenic animals within the genome of which has
been inserted one or several copies of a polynucleotide encoding a
native APM1 protein, or alternatively a mutant APM1 protein.
[0610] In a second preferred embodiment, these transgenic animals
may express a desired polypeptide of interest under the control of
the regulatory polynucleotides of the APM1 gene, leading to good
yields in the synthesis of this protein of interest, and eventually
a tissue specific expression of this protein of interest.
[0611] The design of the transgenic animals of the invention may be
made according to the conventional techniques well known from the
one skilled in the art. For more details regarding the production
of transgenic animals, and specifically transgenic mice, it may be
referred to U.S. Pat. Nos 4,873,191, issued Oct. 10, 1989,
5,464,764 issued Nov. 7, 1995 and 5,789,215, issued Aug. 4, 1998,
these documents being herein incorporated by reference to disclose
methods producing transgenic mice.
[0612] Transgenic animals of the present invention are produced by
the application of procedures which result in an animal with a
genome that has incorporated exogenous genetic material. The
procedure involves obtaining the genetic material, or a portion
thereof, which encodes either a APM1 coding sequence, a APM1
regulatory polynucleotide or a DNA sequence encoding a APM1
antisense polynucleotide such as described in the present
specification.
[0613] A recombinant polynucleotide of the invention is inserted
into an embryonic or ES stem cell line. The insertion is preferably
made using electroporation, such as described by Thomas et al.
(1987). The cells subjected to electroporation are screened (e.g.
by selection via selectable markers, by PCR or by Southern blot
analysis) to find positive cells which have integrated the
exogenous recombinant polynucleotide into their genome, preferably
via an homologous recombination event. An illustrative
positive-negative selection procedure that may be used according to
the invention is described by Mansour et al. (1988).
[0614] Then, the positive cells are isolated, cloned and injected
into 3.5 days old blastocysts from mice, such as described by
Bradley (1987). The blastocysts are then inserted into a female
host animal and allowed to grow to term.
[0615] Alternatively, the positive ES cells are brought into
contact with embryos at the 2.5 days old 8-16 cell stage (morulae)
such as described by Wood et al. (1993) or by Nagy et al. (1993),
the ES cells being internalized to colonize extensively the
blastocyst including the cells which will give rise to the germ
line.
[0616] The offspring of the female host are tested to determine
which animals are transgenic e.g. include the inserted exogenous
DNA sequence and which are wild-type.
[0617] Thus, the present invention also concerns a transgenic
animal containing a nucleic acid, a recombinant expression vector
or a recombinant host cell according to the invention.
[0618] Recombinant Cell Lines Derived From the Transgenic Animals
of the Invention.
[0619] A further object of the invention consists of recombinant
host cells obtained from a transgenic animal described herein. In
one embodiment the invention encompasses cells derived from
non-human host mammals and animals comprising a recombinant vector
of the invention or an APM1 gene disrupted by homologous
recombination with a knock out vector.
[0620] Recombinant cell lines may be established in vitro from
cells obtained from any tissue of a transgenic animal according to
the invention, for example by transfection of primary cell cultures
with vectors expressing onc-genes such as SV40 large T antigen, as
described by Chou (1989) and Shay et al. (1991).
[0621] Method for Producing an APM1 Polypeptide
[0622] It is now easy to produce proteins in high amounts by
genetic engineering techniques through expression vectors such as
plasmids, phages or phagemids. The polynucleotide that codes for
the APM1 protein is inserted in an appropriate expression vector in
order to produce the polypeptide of interest in vitro.
[0623] Thus, the present invention also concerns a method for a
APM1 protein, and especially a polypeptide of SEQ ID No 6, wherein
said method comprises:
[0624] a) culturing, in an appropriate culture medium, a cell host
previously transformed or transfected with the recombinant vector
comprising a nucleic acid encoding the APM1 protein;
[0625] b) harvesting the culture medium thus conditioned or lyse
the cell host, for example by sonication or by an osmotic
shock;
[0626] c) separating or purifying, from the said culture medium, or
from the pellet of the resultant host cell lysate the thus produced
polypeptide of interest; and
[0627] d) optionally characterizing the produced polypeptide of
interest.
[0628] In a specific embodiment of the above method, the nucleic
acid coding for the APM1 protein is inserted in an appropriate
vector, optionally after an appropriate cleavage of this amplified
nucleic acid with one or several restriction endonucleases. In a
preferred embodiment, the nucleic acid encoding for the APM1
protein is selected from a group consisting of SEQ ID No 1 or a
fragment thereof and SEQ ID No 5. In a further embodiment, the
nucleic acid encoding for the APM1 protein comprises an allele of
at least one of the biallelic markers A1 to A26. The nucleic acid
coding for the APM1 protein may be the resulting product of an
amplification reaction using a pair of primers according to the
invention (by SDA, TAS, 3SR NASBA, TMA etc.).
[0629] The polypeptides according to the invention may be
characterized by binding onto an immunoaffinity chromatography
column on which polyclonal or monoclonal antibodies directed to a
polypeptide of SEQ ID No 6 have previously been immobilized.
[0630] The polypeptides or peptides thus obtained may be purified,
for example by high performance liquid chromatography, such as
reverse phase and/or cationic exchange HPLC, as described by
Rougeot et al. (1994). The reason to prefer this kind of peptide or
protein purification is the lack of byproducts found in the elution
samples which renders the resultant purified protein or peptide
more suitable for a therapeutic use.
[0631] Method for Screening Substances Interacting With the
Regulatory Sequences of the APM1 Gene.
[0632] The present invention also concerns a method for screening
substances or molecules that are able to interact with the
regulatory sequences of the APM1 gene, such as for example promoter
or enhancer sequences.
[0633] Nucleic acids encoding proteins which are able to interact
with the regulatory sequences of the APM1 gene, more particularly a
nucleotide sequence selected from the group consisting of the
polynucleotides of SEQ ID Nos 2 and 3 or a fragment or variant
thereof, and preferably a variant comprising one of the biallelic
markers of the invention, may be identified by using a one-hybrid
system, such as that described in the booklet enclosed in the
Matchmaker One-Hybrid System kit from Clontech (Catalog Ref.
n.degree. K1603-1), the technical teachings of which are herein
incorporated by reference. Briefly, the target nucleotide sequence
is cloned upstream of a selectable reporter sequence and the
resulting DNA construct is integrated in the yeast genome
(Saccharomyces cerevisiae). The yeast cells containing the reporter
sequence in their genome are then transformed with a library
consisting of fusion molecules between cDNAs encoding candidate
proteins for binding onto the regulatory sequences of the APM1 gene
and sequences encoding the activator domain of a yeast
transcription factor such as GAL4. The recombinant yeast cells are
plated in a culture broth for selecting cells expressing the
reporter sequence. The recombinant yeast cells thus selected
contain a fusion protein that is able to bind onto the target
regulatory sequence of the APM1 gene. Then, the cDNAs encoding the
fusion proteins are sequenced and may be cloned into expression or
transcription vectors in vitro. The binding of the encoded
polypeptides to the target regulatory sequences of the APM1 gene
may be confirmed by techniques familiar to the one skilled in the
art, such as gel retardation assays or DNAse protection assays.
[0634] Gel retardation assays may also be performed independently
in order to screen candidate molecules that are able to interact
with the regulatory sequences of the APM1 gene, such as described
by Fried and Crothers (1981), Garner and Revzin (1981) and Dent and
Latchman (1993), the teachings of these publications being herein
incorporated by reference. These techniques are based on the
principle according to which a DNA fragment which is bound to a
protein migrates slower than the same unbound DNA fragment.
Briefly, the target nucleotide sequence is labeled. Then the
labeled target nucleotide sequence is brought into contact with
either a total nuclear extract from cells containing transcription
factors, or with different candidate molecules to be tested. The
interaction between the target regulatory sequence of the APM1 gene
and the candidate molecule or the transcription factor is detected
after gel or capillary electrophoresis through a retardation in the
migration.
[0635] Method for Screening Ligands that Modulate the Expression of
the APM1 Gene.
[0636] Another subject of the present invention is a method for
screening molecules that modulate the expression of the APM1
protein. Such a screening method comprises:
[0637] a) cultivating a prokaryotic or an eukaryotic cell that has
been transfected with a nucleotide sequence encoding the APM1
protein or a variant or a fragment thereof, placed under the
control of its own promoter;
[0638] b) bringing into contact the cultivated cell with a molecule
to be tested; and
[0639] c) quantifying the expression of the APM1 protein or a
variant or a fragment thereof.
[0640] In an embodiment, the nucleotide sequence encoding the APM1
protein or a variant or a fragment thereof comprises an allele of
at least one of the biallelic markers A1, A2, A3, A4, A5, A6, A7,
A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21,
A22, A23, A24, A25 and A26, and the complements thereof;
optionally, wherein said APM1-related biallelic marker is selected
from the group consisting of A1, A2, and A7 or the group consisting
of A4 and A8.
[0641] Using DNA recombination techniques well known by the one
skill in the art, the APM1 protein encoding DNA sequence is
inserted into an expression vector, downstream from its promoter
sequence. As an illustrative example, the promoter sequence of the
APM1 gene is contained in the nucleic acid of SEQ ID No 2.
[0642] The quantification of the expression of the APM1 protein may
be realized either at the mRNA level or at the protein level. In
the latter case, polyclonal or monoclonal antibodies may be used to
quantify the amounts of the APM1 protein that have been produced,
for example in an ELISA or a RIA assay.
[0643] In a preferred embodiment, the quantification of the APM1
mRNA is realized by a quantitative PCR amplification of the cDNA
obtained by a reverse transcription of the total mRNA of the
cultivated APM1-transfected host cell, using a pair of primers
specific for APM1.
[0644] The present invention also concerns a method for screening
substances or molecules that are able to increase, or in contrast
to decrease, the level of expression of the APM1 gene. Such a
method may allow the one skilled in the art to select substances
exerting a regulating effect on the expression level of the APM1
gene and which may be useful as active ingredients included in
pharmaceutical compositions for treating patients suffering from
deficiencies in the regulation of expression of the APM1 gene,
particularly patients suffering from obesity.
[0645] The invention also features a method for screening a
candidate substance or molecule for modulation of the expression of
the APM1 gene, comprising:
[0646] a) providing a recombinant cell host containing a nucleic
acid, wherein said nucleic acid comprises a nucleotide sequence of
SEQ ID No 2 or a biologically active fragment or variant thereof
located upstream a polynucleotide encoding a detectable
protein;
[0647] b) obtaining a candidate substance; and
[0648] c) determining the ability of the candidate substance to
modulate the expression levels of the polynucleotide encoding the
detectable protein.
[0649] In a specific embodiment, the nucleic acid comprising a
nucleotide sequence of SEQ ID No 2 or a biologically active
fragment or variant thereof includes a biallelic marker selected
from the group consisting of A1, A2, A3, A4, A5, A6, A7, A8, A9,
A10, A11, A12, A13, A14, A15, A16, A17, A18, A19, A20, A21, A22,
A23, A24, A25 and A26 or the complements thereof; optionally,
wherein said APM1-related biallelic marker is selected from the
group consisting of A1, A2, and A7 or the group consisting of A4
and A8.
[0650] In a further embodiment, the nucleic acid comprising the
nucleotide sequence of SEQ ID No 2 or a biologically active
fragment or variant thereof also includes a 5'UTR region of the
APM1 cDNA of SEQ ID No 5, or one of its biologically active
fragments or variants thereof.
[0651] Among the preferred polynucleotides encoding a detectable
protein, there may be cited polynucleotides encoding beta
galactosidase, green fluorescent protein (GFP) and chloramphenicol
acetyl transferase (CAT).
[0652] The invention also pertains to kits useful for performing
the hereinbefore described screening method. Preferably, such kits
comprise a recombinant vector that allows the expression of a
nucleotide sequence of SEQ ID No 2 or a biologically active
fragment or variant thereof located upstream and operably linked to
a polynucleotide encoding a detectable protein or the APM1 protein
or a fragment or a variant thereof.
[0653] In another embodiment, a method for the screening of a
candidate substance or molecule for modulation of the expression of
the APM1 gene comprises:
[0654] a) providing a recombinant host cell containing a nucleic
acid, wherein said nucleic acid comprises a 5'UTR sequence of the
APM1 cDNA of SEQ ID No 5, or one of its biologically active
fragments or variants, the 5'UTR sequence or its biologically
active fragment or variant being operably linked to a
polynucleotide encoding a detectable protein;
[0655] b) obtaining a candidate substance; and
[0656] c) determining the ability of the candidate substance to
modulate the expression levels of the polynucleotide encoding the
detectable protein.
[0657] In a specific embodiment of the above screening method, the
nucleic acid that comprises a nucleotide sequence selected from the
group consisting of the 5'UTR sequence of the APM1 cDNA of SEQ ID
No 5 or one of its biologically active fragments or variants,
includes a promoter sequence which is endogenous with respect to
the APM1 5'UTR sequence.
[0658] In another specific embodiment of the above screening
method, the nucleic acid that comprises a nucleotide sequence
selected from the group consisting of the 5'UTR sequence of the
APM1 cDNA of SEQ ID No 5 or one of its biologically active
fragments or variants, includes a promoter sequence which is
exogenous with respect to the APM1 5'UTR sequence defined
therein.
[0659] In a further preferred embodiment, the nucleic acid
comprising the 5'-UTR sequence of the APM1 cDNA or SEQ ID NO 5 or
the biologically active fragments thereof includes a biallelic
marker selected from the group consisting of A1, A2, A3, A4, A5,
A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, A16, A17, A18, A19,
A20, A21, A22, A23, A24, A25 and A26 or the complements thereof;
optionally, wherein said APM1-related biallelic marker is selected
from the group consisting of A1, A2, and A7 or the group consisting
of A4 and A8.
[0660] The invention further deals with a kit for the screening of
a candidate substance modulating the expression of the APM1 gene,
wherein said kit comprises a recombinant vector that comprises a
nucleic acid including a 5'UTR sequence of the APM1 cDNA of SEQ ID
No 5, or one of their biologically active fragments or variants,
the 5'UTR sequence or its biologically active fragment or variant
being operably linked to a polynucleotide encoding a detectable
protein.
[0661] For the design of suitable recombinant vectors useful for
performing the screening methods described above, it will be
referred to the section of the present specification wherein the
preferred recombinant vectors of the invention are detailed.
[0662] Expression levels and patterns of APM1 may be analyzed by
solution hybridization with long probes as described in
International Patent Application No. WO 97/05277, which is hereby
incorporated herein by reference in its entirety including any
figures, tables, or references. Briefly, the APM1 cDNA or the APM1
genomic DNA described above, or fragments thereof, is inserted at a
cloning site immediately downstream of a bacteriophage (T3, T7 or
SP6) RNA polymerase promoter to produce antisense RNA. Preferably,
the APM1 insert comprises at least 100 or more consecutive
nucleotides of the genomic DNA sequence or the cDNA sequences. The
plasmid is linearized and transcribed in the presence of
ribonucleotides comprising modified ribonucleotides (i.e.
biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA is
hybridized in solution with mRNA isolated from cells or tissues of
interest. The hybridizations are performed under standard stringent
conditions (40-50.degree. C. for 16 hours in an 80% formamide, 0.4
M NaCl buffer, pH 7-8). The unhybridized probe is removed by
digestion with ribonucleases specific for single-stranded RNA (i.e.
RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP
modification enables capture of the hybrid on a microtitration
plate coated with streptavidin. The presence of the DIG
modification enables the hybrid to be detected and quantified by
ELISA using an anti-DIG antibody coupled to alkaline
phosphatase.
[0663] Quantitative analysis of APM1 gene expression may also be
performed using arrays. As used herein, the term "array" means a
one dimensional, two dimensional, or multidimensional arrangement
of a plurality of nucleic acids of sufficient length to permit
specific detection of expression of mRNAs capable of hybridizing
thereto. For example, the arrays may contain a plurality of nucleic
acids derived from genes whose expression levels are to be
assessed. The arrays may include the APM1 genomic DNA, the APM1
cDNA sequences or the sequences complementary thereto or fragments
thereof, particularly those comprising at least one of the
biallelic markers according the present invention, preferably at
least one of the biallelic markers A1 to A26. Preferably, the
fragments are at least 15 nucleotides in length. In other
embodiments, the fragments are at least 25 nucleotides in length.
In some embodiments, the fragments are at least 50 nucleotides in
length. More preferably, the fragments are at least 100 nucleotides
in length. In another preferred embodiment, the fragments are more
than 100 nucleotides in length. In some embodiments the fragments
may be more than 500 nucleotides in length.
[0664] For example, quantitative analysis of APM1 gene expression
may be performed with a complementary DNA microarray as described
by Schena et al. (1995 and 1996). Full length APM1 cDNAs or
fragments thereof are amplified by PCR and arrayed from a 96-well
microtiter plate onto silylated microscope slides using high-speed
robotics. Printed arrays are incubated in a humid chamber to allow
rehydration of the array elements and rinsed, once in 0.2% SDS for
1 min, twice in water for 1 min and once for 5 min in sodium
borohydride solution. The arrays are submerged in water for 2 min
at 95.degree. C., transferred into 0.2% SDS for 1 min, rinsed twice
with water, air dried and stored in the dark at 25.degree. C.
[0665] Cell or tissue mRNA is isolated or commercially obtained and
probes are prepared by a single round of reverse transcription.
Probes are hybridized to 1 cm.sup.2 microarrays under a 14.times.14
mm glass coverslip for 6-12 hours at 60.degree. C. Arrays are
washed for 5 min at 25.degree. C. in low stringency wash buffer
(1.times. SSC/0.2% SDS), then for 10 min at room temperature in
high stringency wash buffer (0.1.times. SSC/0.2% SDS). Arrays are
scanned in 0.1.times. SSC using a fluorescence laser scanning
device fitted with a custom filter set. Accurate differential
expression measurements are obtained by taking the average of the
ratios of two independent hybridizations.
[0666] Quantitative analysis of APM1 gene expression may also be
performed with full length APM1 cDNAs or fragments thereof in
complementary DNA arrays as described by Pietu et al. (1996). The
full length APM1 cDNA or fragments thereof is PCR amplified and
spotted on membranes. Then, mRNAs originating from various tissues
or cells are labeled with radioactive nucleotides. After
hybridization and washing in controlled conditions, the hybridized
mRNAs are detected by phospho-imaging or autoradiography. Duplicate
experiments are performed and a quantitative analysis of
differentially expressed mRNAs is then performed.
[0667] Alternatively, expression analysis using the APM1 genomic
DNA, the APM1 cDNA, or fragments thereof can be done through high
density nucleotide arrays as described by Lockhart et al. (1996)
and Sosnowsky et al. (1997). Oligonucleotides of 15-50 nucleotides
from the sequences of the APM1 genomic DNA, the APM1 cDNA sequences
particularly those comprising at least one of biallelic markers
according the present invention, preferably at least one biallelic
marker selected from the group consisting of A1 to A26, or the
sequences complementary thereto, are synthesized directly on the
chip (Lockhart et al., supra) or synthesized and then addressed to
the chip (Sosnowski et al., supra). Preferably, the
oligonucleotides are about 20 nucleotides in length.
[0668] APM1 cDNA probes labeled with an appropriate compound, such
as biotin, digoxigenin or fluorescent dye, are synthesized from the
appropriate mRNA population and then randomly fragmented to an
average size of 50 to 100 nucleotides. The said probes are then
hybridized to the chip. After washing as described in Lockhart et
al., supra and application of different electric fields (Sosnowsky
et al., 1997)., the dyes or labeling compounds are detected and
quantified. Duplicate hybridizations are performed. Comparative
analysis of the intensity of the signal originating from cDNA
probes on the same target oligonucleotide in different cDNA samples
indicates a differential expression of APM1 mRNA.
[0669] Methods for Inhibiting the Expression of an APM1 Gene
[0670] Other therapeutic compositions according to the present
invention comprise advantageously an oligonucleotide fragment of
the nucleic sequence of APM1 as an antisense tool or a triple helix
tool that inhibits the expression of the corresponding APM1 gene. A
preferred fragment of the nucleic sequence of APM1 comprises an
allele of at least one of the biallelic markers A1 to A26.
[0671] Antisense Approach
[0672] Preferred methods using antisense polynucleotide according
to the present invention are the procedures described by Sczakiel
et al. (1995).
[0673] Preferably, the antisense tools are chosen among the
polynucleotides (15-200 bp long) that are complementary to the
5'end of the APM1 mRNA. In another embodiment, a combination of
different antisense polynucleotides complementary to different
parts of the desired targeted gene are used.
[0674] Preferred antisense polynucleotides according to the present
invention are complementary to a sequence of the mRNAs of APM1 that
contains either the translation initiation codon ATG or a splicing
donor or acceptor site.
[0675] The antisense nucleic acids should have a length and melting
temperature sufficient to permit formation of an intracellular
duplex having sufficient stability to inhibit the expression of the
APM1 mRNA in the duplex. Strategies for designing antisense nucleic
acids suitable for use in gene therapy are disclosed in Green et
al., (1986) and Izant and Weintraub, (1984), the disclosures of
which are incorporated herein by reference.
[0676] In some strategies, antisense molecules are obtained by
reversing the orientation of the APM1 coding region with respect to
a promoter so as to transcribe the opposite strand from that which
is normally transcribed in the cell. The antisense molecules may be
transcribed using in vitro transcription systems such as those
which employ T7 or SP6 polymerase to generate the transcript.
Another approach involves transcription of APM1 antisense nucleic
acids in vivo by operably linking DNA containing the antisense
sequence to a promoter in a suitable expression vector.
[0677] Alternatively, suitable antisense strategies are those
described by Rossi et al. (1991), in the International Applications
Nos. WO 94/23026, WO 95/04141, WO 92/18522 and in the European
Patent Application No. EP 0 572 287 A2
[0678] An alternative to the antisense technology that is used
according to the present invention consists in using ribozymes that
will bind to a target sequence via their complementary
polynucleotide tail and that will cleave the corresponding RNA by
hydrolyzing its target site (namely "hammerhead ribozymes").
Briefly, the simplified cycle of a hammerhead ribozyme consists of
(1) sequence specific binding to the target RNA via complementary
antisense sequences; (2) site-specific hydrolysis of the cleavable
motif of the target strand; and (3) release of cleavage products,
which gives rise to another catalytic cycle. Indeed, the use of
long-chain antisense polynucleotide (at least 30 bases long) or
ribozymes with long antisense arms are advantageous. A preferred
delivery system for antisense ribozyme is achieved by covalently
linking these antisense ribozymes to lipophilic groups or to use
liposomes as a convenient vector. Preferred antisense ribozymes
according to the present invention are prepared as described by
Sczakiel et al. (1995), the specific preparation procedures being
referred to in said article being herein incorporated by
reference.
[0679] Triple Helix Approach
[0680] The APM1 genomic DNA may also be used to inhibit the
expression of the APM1 gene based on intracellular triple helix
formation.
[0681] Triple helix oligonucleotides are used to inhibit
transcription from a genome. They are particularly useful for
studying alterations in cell activity when it is associated with a
particular gene.
[0682] Similarly, a portion of the APM1 genomic DNA can be used to
study the effect of inhibiting APM1 transcription within a cell.
Traditionally, homopurine sequences were considered the most useful
for triple helix strategies. However, homopyrimidine sequences can
also inhibit gene expression. Such homopyrimidine oligonucleotides
bind to the major groove at homopurine:homopyrimidine sequences.
Thus, both types of sequences from the APM1 genomic DNA are
contemplated within the scope of this invention.
[0683] To carry out gene therapy strategies using the triple helix
approach, the sequences of the APM1 genomic DNA are first scanned
to identify 10-mer to 20-mer homopyrimidine or homopurine stretches
which could be used in triple-helix based strategies for inhibiting
APM1 expression. Following identification of candidate
homopyrimidine or homopurine stretches, their efficiency in
inhibiting APM1 expression is assessed by introducing varying
amounts of oligonucleotides containing the candidate sequences into
tissue culture cells which express the APM1 gene.
[0684] The oligonucleotides can be introduced into the cells using
a variety of methods known to those skilled in the art, including
but not limited to calcium phosphate precipitation, DEAE-Dextran,
electroporation, liposome-mediated transfection or native
uptake.
[0685] Treated cells are monitored for altered cell function or
reduced APM1 expression using techniques such as Northern blotting,
RNase protection assays, or PCR based strategies to monitor the
transcription levels of the APM1 gene in cells which have been
treated with the oligonucleotide.
[0686] The oligonucleotides which are effective in inhibiting gene
expression in tissue culture cells may then be introduced in vivo
using the techniques described above in the antisense approach at a
dosage calculated based on the in vitro results, as described in
antisense approach.
[0687] In some embodiments, the natural (beta) anomers of the
oligonucleotide units can be replaced with alpha anomers to render
the oligonucleotide more resistant to nucleases. Further, an
intercalating agent such as ethidium bromide, or the like, can be
attached to the 3' end of the alpha oligonucleotide to stabilize
the triple helix. For information on the generation of
oligonucleotides suitable for triple helix formation see Griffin et
al. (1989), which is hereby incorporated by this reference.
[0688] Throughout the application it is specifically contemplated
that in each case of a list of biallelic markers, or probes, or
primers, that list is also envisioned to include all but any one of
its members, or all but any two, or all but any three, until there
is only one remaining member. The examples that follow are
exemplary only, and not to be taken as meant to limit the invention
in any way.
EXAMPLES
Example 1
[0689] Identification of Biallelic Markers--DNA Extraction
[0690] Donors were unrelated and healthy. They presented a
sufficient diversity for being representative of a French
heterogeneous population. The DNA from 100 individuals was
extracted and tested for the detection of the biallelic
markers.
[0691] 30 mL of peripheral venous blood were taken from each donor
in the presence of EDTA. Cells (pellet) were collected after
centrifugation for 10 minutes at 2000 rpm. Red cells were lysed by
a lysis solution (50 mL final volume: 10 mM Tris pH7.6; 5 mM
MgCl.sub.2; 10 mM NaCl). The solution was centrifuged (10 minutes,
2000 rpm) as many times as necessary to eliminate the residual red
cells present in the supernatant, after resuspension of the pellet
in the lysis solution.
[0692] The pellet of white cells was lysed overnight at 42.degree.
C. with 3.7 ml of lysis solution composed of:
[0693] 3 mL TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0 4 M
[0694] 200 .mu.L SDS 10%
[0695] 500 .mu.L K-proteinase (2 mg K-proteinase in TE 10-2/NaCl
0.4 M).
[0696] For the extraction of proteins, 1 mL saturated NaCl (6M)
(1/3.5 v/v) was added. After vigorous agitation, the solution was
centrifuged for 20 minutes at 10000 rpm.
[0697] For the precipitation of DNA, 2 to 3 volumes of 100% ethanol
were added to the previous supernatant, and the solution was
centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed
three times with 70% ethanol to eliminate salts, and was
centrifuged for 20 minutes at 2000 rpm. The pellet was dried at
37.degree. C., and was resuspended in 1 mL TE 10-1 or 1 mL water.
The DNA concentration was evaluated by measuring the OD at 260 nm
(1 unit OD=50 .mu.g/mL DNA).
[0698] To determine the presence of proteins in the DNA solution,
the OD 260/OD 280 ratio was determined. Only DNA preparations
having a OD 260/OD 280 ratio between 1.8 and 2 were used in the
subsequent examples described below.
[0699] The pool was constituted by mixing equivalent quantities of
DNA from each individual.
Example 2
[0700] Identification of Biallelic Markers: Amplification of
Genomic DNA by PCR
[0701] The amplification of specific genomic sequences of the DNA
samples of example 1 was carried out on the pool of DNA obtained
previously. In addition, 50 individual samples were similarly
amplified.
[0702] PCR assays were performed using the following protocol:
4 Final volume 25 .mu.L DNA 2 ng/.mu.L MgCl.sub.2 2 mM dNTP (each)
200 .mu.M primer (each) 2.9 ng/.mu.L Ampli Taq Gold DNA polymerase
0.05 unit/.mu.L PCR buffer (10x = 0.1 M TrisHCl pH8.3 0.5M KCl)
1x
[0703] Each pair of first primers was designed using the sequence
information of the APM1 gene disclosed herein and the OSP software
(Hillier & Green, 1991). This first pair of primers was about
20 nucleotides in length and had the sequence corresponding to the
SEQ ID positions disclosed in Table 1 in the columns labeled PU and
RP.
5TABLE 1 Position of the amplicon in SEQ Position of PU Position of
RP Amplicon ID 1 PU primer in SEQ ID 1 RP primer in SEQ ID 1 9-27
3528-3946 B1 3528-3545 C1 3928-3946 9-28 3892-4321 B2 3892-3911 C2
4303-4321 99-14402 4155-4602 B3 4155-4175 C3 4584-4602 9-29
4223-4642 B4 4223-4242 C4 4623-4642 9-30 4599-5027 B5 4599-4618 C5
5008-5027 99-14387 10990-11442 B6 10990-11008 C6 11423-11442
99-14389 12472-12966 B7 12472-12491 C7 12946-12966 9-12 15073-15520
B8 15073-15092 C8 15503-15520 9-13 15131-15551 B9 15131-15150 C9
15532-15551 99-14405 15759-16211 B10 15759-15776 C10 16191-16211
9-14 16233-16652 B11 16233-16251 C11 16633-16652 9-15 16604-17025
B12 16604-16621 C12 17006-17025 9-16 16982-17402 B13 16982-17001
C13 17384-17402 9-17 17216-17517 B14 17216-17233 C14 17498-17517
9-18 17300-17503 B15 17300-17317 C15 17486-17503 17-30 730-1137 B16
730-752 C16 1117-1137 17-31 4798-5385 B17 4798-4819 C17 5364-5385
17-32 10614-11114 B18 10614-10635 C18 11093-11114 17-33 13843-14517
B19 13843-13865 C19 14496-14517 17-34 13843-14859 C20 14839-14859
17-35 14745-15219 B20 14745-14766 C21 15199-15219 17-36 15381-15987
B21 15381-15402 C22 15966-15987 17-37 17201-18261 B22 17201-17222
C23 18240-18261 17-38 18141-19336 B23 18141-18163 C24
19314-19336
[0704] Preferably, the primers contained a common oligonucleotide
tail upstream of the specific bases targeted for amplification
which was useful for sequencing.
[0705] Primers PU contain the following additional PU 5' sequence:
TGTAAAACGACGGCCAGT; primers RP contain the following RP 5'
sequence: CAGGAAACAGCTATGACC. The primer containing the additional
PU 5' sequence is listed in SEQ ID No 7. The primer containing the
additional RP 5' sequence is listed in SEQ ID No 8.
[0706] The synthesis of these primers was performed following the
phosphoramidite method, on a GENSET UFPS 24.1 synthesizer.
[0707] DNA amplification was performed on a Genius II thermocycler.
After heating at 95.degree. C. for 10 min, 40 cycles were
performed. Each cycle comprised: 30 sec at 95.degree. C.,
54.degree. C. for 1 min, and 30 sec at 72.degree. C. For final
elongation, 10 min at 72.degree. C. ended the amplification. The
quantities of the amplification products obtained were determined
on 96-well microtiter plates, using a fluorometer and Picogreen as
intercalant agent (Molecular Probes).
Example 3
[0708] Identification of Biallelic Markers--Sequencing of Amplified
Genomic DNA and Identification of Polymorphisms
[0709] The sequencing of the amplified DNA obtained in example 2
was carried out on ABI 377 sequencers. The sequences of the
amplification products were determined using automated dideoxy
terminator sequencing reactions with a dye terminator cycle
sequencing protocol. The products of the sequencing reactions were
run on sequencing gels and the sequences were determined using gel
image analysis (ABI Prism DNA Sequencing Analysis software (2.1.2
version) and the above mentioned proprietary "Trace"
basecaller).
[0710] The sequence data were further evaluated using the above
mentioned polymorphism analysis software designed to detect the
presence of biallelic markers among the pooled amplified fragments.
The polymorphism search was based on the presence of superimposed
peaks in the electrophoresis pattern resulting from different bases
occurring at the same position as described previously.
[0711] 15 fragments of amplification were analyzed. In 5 of these
segments, 8 biallelic markers were detected. The localization of
these biallelic markers are as shown in Table 2.
6TABLE 2 Biallelic Localization in Marker position in Amplicon
marker Marker Name APM1 gene Polymorphism SEQ ID No 1 9-27 A1
9-27/261 5` regulatory Allele 1: G 3787 region Allele 2: C 99-14387
A2 99-14387/129 Intron 1 Allele 1: A 11118 Allele 2: C 9-12 A3
9-12/48 Intron 1 Allele 1: T 15120 Allele 2: C 9-12 and A4 9-12/124
or Exon 2 Allele 1: T 15196 9-13 9-13/66 Allele 2: G 9-12 and A5
9-12/355 or Intron 2 Allele 1: G 15427 9-13 9-13/297 Allele 2: T
9-12 and A6 9-12/428 or Intron 2 Allele 1: A 15500 9-13 9-13/370
Allele 2: G 99-14405 A7 99-14405/105 Intron 2 Allele 1: G 15863
Allele 2: A 9-16 A8 9-16/189 Exon 3 Allele 1: A 17170 Allele 2: Del
17-30 A9 17-30-216 5` regulatory Allele 1: A 945 region Allele 2: G
9-27 A10 9-27-211 5` regulatory Allele 1: A 3738 region Allele 2: G
9-27 A11 9-27-246 5` regulatory Allele 1: A 3773 region Allele 2: G
17-31 A12 17-31-298 Intron 1 Allele 1: A 5095 Allele 2: G 17-31 A13
17-31-413 Intron 1 Allele 1: C 5210 Allele 2: T 17-32 A14 17-32-24
Intron 1 Allele 1: T 10637 Allele 2: C 99-14387 A15 99-14387-50
Intron 1 Allele 1: A 11039 Allele 2: C 99-14387 A16 99-14387-199
Intron 1 Allele 1: A 11188 Allele 2: G 17-33 A17 17-33- Intron 1
Allele 1: no 13973 TGAGACT insert Allele 2: TGAGACT insert 17-34
A18 17-34-860 Intron 1 Allele 1: A 14702 Allele 2: G 17-34 A19
17-34-915 Intron 1 Allele 1: A 14757 Allele 2: G 17-35 A20 17-35-71
Intron 1 Allele 1: C 14815 Allele 2: T 17-35 A21 17-35-306 Intron 1
Allele 1: G 15050 Allele 2: T 17-36 A22 17-36-47 Intron 2 Allele 1:
G 15680 Allele 2: C 17-36 A23 17-36-120 Intron 2 Allele 1: C 15790
Allele 2: T 17-37 A24 17-37-629 Exon 3 Allele 1: A 17829 Allele 2:
G 17-37 A25 17-37-811 Exon 3 Allele 1: A 18011 Allele 2: G 17-38
A26 17-38-349 Exon 3 Allele 1: C 18489 Allele 2: T
Example 4
[0712] Validation of the Polymorphisms Through Microsequencing
[0713] The biallelic markers identified in example 3 were further
confirmed and their respective frequencies were determined through
microsequencing. Microsequencing was carried out for each
individual DNA sample described in Example 1.
[0714] Amplification from genomic DNA of individuals was performed
by PCR as described above for the detection of the biallelic
markers with the same set of PCR primers (Table 1).
[0715] The preferred primers used in microsequencing were about 19
nucleotides in length and hybridized just upstream of the
considered polymorphic base. According to the invention, the
primers used in microsequencing are detailed in Table 3.
7TABLE 3 Position of mis 1 in Position of mis 2 In Marker Name
Marker Mis. 1 SEQ ID No 1 Mis. 2 SEQ ID No 1 9-27/261 A1 D1
3768-3786 E1 3788-3806 99-14387/129 A2 D2 11099-11117 E2
11119-11137 9-12/48 A3 D3 15101-15119 E3 15121-15139 9-12/124 or A4
D4 15177-15195 E4 15197-15215 9-13/66 9-12/355 or A5 D5 15408-15426
E5 15428-15446 9-13/297 9-12/428 or A6 D6 15481-15499 E6
15501-15519 9-13/370 99-14405/105 A7 D7 15844-15862 E7 15864-15882
9-16/189 A8 D8 17151-17169 E8 17171-17189 17-30-216 A9 D9 926-944
E9 946-964 9-27-211 A10 D10 3719-3737 E10 3739-3757 9-27-246 A11
D11 3754-3772 E11 3774-3792 17-31-298 A12 D12 5076-5094 E12
5096-5114 17-31-413 A13 D13 5191-5209 E13 5211-5229 17-32-24 A14
D14 10618-10636 E14 10638-10656 99-14387-50 A15 D15 11020-11038 E15
11040-11058 99-14387-199 A16 D16 11169-11187 E16 11189-11207 17-33-
A17 D17 13954-13972 E17 13974-13992 TGAGACT 17-34-860 A18 D18
14683-14701 E18 14703-14721 17-34-915 A19 D19 14738-14756 E19
14758-14776 17-35-71 A20 D20 14796-14814 E20 14816-14834 17-35-306
A21 D21 15031-15049 E21 15051-15069 17-36-47 A22 D22 15661-15679
E22 15681-15699 17-36-120 A23 D23 15771-15789 E23 15791-15809
17-37-629 A24 D24 17810-17828 E24 17830-17848 17-37-811 A25 D25
17992-18010 E25 18012-18030 17-38-349 A26 D26 18470-18488 E26
18490-18508
[0716] Mis 1 and Mis 2 refer to microsequencing primers that
hybridize with the non-coding strand of the APM1 gene and with the
coding strand of the APM1 gene, respectively.
[0717] The microsequencing reaction was performed as follows:
[0718] After purification of the amplification products, the
microsequencing reaction mixture was prepared by adding, in a 20
.mu.L final volume: 10 pmol microsequencing oligonucleotide, 1 U
Thermosequenase (Amersham E79000G), 1.25 .mu.L Thermosequenase
buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl.sub.2), and the two
appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set
401095) complementary to the nucleotides at the polymorphic site of
each biallelic marker tested, following the manufacturer's
recommendations. After 4 minutes at 94.degree. C., 20 PCR cycles of
15 sec at 55.degree. C., 5 sec at 72.degree. C., and 10 sec at
94.degree. C. were carried out in a Tetrad PTC-225 thermocycler (MJ
Research). The unincorporated dye terminators were then removed by
ethanol precipitation. Samples were finally resuspended in
formamide-EDTA loading buffer and heated for 2 min at 95.degree. C.
before being loaded on a polyacrylamide sequencing gel. The data
were collected by an ABI PRISM 377 DNA sequencer and processed
using the GENESCAN software (Perkin Elmer).
[0719] Following gel analysis, data were automatically processed
with software that allows the determination of the alleles of
biallelic markers present in each amplified fragment.
[0720] The software evaluates such factors as whether the
intensities of the signals resulting from the above microsequencing
procedures are weak, normal, or saturated, or whether the signals
are ambiguous. In addition, the software identifies significant
peaks (according to shape and height criteria). Among the
significant peaks, peaks corresponding to the targeted site are
identified based on their position. When two significant peaks are
detected for the same position, each sample is categorized
classification as homozygous or heterozygous type based on the
height ratio.
[0721] Oligonucleotide probes may be used in genotyping biallelic
markers by hybridization assays. The nucleic acid sample is
contacted with one or more allele specific oligonucleotide probes
which, specifically hybridize to one or more alleles associated
with a detectable phenotype. The probes are 25-mers with an
APM1-related biallelic marker in the center position. Probes used
in the hybridization assay may include the probes listed in Table
4.
8 TABLE 4 Position range of probes in SEQ BM Marker Name ID No
genomic Probes A1 9-27/261 3775 3799 P1 A2 99-14387/129 11106 11130
P2 A3 9-12/48 15108 15132 P3 A4 9-12/124 15184 15208 P4 A5 9-12/355
15415 15439 P5 A6 9-12/428 15488 15512 P6 A7 99-14405/105 15851
15875 P7 A8 9-16/189 17158 17182 P8 A9 17-30-216 933 957 P9 A10
9-27-211 3726 3750 P10 A11 9-27-246 3761 3785 P11 A12 17-31-298
5083 5107 P12 A13 17-31-413 5198 5222 P13 A14 17-32-24 10625 10649
P14 A15 99-14387-50 11027 11051 P15 A16 99-14387-199 11176 11200
P16 A17 17-33-TGAGACT 13961 13985 P17 A18 17-34-860 14690 14714 P18
A19 17-34-915 14745 14769 P19 A20 17-35-71 14803 14827 P20 A21
17-35-306 15038 15062 P21 A22 17-36-47 15668 15692 P22 A23
17-36-120 15778 15802 P23 A24 17-37-629 17817 17841 P24 A25
17-37-811 17999 18023 P25 A26 17-38-349 18477 18501 P26
Example 5
[0722] Preparation of Antibody Compositions to the 56-Glu Variant
of APM1
[0723] Substantially pure protein or polypeptide is isolated from
transfected or transformed cells containing an expression vector
encoding the APM1 protein or a portion thereof. The concentration
of protein in the final preparation is adjusted, for example, by
concentration on an Amicon filter device, to the level of a few
micrograms/mL. Monoclonal or polyclonal antibody to the protein can
then be prepared as follows:
[0724] A. Monoclonal Antibody Production by Hybridoma Fusion
[0725] Monoclonal antibody to epitopes in the APM1 protein or a
portion thereof can be prepared from murine hybridomas according to
the classical method of Kohler, G. and Milstein, C., Nature 256:495
(1975) or derivative methods thereof. Also see Harlow, E., and D.
Lane. 1988. Antibodies A Laboratory Manual. Cold Spring Harbor
Laboratory. pp. 53-242.
[0726] Briefly, a mouse is repetitively inoculated with a few
micrograms of the APM1 protein or a portion thereof over a period
of a few weeks. The mouse is then sacrificed, and the antibody
producing cells of the spleen isolated. The spleen cells are fused
by means of polyethylene glycol with mouse myeloma cells, and the
excess unfused cells destroyed by growth of the system on selective
media comprising aminopterin (HAT media). The successfully fused
cells are diluted and aliquots of the dilution placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as ELISA, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Davis, L. et al. Basic Methods
in Molecular Biology Elsevier, N.Y. Section 21-2.
[0727] B. Polyclonal Antibody Production by Immunization
[0728] Polyclonal antiserum containing antibodies to heterogeneous
epitopes in the APM1 protein or a portion thereof can be prepared
by immunizing suitable non-human animal with the APM1 protein or a
portion thereof, which can be unmodified or modified to enhance
immunogenicity. A suitable non-human animal is preferably a
non-human mammal is selected, usually a mouse, rat, rabbit, goat,
or horse. Alternatively, a crude preparation which has been
enriched for APM1 concentration can be used to generate antibodies.
Such proteins, fragments or preparations are introduced into the
non-human mammal in the presence of an appropriate adjuvant (e.g.
aluminum hydroxide, RIBI, etc.) which is known in the art. In
addition the protein, fragment or preparation can be pretreated
with an agent which will increase antigenicity, such agents are
known in the art and include, for example, methylated bovine serum
albumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface
antigen, and keyhole limpet hemocyanin (KLH). Serum from the
immunized animal is collected, treated and tested according to
known procedures. If the serum contains polyclonal antibodies to
undesired epitopes, the polyclonal antibodies can be purified by
immunoaffinity chromatography.
[0729] Effective polyclonal antibody production is affected by many
factors related both to the antigen and the host species. Also,
host animals vary in response to site of inoculations and dose,
with both inadequate or excessive doses of antigen resulting in low
titer antisera. Small doses (ng level) of antigen administered at
multiple intradermal sites appears to be most reliable. Techniques
for producing and processing polyclonal antisera are known in the
art, see for example, Mayer and Walker (1987). An effective
immunization protocol for rabbits can be found in Vaitukaitis, J.
et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).
[0730] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of
Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc.
For Microbiol., Washington, D.C. (1980).
[0731] Antibody preparations prepared according to either the
monoclonal or the polyclonal protocol are useful in quantitative
immunoassays which determine concentrations of antigen-bearing
substances in biological samples; they are also used
semi-quantitatively or qualitatively to identify the presence of
antigen in a biological sample. The antibodies may also be used in
therapeutic compositions for killing cells expressing the protein
or reducing the levels of the protein in the body.
Example 6
[0732] Association Between Apm1 Markers and Characteristics in an
Obese Population
[0733] Materials and Methods:
[0734] Patients
[0735] Subjects of this study were unrelated and lived in the
region of Paris. Obese girls were severely obese since early
childhood and exceeded the 98.sup.th percentile of normal growth
curves. Blood sampling and testing of these subjects were performed
prior to any weight reduction treatment. At the time of admission,
weights and heights were recorded, blood samples were collected,
the buffy coat was isolated for DNA preparation and the plasma was
separated for biochemical analysis. A summary of their biochemical
characteristics is listed in Table 5. In this first study, genotype
analysis was performed for markers 9-27/261, 99-14387/129,
9-12/124, 99-14405/105, and 9-16/189.
9TABLE 5 Characteristics of Obese Adolescent Girls used in Study 1
Parameter Value n 159 Mean .+-. SEM Age (yrs) 12.1 .+-. 0.3 Body
mass index (kg/m.sup.2) 30.5 .+-. 0.5 Cholesterol (mg/dl) 172 .+-.
3.0 FFA (mM) 0.612 .+-. 0.022 Glucose (mg/dl) 76.3 .+-. 0.81
Insulin (.mu.U/ml) 16.4 .+-. 0.67 Leptin (ng/ml) 35.7 .+-. 1.57
[0736] A second group of both obese girls and boys was also used to
confirm some results of the first study (Table 6). Genotype
analysis was performed for markers 9-12/48, 9-12/124, 9-12/355,
99-14405/105, and 9-16/189.
[0737] All parents of obese children provided informed consent for
biological testing and the use of DNA for genetic analysis. The
study protocol was approved by the Comit Consultatif de Protection
des Personnes Participants la Recherche Clinique.
10TABLE 6 Characteristics of Obese Adolescent Boys and Girls used
in Study 2 Parameter Value n 155 Boys 55 Girls 100 Mean .+-. SEM
Age (yrs) 12.0 .+-. 0.3 Body mass index (kg/m.sup.2) 29.2 .+-. 0.5
Cholesterol (mg/dl) 171 .+-. 0.02 FFA (mM) 0.592 .+-. 0.021 Glucose
(mg/dl) 74.5 .+-. 0.59 Insulin (.mu.U/ml) 14.8 .+-. 0.72 Leptin
(ng/ml) 31.2 .+-. 1.62
[0738] DNA Extraction
[0739] Blood samples were centrifuged 20 min at 913.times.g. The
middle leukocyte layer was removed and washed twice in large
volumes of 10 mM Tris HCl, pH 7.6 containing 5 mM MgCl.sub.2 and 10
mM NaCl. To the cell pellet was added 3 ml of 10 mM Tris HCl, pH
7.6 containing 1 mM EDTA and 0.4 mM NaCl, 200 .mu.l 10% (w/v) SDS,
and 500 .mu.l proteinase K (Sigma, St. Louis, Mo.; 1 mg/ml). Tubes
were placed in a shaking water bath at 42.degree. C. for 5 h. Tubes
were then chilled on ice for 10 min. To precipitate proteins, 1 ml
of 5 M NaCl was added and the precipitates were pelleted, and the
supernatant removed. To precipitate the DNA, isopropanol (5 ml) was
added, followed by recentrifugation at 3210.times.g for 20 min. The
supernatant was discarded and 5 ml of 70% ethanol was added to the
DNA pellet. After incubating 6 h or overnight at 4.degree. C., the
samples were spun at 2800.times.g for 5 min. The supernatant was
poured off and discarded, and the pellet left to air dry. Once dry,
1.5 ml 10 mM Tris HCl containing 10 mM EDTA was added and incubated
at room temperature on a rocker platform to rehydrate the DNA. DNA
concentration was measured and the DNA was stored at -20.degree.
C.
[0740] Single Nucleotide Polymorphism (SNP) Identification
[0741] Amplicons investigated covered the APM1 gene. Random markers
were generated from amplicons derived from BAC sequence positioned
in the indicated genomic regions (Table 7). The PCR primers were
then used to amplify the corresponding genomic sequence in a pool
of DNA from 100 unrelated individuals (blood donors of French
origin). PCR reactions (25 ml) contained 2 ng/.mu.l pooled DNA, 2
mM MgCl.sub.2, 200 .mu.M of each dNTP, 2.9 ng/.mu.l each primer,
0.05 unit/.mu.l Ampli Taq Gold DNA polymerase (Perkin Elmer, Foster
City, Calif.) and 1.times. PCR buffer (10 mM Tris HCl pH 8.3, 50 mM
KCl). Amplification reactions were performed in a PTC200 MJ
Research Thermocycler, with initial denaturation at 95.degree. C.
for 30 sec, annealing at 54.degree. C. for 1 min, and extension at
72.degree. C. for 30 sec. After cycling, a final elongation step
was performed at 72.degree. C. for 10 min. Amplification products
from pooled DNA samples were sequenced on both strands by
fluorescent automated sequencing on ABI 377 sequences (Perkin
Elmer), using a dye-primer cycle analysis and DNA sequence
extraction with ABI Prism DNA sequencing Analysis software.
Sequence data analysis was automatically processed with AnaPolys
(Genset, Paris, France), a software designed to detect the presence
of SNPs among pooled amplified fragments. The polymorphism search
is based on the presence of superimposed peaks in the
electrophoresis pattern from both strands, resulting from two bases
occurring at the same position. The detection limit for the
frequency of SNPs detected by microsequencing pools of 100
individuals is about 10% for the minor allele, as verified by
sequencing pool of known allelic frequencies. However, more than
90% of the SNPs detected by the pooling method have a frequency for
minor allele higher than 20%.
11TABLE 7 Characteristics of Random SNPs* Allele Hardy-Weinberg
Random Chromosomal Allelic Frequency Equilibrium SNPs Localization
variation (%) .chi..sup.2 A 7p12-p14 T .fwdarw. C 65 0.252 B 13q22
T .fwdarw. C 74 1.194 C 14q24.1 A .fwdarw. G 54 0.027 D 14q31 T
.fwdarw. C 62 0.322 E 14q31 G .fwdarw. C 64 0.092 F 14q22-q23 T
.fwdarw. A 79 0.594 G 16q22-q24 G .fwdarw. A 54 1.166 H 16q24 A
.fwdarw. G 62 0.656 I 18p11-p31 A .fwdarw. G 51 0.319 J 21q22.8 A
.fwdarw. G 56 0.054 K 21q22 C .fwdarw. T 59 1.475 L 21q22.3 A
.fwdarw. G 70 2.070 M 21q22.3 T .fwdarw. C 60 1.709 N 21q22,1 A
.fwdarw. G 56 1.060 *SNPs were identified using a pool of 100 DNA
clones, as described in the Experimental Procedures. The allele
frequency and Hardy-Weinberg equilibrium were measured for each
marker.
[0742] Genotyping
[0743] Genotyping of individual DNA samples was performed using
microsequencing procedure as follows. Amplification products
containing the SNPs were obtained by performing PCR reactions
similar as those described for SNP identification. After
purification of the amplification products, the microsequencing
reaction mixture was prepared by adding in a 20 .mu.l final volume:
10 pmol microsequencing primer (which hybridize just upstream of
the polymorphic base), 1 U of Thermosequenase (Amersham Pharmacia
Biotech, Piscataway, N.J.) or TaqFS (Perkin Elmer) and the 2
appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set)
complementary to the nucleotides at the polymorphic site of each
SNP tested. After 4 minutes at 94.degree. C.; 20 microsequencing
cycles of 15 sec at 55.degree. C., 5 sec at 72.degree. C., and 10
sec at 94.degree. C. were carried out in a GeneAmp PCR System 9700
(PE Applied Biosystem). After reaction, the 3'-extended primers
were precipitated to remove the unincorporate fluorescent ddNTPs
and analysed by electrophoresis on ABI 377 sequencers. Following
gel analysis with GENESCAN software (Perkin Elmer), data were
automatically processed with AnaMIS (Genset). Genotype data were
compiled and checked for scoring accuracy with 32 duplicate
samples.
[0744] Biochemical Analysis
[0745] Plasma biological parameters were determined using
commercially available kits and following manufacturer
instructions: (triglycerides, total cholesterol, and glucose: Roche
Molecular Biochemicals; FFA: Wako Chemical, Neuss, Germany; leptin
and insulin: RIA from Linco, St. Charles, Mo.).
[0746] Statistical Analysis.
[0747] Allelic frequencies and .chi.2 test of Hardy Weinberg
proportions were performed as data were collected (1-3). ANOVA was
used for comparison of difference in time series. Two tailed t-test
was used to compare the difference at each time point and .chi.2
analysis was used for comparison of proportions.
[0748] Results
[0749] In this example, we refer to 9-27/261 as SNP1, 99-14387/129
as SNP#2, 9-12/124 as SNP#3, 99-14405/105 as SNP#4, and 9-16/189 as
SNP#5. The approximate location of the markers on a schematic (not
to scale) drawing of the genomic structure of the Apm1 gene is
provided in FIG. 1. The exact location of the markers in the
genomic sequence of Apm1 is given in the sequence listing and in
tables 1-4.
[0750] The effect of Apm1 polymorphisms on plasma lipid values in
obese adolescent girls was examined by separating the study
population into 2 groups based on their mean lipid value: one group
with values above the mean, and the second group with values below
the mean. The genotype frequencies of the two sample groups were
then measured and analyzed for statistical significance using the
.chi.2 test for each lipid parameter. Similar analyses were
performed for 14 random markers generated, where the mean and
99.99% confidence interval are indicated as a solid and dotted
line, respectively. This served as our negative control.
[0751] The results show that the genotype frequencies for SNP# 3-5
are significantly different for obese adolescent girls with low
(i.e., below the mean) or high (i.e., above the mean) free fatty
acids (FFA) levels (FIG. 2). The effect of Apm1 polymorphism on FFA
in obese adolescents girls was also assessed in study I by
comparing the mean values of FFA between the homozygote
populations, with the heterozygotes included with either of the
homozygotes. The significance of the difference in FFA levels is
shown for SNP# 4 and 5 in FIG. 4. This indicates that high FFA
levels are associated with a specific genotype. The results
presented in Table 8 show that the significant relationship between
plasma FFA and genotype of Apm1 SNP#4 that was observed in a
population of obese adolescent girls was not observed with any
other parameters. The n value is reduced since all patients in
which FFA were not determined were eliminated.
12TABLE 8 Effect of ACRP30 SNP #4 (99-14405/105) on Clinical and
Biochemical Parameters in Obese Adolescent Girls in Study 1 Total
p-value Parameter Population GG AG + AA (GG vs AG + AA) n 106 37 69
-- Mean .+-. SEM Age (yrs) 11.3 .+-. 0.3 11.2 .+-. 0.4 11.4 .+-.
0.4 NS Body mass index (kg/m.sup.2) 29.5 .+-. 0.5 29.4 .+-. 0.7
29.6 .+-. 0.7 NS Leptin (ng/ml) 34.2 .+-. 1.5 33.9 .+-. 2.6 34.4
.+-. 1.8 NS Insulin (.mu.U/ml) 16.9 .+-. 0.8 17.0 .+-. 1.1 16.8
.+-. 1.1 NS Glucose (mg/dl) 74.6 .+-. 0.6 74.7 .+-. 1.1 74.6 .+-.
0.8 NS Triglycerides (mg/dl) 106.7 .+-. 5.4 96.8 .+-. 6.4 112.0
.+-. 7.5 NS Cholesterol (mg/dl) 172.0 .+-. 3.6 165.3 .+-. 4.3 176.5
.+-. 5.0 NS FFA (mM) 0.612 .+-. 0.022 0.525 .+-. 0.031 0.659 .+-.
0.029 0.0037
[0752] The comparison of genotype and biochemical characteristics
of the study 2 population (mixture of boys and girls) is shown in
Table 9. As in Table 8, the FFA are significantly different in the
2 sample groups (AG+AA versus GG). As for Table 8, only those
patients with data on FFA were kept in this analysis.
[0753] The effect of APM1 polymorphism on respiratory quotient in
obese adolescents was determined in study 2, where the respiratory
quotient was measured and compared to the genotype profile of the
SNP#4 and 5, as in FIG. 4. A low respiratory quotient is associated
with the same genotype that indicates a high FFA level, and vice
versa (FIG. 5). This is true for both markers #4 and 5. These
results are also shown in Table 9.
13TABLE 9 Effect of ACRP30 SNP 99-14405/105 on Clinical and
Biochemical Parameters in Obese Adolescent Boys and Girls Total
p-value Parameter Population GG AG + AA (GG vs AG + AA) n 97 37 60
-- Boys 32 13 19 -- Girls 65 24 41 Mean .+-. SEM Age (yrs) 11.4
.+-. 0.3 11.1 .+-. 0.5 11.5 .+-. 0.4 NS Body mass index
(kg/m.sup.2) 29.7 .+-. 0.6 29.4 .+-. 0.8 29.9 .+-. 0.8 NS Leptin
(ng/ml) 31.3 .+-. 1.5 29.1 .+-. 2.6 32.6 .+-. 1.9 NS Insulin
(.mu.U/ml) 16.2 .+-. 0.8 15.5 .+-. 0.9 16.6 .+-. 1.4 NS Glucose
(mg/dl) 74.9 .+-. 0.6 75.1 .+-. 1.0 74.8 .+-. 0.8 NS Triglycerides
(mg/dl) 110.5 .+-. 0.1 106.7 .+-. 0.1 112.7 .+-. 0.1 NS Cholesterol
(mg/dl) 167.7 .+-. 0.03 163.5 .+-. 0.04 170.5 .+-. 0.03 NS FFA (mM)
0.599 .+-. 0.021 0.545 .+-. 0.033 0.633 .+-. 0.027 0.046
Respiratory quotient 0.834 .+-. 0.005 0.848 .+-. 0.009 0.826 .+-.
0.005 0.026
[0754] The effect of Apm1 polymorphisms on the leptin/BMI
relationship in obese adolescents girls was also tested using a
similar analysis as for the lipid values reported in FIG. 2, but
using leptin/BMI ratio as the parameter. FIG. 3A shows the
significant correlation between leptin levels and BMI; this has
previously been reported. FIG. 3B shows a significant difference in
genotype frequencies for SNP# 1-2.
[0755] The effect of APM1 on leptin/BMI ratio in obese adolescents
girls was further analyzed using a similar analysis as that
described for FIG. 5 using leptin/BMI ratios calculated from those
values measured in study 1. The results indicate a significant
difference in leptin/BMI ratio between the 2 homozygote populations
(FIG. 6). There was a less significant difference (p=0.07), if the
heterozygote population was added to the AA population. This
increased the n value significantly versus the CC population, and
hence, reduced the power of the test. We would expect that with a
bigger population size, this may become significant.
[0756] The effect of Apm1 polymorphism on glucose tolerance in
obese adolescent girls was also determined. The difference in
glucose tolerance, calculated as shown on the y-axis, was highly
significant between the two homozygote populations of SNP#2 in
obese adolescent girls (FIG. 7).
[0757] Apm1 function was predicted from polymorphism and in vivo
analysis (FIG. 8). Based on the analysis of the polymorphisms, we
would expect that this protein is directly implicated in the
regulation of FFA metabolism. In vivo studies indicate that an
active form of APM1 does decrease FFA in the circulation. The
parallel decrease of plasma triglycerides suggests that the FFA are
not being converted to triglycerides, but rather oxidized. The
correlation of FFA concentrations with insulin resistance would
suggest that insulin resistance would be decreased with lower
circulating FFA. This, in turn, would create an environment more
responsive to insulin, and hence, improve glucose tolerance.
[0758] Overall, these results demonstrate the utility of APM1
markers in assays for detecting a patient's ability or inability to
oxidize FFA, particularly those derived from dietary lipid. This
inability to oxidize FFA would contribute to increased accumulation
of FFA in storage by the adipose tissue, which would lead to the
eventual development of obesity.
[0759] The presence of FFA is also directly related to insulin
resistance. Therefore this would also reflect a patient's ability
to manage high levels of glucose, and his/her susceptibility
towards the development of type II diabetes.
[0760] While preferred embodiments of the invention have been
illustrated and described, it will be appreciated that various
changes can be made therein by one skilled in the art without
departing from the spirit and scope of the invention.
References
[0761] Abbondanzo S. J. et al. (1993) Methods in Enzymology,
Academic Press, New York. pp. 803-823.
[0762] Ajioka R. S. et al. (1997) Am. J. Hum. Genet.
60:1439-1447.
[0763] Anton M. (1995) et al., J. Virol. 69: 4600-4606.
[0764] Araki K et al. (1995) Proc. Natl. Acad. Sci. USA.
92(1):160-4.
[0765] Ausubel et al. (1989) Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley Interscience,
N.Y.
[0766] Bates G. P. et al. (1997a) Hum. Mol. Genet.
6(10):633-1637.
[0767] Bates G P et al. (1997b) Molecular Medicine today,
508:515.
[0768] Baubonis W. (1993) Nucleic Acids Res. 21(9):2025-9.
[0769] Beaucage et al. (1981) Tetrahedron Lett. 22:1859-1862.
[0770] Bradley A., (1987) Production and analysis of chimeric mice.
In: E. J. Robertson (Ed.), Teratocarcinomas and embryonic stem
cells: A practical approach. IRL Press, Oxford, pp. 113.
[0771] Brown E. L., Belagaje R., Ryan M. J., Khorana H. G. (1979)
Methods Enzymol. 68:109-151.
[0772] Burright et al. (1997) Brain Pathology. 7:965-977.
[0773] Chai H. et al. (1993) Biotechnol. Appl.
Biochem.18:259-273.
[0774] Chee et al. (1996) Science. 274:610-614.
[0775] Chen et al. (1987) Mol. Cell. Biol. 7:2745-2752.
[0776] Chen and Kwok (1997) Nucleic Acids Research. 25:347-353.
[0777] Chen et al. (1997) Proc. Natl. Acad Sci. USA.
94(20):10756-10761.
[0778] Chou J. Y. (1989) Mol. Endocrinol. 3:1511-1514.
[0779] Clark A. G. (1990) Mol. Biol. Evol. 7:111-122.
[0780] Coles R., Caswell R., and Rubinsztein D. C. (1998) Hum. Mol.
Genet. 7(5):791-800.
[0781] Compton J. (1991) Nature. 350(6313):91-92.
[0782] Davies S. W., Turmaine M., Cozens B. A., DiFiglia M., Sharp
A. H., Ross C. A., Scherzinger E., Feldman and Steg. (1996)
Medecine/Sciences. 12:47-55.
[0783] Dempster et al., (1977) J R. Stat. Soc., 39B:1-38.
[0784] Dent D. S. and Latchman D. S. (1993) The DNA mobility shift
assay. In: Transcription Factors: A Practical Approach (Latchman D
S, ed.) Oxford: IRL Press. pp1-26.
[0785] Eckner R. et al. (1991) EMBO J. 10:3513-3522.
[0786] Excoffier L. and Slatkin M. (1995) Mol. Biol. Evol., 12(5):
921-927.
[0787] Flotte et al. (1992) Am. J. Respir. Cell Mol. Biol.
7:349-356.
[0788] Fodor et al. (1991) Science 251:767-777.
[0789] Fraley et al. (1979) Proc. Natl. Acad. Sci. USA.
76:3348-3352.
[0790] Fried M. and Crothers D. M. (1981) Nucleic Acids Res.
9:6505-6525.
[0791] Fuller S. A. et al. (1996) Immunology in Current Protocols
in Molecular Biology, Ausubel et al. Eds, John Wiley & Sons,
Inc., USA.
[0792] Furth P. A. et al. (1994) Proc. Natl. Acad. Sci USA.
91:9302-9306.
[0793] Garner M. M. and Revzin A. (1981) Nucleic Acids
Res.9:3047-3060.
[0794] Ghosh and Bacchawat (1991) Targeting of liposomes to
hepatocytes, IN: Liver Diseases, Targeted diagnosis and therapy
using specific receptors and ligands. Wu et al. Eds., Marcel
Dekeker, N.Y., pp. 87-104.
[0795] Gopal (1985) Mol. Cell Biol, 5:1188-1190.
[0796] Gossen M. et al. (1992) Proc. Natl. Acad. Sci. USA.
89:5547-5551.
[0797] Gossen M. et al. (1995) Science. 268:1766-1769.
[0798] Graham et al. (1973) Virology 52:456-457.
[0799] Green et al. (1986) Ann. Rev. Biochem. 55:569-597.
[0800] Griffin et al. (1989) Science. 245:967-971.
[0801] Grompe, M. et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86:5855-5892.
[0802] Grompe, M. (1993) Nature Genetics. 5:111-117.
[0803] Gu H. et al. (1993) Cell 73:1155-1164.
[0804] Gu H. et al. (1994) Science 265:103-106.
[0805] Guatelli J C et al. Proc. Natl. Acad. Sci. USA.
35:273-286.
[0806] Gura. (1997) Science 275:751.
[0807] Hacia J. G., et al. (1996) Nat. Genet. 14(4):441-447.
[0808] Haff L. A. and Smirnov I. P. (1997) Genome Research,
7:378-388.
[0809] Hames B. D. and Higgins S. J. (1985) Nucleic Acid
Hybridization: A Practical Approach. Hames and Higgins Ed., IRL
Press, Oxford.
[0810] Harju L. et al. (1993) Clin Chem., 39(11 Pt
1):2282-2287.
[0811] Harland et al. (1985) J. Cell. Biol. 101: 1094-1095.
[0812] Hawley M. E. et al. (1994) Am. J. Phys. Anthropol.
18:104.
[0813] Hillier L. and Green P. (1991) Methods Appl. 1:124-8.
[0814] Hoess et al. (1986) Nucleic Acids Res. 14:2287-2300.
[0815] Hu E., Liang P., and Spiegelman B. M. (1 996) J. Biol. Chem.
271:10697-10703.
[0816] Huang L. et al. (1996) Cancer Res 56(5):1137-1141.
[0817] Huygen et al. (1996) Nature Medicine. 2(8):893-898.
[0818] Izant J. G. and Weintraub H. (1984) Cell
36(4):1007-1015.
[0819] Julan et al. (1992) J. Gen. Virol. 73:3251-3255.
[0820] Kanegae Y. et al., Nucl. Acids Res. 23:3816-3821.
[0821] Khoury J. et al. (1993) Fundamentals of Genetic
Epidemiology, Oxford University Press, N.Y.
[0822] Kim U -J. et al. (1996) Genomics 34:213-218.
[0823] Klein et al. (1987) Nature. 327:70-73.
[0824] Koller et al. (1992) Annu. Rev. Immunol. 10:705-730.
[0825] Kopp M. U., Mello A. J., Manz A., (1998) Science.
280(5366):1046-1048.
[0826] Kozal M. J. et al. (1996) Nat. Med. 2(7):753-759.
[0827] Landegren U. et al. (1998) Genome Research, 8:769-776.
[0828] Lander and Schork (1994) Science. 265:2037-2048.
[0829] Lange K. (1997) Mathematical and Statistical Methods for
Genetic Analysis. Springer, N.Y.
[0830] Lenhard T. et al. (1996) Gene. 169:187-190.
[0831] Lin M. W. et al. (1997) Hum. Genet. 99(3): 417-420.
[0832] Linton M. F. et al. (1993) J. Clin. Invest.
92:3029-3037.
[0833] Liu Z. et al. (1994) Proc. Natl. Acad. Sci. USA. 91:
4528-4262.
[0834] Livak K. J. and Hainer J. W. (1994) Hum. Mutat.
3(4):379-385.
[0835] Lockhart et al. (1996) Nature Biotechnology
14:1675-1680.
[0836] Mackey K., Steinkamp A., and Chomczynski P. (1998) Mol
Biotechnol. 9(1):1-5.
[0837] Maeda et al. (1996) Biochem. Biophys. Res. Comm.
221:286-289.
[0838] Mangiarini L., Sathasivam K., Mahal A., Mott R., Seller M.,
and Bates G. P. (1997) Nat. Genet. 15(2):197-200.
[0839] Mansour S. L. et al. (1988) Nature. 336:348-352.
[0840] Manz et al. (1993) Adv. in Chromatogr. 33:1-66.
[0841] Marshall R. L. et al. (1994) PCR Methods and Applications.
4:80-84.
[0842] McCormick et al. (1994) Genet. Anal. Tech. Appl.
11:158-164.
[0843] McLaughlin B. A. et al. (1996) Am. J. Hum. Genet.
59:561-569.
[0844] Montague et al. (1997) Nature. 387:903.
[0845] Morton N. E. (1955) Am. J. Hum. Genet. 7:277-318.
[0846] Muzyczka et al. (1992) Curr. Topics in Micro. and Immunol.
158:97-129.
[0847] Nada S. et al. (1993) Cell 73:1125-1135.
[0848] Nagy A. et al. (1993) Proc. Natl. Acad. Sci. USA. 90:
8424-8428.
[0849] Narang S. A., Hsiung H. M. (1979) Brousseau R., Methods
Enzymol. 68:90-98.
[0850] Neda et al. (1991) J. Biol. Chem. 266:14143-14146.
[0851] Newton et al. (1989) Nucleic Acids Res. 17:2503-2516.
[0852] Nickerson D. A. et al. (1990) Proc. Natl. Acad. Sci. U.S.A.
87:8923-8927.
[0853] Nicolau et al. (1982) Biochim. Biophys. Acta.
721:185-190.
[0854] Nyren P. et al. (1993) Anal. Biochem. 208(1): 171-175.
[0855] O'Reilly et al. (1992) Baculovirus Expression Vectors: A
Laboratory Manual. W. H. Freeman and Co., New York.
[0856] Ohno et al. (1994) Science. 265:781-784.
[0857] Orita et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:
2776-2770.
[0858] Ott J. (1991) Analysis of Human Genetic Linkage. John
Hopkins University Press, Baltimore.
[0859] Pastinen et al. (1997) Genome Research. 7:606-614.
[0860] Pease S. and William R. S. (1990) Exp. Cell. Res.
190:09-211.
[0861] Perlin et al. (1994) Am. J. Hum. Genet. 55:777-787.
[0862] Peterson et al. (1993) Proc. Natl. Acad. Sci. USA. 90:
7593-7597.
[0863] Pietu et al. (1996) Genome Research.6:492-503.
[0864] Potter et al. (1984) Proc. Natl. Acad. Sci. U.S.A.
81(22):7161-7165.
[0865] Reid L. H. et al. (1990) Proc. Natl. Acad. Sci. U.S.A.
87:4299-4303.
[0866] Risch, N. and Merikangas, K. (1996) Science.
273:1516-1517.
[0867] Robertson E. (1987) "Embryo-Derived Stem Cell Lines." In: E.
J. Robertson Ed. Teratocarcinomas And Embryonic Stem Cells: A
Practical Approach. IRL Press, Oxford, pp. 71.
[0868] Rossi et al. (1991) Pharmacol. Ther. 50:245-254.
[0869] Roth J. A. et al. (1996) Nature Medicine. 2(9):985-991.
[0870] Rougeot, C. et al. Eur. J. Biochem. 219(3):765-773.
[0871] Roux et al. (1989) Proc. Natl. Acad. Sci. U.S.A.
86:9079-9083.
[0872] Ruano et al. (1990) Proc. Natl. Acad. Sci. U.S.A.
87:6296-6300.
[0873] Sambrook, J., Fritsch, E. F., and T. Maniatis. (1989)
Molecular Cloning: A Laboratory Manual. 2ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
[0874] Samson M, et al. (1996) Nature, 382(6593):722-725.
[0875] Samulski et al. (1989) J. Virol. 63:3822-3828.
[0876] Sanchez-Pescador R. (1988) J. Clin. Microbiol.
26(10):1934-1938.
[0877] Sandou et al. (1994) Science. 265:1875-1878.
[0878] Sarkar, G. and Sommer S. S. (1991) Biotechniques.
[0879] Sauer B. et al. (1988) Proc. Natl. Acad. Sci. U.S.A.
85:5166-5170.
[0880] Schaid D. J. et al. (1996) Genet. Epidemiol. 13:423-450.
[0881] Schedl A. et al. (1993a) Nature. 362:258-261.
[0882] Schedl et al. (1993b) Nucleic Acids Res. 21:4783-4787.
[0883] Schena et al. (1995) Science. 270:467-470.
[0884] Schena et al. (1996) Proc. Natl. Acad. Sci. U.S.A.
93(20):10614-10619.
[0885] Schneider et al. (1997) Arlequin: A Software For Population
Genetics Data Analysis. University of Geneva.
[0886] Sczakiel G. et al. (1995) Trends Microbiol.
3(6):213-217.
[0887] Shay J. W. et al. (1991) Biochem. Biophys. Acta.
1072:1-7.
[0888] Sheffield, V. C. et al. (1991) Proc. Natl. Acad. Sci. U.S.A.
49:699-706.
[0889] Shizuya et al. (1992) Proc. Natl. Acad. Sci. U.S.A.
89:8794-8797.
[0890] Shoemaker D. D. et al. (1996) Nat. Genet. 14:450-456.
[0891] Smith (1957) Ann. Hum. Genet. 21:254-276.
[0892] Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165.
[0893] Sosnowski R. G. et al. (1997) Proc. Natl. Acad. Sci. U.S.A.
94:1119-1123.
[0894] Spielmann S. et al. (1993) Am. J. Hum. Genet.
52:506-516.
[0895] Spielmann S. and Ewens W. J. (1998) Am. J. Hum. Genet.
62:450-458.
[0896] Stemberg N. L. (1992) Trends Genet. 8:1-16.
[0897] Stemberg N. L. (1994) Mamm. Genome. 5:397-404.
[0898] Syvanen A. C. et al. (1994) Clin. Chim. Acta.
226(2):225-236.
[0899] Tacson et al. (1996) Nature Medicine. 2(8):888-892.
[0900] Te Riele et al. (1990) Nature. 348:649-651.
[0901] Terwilliger J. D. and Ott J. (1994) Handbook of Human
Genetic Linkage. John Hopkins University Press, London.
[0902] Thomas K. R. et al. (1986) Cell. 44:419-428.
[0903] Thomas K. R. et al. (1987) Cell. 51:503-512.
[0904] Tur-Kaspa et al. (1986) Mol. Cell. Biol. 6:716-718.
[0905] Tyagi et al. (1998) Nature Biotechnology. 16:49-53.
[0906] Urdea M. S. (1988) Nucleic Acids Research. 11:4937-4957.
[0907] Urdea M. S. et al. (1991) Nucleic Acids Symp. Ser.
24:197-200.
[0908] Van der Lugt et al. (1991) Gene. 105:263-267.
[0909] Vlasak R. et al. (1983) Eur. J. Biochem. 135:123-126.
[0910] Wabiko et al. (1986) DNA. 5(4):305-314.
[0911] Walker et al. (1996) Clin. Chem. 42:9-13.
[0912] Wanker E. E., Mangiarini L., and Bates G. P. (1997) Cell.
90(3):537-48.
[0913] Weir, B. S. (1996) Genetic data Analysis II: Methods for
Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland,
Mass., U.S.A.
[0914] White, M. B. et al. (1992) Genomics. 12:301-306.
[0915] White, M. B. et al. (1997) Genomics. 12:301-306.
[0916] Wong et al. (1980) Gene. 10:87-94.
[0917] Wood S. A. et al. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:4582-4585.
[0918] Wu and Wu (1987) J. Biol. Chem. 262:4429-4432.
[0919] Wu and Wu (1988) Biochemistry. 27:887-892.
[0920] Wu et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:2757.
[0921] Yagi T. et al. (1990) Proc. Natl. Acad. Sci. U.S.A.
87:9918-9922.
[0922] Zhao et al. (1998) Am. J. Hum. Genet. 63:225-240.
[0923] Zou Y. R. et al. (1994) Curr. Biol. 4:1099-1103.
[0924] Hill, W. G. (1974) in Heredity, (Edinburgh), pp.
229-239.
[0925] Terwilliger, J. O. (1994) Handbook for Human Genetic Linkage
(John Hopkins University Press, Baltimore).
[0926] Schneider, S., Kueffer, J. M., Roessli, D., & Excofier,
L. (1997) Arlequin: A software for population genetic data
analysis, 1.1 edition (Genetics and Biometry Laboratory, Department
of Anthropology, University of Geneva, Geneva).
Sequence CWU 1
1
8 1 20966 DNA Homo sapiens misc_feature (1)..(4811) 5' regulatory
region 1 gctgatctgc tgcctcagcc ttcccaaagt gctgtaattt attaggcata
agccactgtg 60 cctgcctagt gttgtacatt ctgtgggttt tgacaattgt
atgcatctac atgtatgtac 120 catttatagt attcctgttt ttaattttag
ccattctagt aggcatgtag tgatatctca 180 tggtgatttt aatttgcgtt
tccgtaatgg ttaataatgc tgaacatctt tgcatgtgct 240 tgtttgtcat
ttgtgtttcc tacttggtga aataattgtt catgtccttt gtccattttc 300
taattgaatt tttttttacc atttagtttt gagatttctt tatacaatct agatccaaat
360 ctcttgtctc aaatatggtt tgcaaataca ttcctctaat tcatatattg
ccttttcctc 420 ctcttaacag gatgtttcac agagcaaaag ttttagtttt
gttgaaatct cacttttcat 480 ttttttcttt agtggattgt gcttttgttg
tcatatgtaa gaactcttca ctggccctag 540 atccttgtat tggtttccta
agattgccat agcaaatcac catgaactta gtgacaaaaa 600 gacagaaatt
tattttcact tcctactgtg ggcagactag acgttaatta ttttcatgta 660
tgctcattcc tatgacatct ttctgatata ataattatag ttattcttaa gcttcaccct
720 tttttctatt agctttgtta ccttgggtgt cactttttct tttttgacat
tgtgacctat 780 gccagatcat gtctgttagt acttagccct ccattcacct
ctccataatc ccttttgtat 840 tcctggagct tgatgcctga aatgacacat
cctacattcc tttgccagat gggtaccagt 900 tagcttgtgc acatgggaga
caaccgtgaa aagactgaag tgggraagaa gggaggagct 960 gttgtgtttc
agtgagcgcc cttggcagtg gcggtgacag tggctcctgt tcagtggcaa 1020
tggtggagca gctagcaaga catgcagtaa gcgcaggctc ataggctatg gtccaggagc
1080 agtcaccgat tcctggtctt taggcaatat catctccctt tgcttctcca
gcctttctaa 1140 aattattgta ccttgactag tacaattttt tagtattggg
ggtagtccaa ggacacaggc 1200 tttaaaaagt atgaattcag ggttgcctac
ctgcattgac tgcgcttgaa tcatgatggc 1260 cttctggtcg gtggcaggag
gtgacagtcc aaatcatgca gtagcaaacc agatacttaa 1320 attatcatct
gagatacttc agaagtacag ccgtagccat accttcagaa gagataaaga 1380
aatgttctcc tggccaggcg cggtggctca cgcctgtcat tccagcactt tgggaggccg
1440 agggggtgga tcacctgagg tcgggagttc gagaccagcc tgaccaacat
ggggaaaccc 1500 tgtctctact aaaaatacaa aattagcggg gcgtggtggc
acatgcccat aatcccagct 1560 actcgggagg ctaaggcagg ataatcgctt
gaacctgaga ggcagaggtt gcggtgaact 1620 gagatcatgc catagtactc
cagcctgggc aacaagagtg aaactccatc tcaaaaaaaa 1680 aaaaaagaaa
aaaagataaa gaaatgttct cctttcttgc catttctagg ggtttgggga 1740
tggcgtacat tgctgcaggg cgtgctcact ctaccatctt gctccaatct ttatttttca
1800 aaatacagtg cttatgcttg gttacttcag ttaagattat ttttaaaaat
cataattaag 1860 caaaaatata tggccatgct taaacatatt taagataaat
taagtgattt ggcctgtttc 1920 agtatcccaa ctcacatgct aacaggggct
tgacctgtag ctacggtacc ctggaggaaa 1980 tgatcgcatt tatttggtta
tttcggtcta agtagtaata gttctgtcct gggaaaaaga 2040 ctagcctcaa
ggcatttctg attgaatgtt tttcaattac agtctttaaa ccagtatgcc 2100
acagaactgg ctctttccac atgacggcct ttgtggtggg tggcagattg ccctgaggcc
2160 tcgcaaaatg ctaggctttc acaatgtcac tgactgacag ccaggcccag
cacagtcttg 2220 gtgtgattgt ggggctaaag ttattccacc ttgtgcaata
gctacagcct tctctaacca 2280 gctgcattct tataaagtta gaagaaaata
cttttttttt tttgagatgg attctcgctc 2340 tgttgcccag gctggagtgc
aatggtgcga tctcggctcg ctgcaacctc cgcctcctgg 2400 gttcaaacga
ttctcctccc tcagaccccc gagtagctgg gattgcaggt gcctgccacc 2460
acgcccggct aacttttttg tatttttagt ggagacgggg tttcaccatc ttcgtcaggc
2520 tggtctcaga ctcctgacct caagtgatct gcccgcctca gcctcccaaa
atgctgggat 2580 tacaggcatg agctactgtg cccggccaaa gaaaatactt
tttatgccag ccctgaaact 2640 accctgaagc acatacatca accttgaggc
ctcacactcc atcaagaggg gtgaagggca 2700 tgaggaatta gaaagcatag
ggatttttag ttagacagat ctggttcaaa tcctagactt 2760 gtgccttgaa
caaattattt accctcattg aactctagat tcattatttg taaaatgaaa 2820
gacaataata gttatctcca aaggaaagtt gaatatgatc attcatttat tcattaattc
2880 aacatttatt attgcctact ttgtgccagg ttctattcta ggaactaagg
gatacaactt 2940 tgaataggca aaatctctgc tctcctgaag tttacttttt
tttttttttt ttgagacaga 3000 gtttcactct tgtcacccag gctggagcgc
aatggtgctc ttggctcact gcaacctcca 3060 cctcctgggt tcaagtgatt
ctcttgtctc agcctcccaa gtagctggga ctacaggtat 3120 gtgccaccac
gcccggctat ttctgcattt ttagtagaga tggggtttca ccatgttggc 3180
cagactggtc tcaaactcct gatctcaggt gatatgcctg tcttggcctt ccaaagtact
3240 gggattacag gcctgagcca ctgcacctga cctgaagttt atgttctatt
aaatagcaac 3300 agacagtaac ataaaccaaa aataaatagg aaaacaccat
aacaaaaatc aaacagtgat 3360 ataattgaga gttgcttcta tttctttttg
ttgtcttctt ggttcaatca gcctgctaaa 3420 ctatatggaa cctcattttc
atgggccact tatttaagcc gggggacctt ggaaagtctc 3480 tcatgtctct
catctcaacg gcctaatgtg acttctcttg aaatatttgg acattagcag 3540
gaagctgagg ctttacatca gatctttact ttaatggtgg acttgacttt actggtagat
3600 ttttaggctc tgtgtggact gtggagatga tatctggggg gcaggcagac
acttgccctg 3660 cctctgtctg agaaaattct gttttggatg tcttgttgaa
gttggtgctg gcatcctaag 3720 cccttgctgg ggtcgtartt taattcatca
gaatgtgtgg cttgcaagaa ccrgctcaga 3780 tcctgcsctt caaaaacaaa
acatgagcgt gccaagaaag tccaaggtgt tgaatgttgc 3840 cacttcaagc
ctaaactttc taggaacacc taagtgggtg gcagcttcca gttctccagg 3900
ctgcttctag gccagagctg ggttccacaa gagacagaat aggcatatat atgcttaagg
3960 aactggaaaa acaggctctc tctctctcac aaacacacac acacacatac
caaggtagct 4020 gtcaaaatgt tatccgaaat tttggaacca aaaaatcttg
aaagatggta ttccaatatc 4080 acattttatg taagttttct attatattag
attcaaatta cgattcgagg ccacaagctt 4140 taagaattca gggccttttt
aacttgccaa gccccacacc actccaggaa cttccccaca 4200 ccccagttct
cagaattcat gtgcaaggtc tttcctaaat ccagggtcca ggtcagagag 4260
tggaggatgt gctctatttc ttacctgatt gcagacccct ctgacagtgc tcccttctga
4320 agcactcact gtctgaacgt acacagtctc agacttaatc atgcacagtg
agcaagactg 4380 tggtgtgata attggcgtcc ctgacttatt agggcaaatc
tatgggaggg ggagacctcc 4440 tggaccactg agcaattaat tcatttacat
taggaagttt ctccgtcaga tgcaggaaaa 4500 aaatcttgtt ttcctgctgt
ggttttgact tttgccccat cttctgttgc tgttgtagga 4560 ggcaaaataa
gggtcaaggc ctggaaacac aagtgctttg actgaagctc cacttggctt 4620
ccgaagccca agctgggttg taccaggttc cctagggtgc aggctgtggg caactgccag
4680 ggacatgtgc ctgcccaccg gcctctggcc ctcactgagt tggccaatgg
gaaatgacaa 4740 ttgtgaggtg gggactgcct gcccccgtga gtaccaggct
gttgaggctg ggccatctcc 4800 tcctcacttc c att ctg act gca gtc tgt ggt
tct gat tcc ata cca gag 4850 Ile Leu Thr Ala Val Cys Gly Ser Asp
Ser Ile Pro Glu 1 5 10 g gtaagagcaa ttctgtgaag ttccaggctg
ggtgggggat gcatgcatag 4901 cctctggctg ggatcaccca ggctctcccg
tccgtagtag tgtgggagtg gatacaggtg 4961 gatactctgg tcagagcagc
actggtggag gcagatatgc actgggcttc ttcctccgtt 5021 ctcccacagc
cccaagagag aaagggttat ttcagacatt ccttctaaga tgcatggaac 5081
cattctgaat tttrcccagt tcgctctgta gcaggatacc tattgagaaa aagttagggt
5141 cagtaaggtg gaagggtctg tccacagatg aagtccaatt cgattaaggg
ggataaggga 5201 atacattgyc tcttagcttg accaggtagg gcaaaggaag
aagcatatat gaaggcagct 5261 tcagaaaagt caagctgagc actgacttca
gactggaatt aggaatccag ctctgccact 5321 ttattctact cagcaaatat
ttactgagca aattctatgg gctagacagt ggattgggtt 5381 cacaagatac
aatgagtgtg acatggttgt tgtctatgga tttggggata tatgtaggta 5441
tagggatatc ttacaaggta atcaagaggt tctaatgagg ccagccatgg tggctcacac
5501 ctgtaatccc agcaatttgg gagaccgagg cgggtggatc acctgaggtc
aggagttcca 5561 gactagcctg accaacatgg tgaaaccccg cctctaccaa
aaatacaaaa attagttggg 5621 cgtgatggca ggtgcctgta atcccagctt
ctcgggaggc tgaggcagga gaattgtctg 5681 aacctgggag gcagaggttg
cagtgagccg agattgttgc cactgcattc cagcctgggt 5741 gacagagcga
gactttgtgt caaaaaaaaa aaaaaaaaga aagaaaagaa aaagaggctc 5801
taatgagata aaatgagaaa agcctggcat gtagtggcaa cttatgaaaa attgtaatta
5861 aaaaaaaaca ttttctgaca gaagaaactg gatctacctg gtttttctga
agcctaatcc 5921 tgctcgcccc agtgagtgct gtttctgagg catcctggtt
gttttgagct gtggatgctg 5981 aaggttagag tgggagggat tttagaggtt
aggtctgccc ctcttgtgtt agaggacatg 6041 gatccctggt ctggagaggt
tctggttttt ggatcaagcc tcacaagggg tggcaccaac 6101 tcactcctag
gaactccgct agaaggaagg ccagctctgc ctaattcggt tggggagatg 6161
ggggtccctt tatgctagca gaatatgtcc gaaggagcat gatggtgtca gctttgttca
6221 tgaaggccag tggtacacag ggagcccggc agcttcctca gcagtccctg
ctgccactct 6281 tccttaagtc ttgaggagtc tttttttggc acaatctcag
ctcactgcaa cctccgcctc 6341 ccaggttcaa gcgattctcc tgcctcagtc
tcccaagtag ctgagactac aggcatgcgc 6401 caccacgccc agctaatttt
tatattttta gtagagatgg ggttcaccat attggccagg 6461 atggtctcga
tctcttgacc tcatattcca cctgcctcgg cctcccaaag tgctggtatt 6521
acaggtgtga gccactgcgc ctggccgagg agtcttaagc tgagatcaca gcattgcact
6581 ccagcctggg caaaaagagc aaaactccat ctcaaaaaaa aaaaaaaaat
agacacaaga 6641 ctggctcctt gtcttttttg gggacagggt ctcactctat
cacccaggct ggagtgcagt 6701 ggtgcaatca cagctcactg cagcctcgat
ttcccaggct caagtgaccc tcccatctta 6761 gcctcctgag tagctgggac
tacaggtgtg tgcaaccatg cctggctaat ttttaaaaat 6821 tttttgtaga
gatgaggtct cactatattg gctggggggc ctcaaactcc tgggctcagc 6881
agtcctccca cctcagcctc ccaaaaggct gggattatat gcttgctctt tttaaggtgg
6941 ctgtagggac aaactttcca cctactcctt gtcaagccag tggaccggtg
gtcccagaca 7001 tacggctaaa gtcaagaggt gatgtctttt ggagagatac
tttcaatcag gaatttcaat 7061 cagaaattca atcatgtgga gagagactta
tcctaaaaat gtggtggtgc gtgggatgct 7121 ctgttttatt agttccttga
cagtatgtat gtgtgtgagt gtgtgtgtgt gcgcgcgcac 7181 actcatttgg
atgggtgtgt atgtgtgtgg gggggtggtg cgtacgtatg tggatgtgtg 7241
gatgtggtgt gtgggtgtgc gcgtgcatag gtggaggtgt gtgtatgggt gcgggtatgt
7301 gtgtgtgttg ggcatggaga tattgacagc tctcccaggg ctgagtgaag
gctttcgggc 7361 aaagctcctg ggagctaggc aaagctgagt tgattcctgg
ttatgccatt tattattggg 7421 ttgcaccgtg tgaaactgcc aatattctac
actttgactt ttatttattt ttatttttat 7481 tttttttgag acagagtttc
acacttgtca cctaggctgg agtgcagtgg cgcgatctca 7541 gctcactgca
acctctgcct catggattca agtgattctc ctgcctcagc ctcccaagta 7601
gctggaatta caggtgcccg ctaccacgcc tgactaattt ttgtattttt ggtagagacg
7661 ggatttcacc atgttgtcca ggctggtctg aaactcctga catcaggtaa
tccacccacc 7721 tcagcctccc aaagtgctgg gattacaggc atgagccact
gcgcccggcc cattttgact 7781 tttaaaaatg ggagtttgat ataattcaat
ccagtggttg aattagctag catcgttccc 7841 tctccaagtc tcaggttctc
ctacacgtta gagtcaaaag cagggctatg ggaagattaa 7901 gtaaaataaa
ttttgaaaat gccttatgaa aattacactc caaagaactc gcgccagtgt 7961
cagtgttctc atgttcctca tctcacatga tcacatttcg cggattagga agctgagtct
8021 gagaagctcc gtgtagtgct ttttcggagg caccgtgatg tgatggaagg
ctcactcgtt 8081 aggaagtcag aacagagtct ctgagggatc atttccttaa
tctgtcagtt tcctcatctc 8141 tgaagttggg ctcatttcct tccttcatgg
agttattgta aagatgaaga taaataacgt 8201 gtaaaatcta gcatgggaac
tggcttctat aaggttctaa taagtgcatt cctactcctt 8261 cccctcagcc
ttcccatttg taaaagcaag gcaggggtga ggtgatttct ggggctcctt 8321
ttggctctga catttgagga ttttgtatcc tttttttttt cagagtcttg ctctgtcacc
8381 caggttggag tgcagctcaa tgcaaattcc gcctcccagg ctcaagcaat
tcttatgtct 8441 cagcctcctg agtacctggg attacaggca ggcaccacca
cccccagcta attttttgta 8501 ttttcagtag agacggggtt ttgccatatt
ggccaggctg gtcttgaact cctgacttca 8561 tgtgacccac ccatctcagc
ctcccaaagt gctgagatga caggtgtgag ctaccgtgcc 8621 tggccaattt
tgtgtgcttt aatgcccttt tctgctggaa gagttggcac caggtttggt 8681
gatctctttc ccccacacgg ctctgcctcc tgccagtccc agaggggacc ctgtccttgc
8741 atttcacagg attctgctgt tgcaactgaa attccagtag gtcaaagtga
aatttctcat 8801 acactttaac atgaagataa atgatcacag tatggccctt
taggatcctg agaacatcac 8861 ggtcatcccc tggtataatt ttaaaagcag
atgaatccat gcctgtgcga ggtttgccag 8921 gaaagccagt gctgggatta
cagtggaagt ctttttatgc tacttttttc ttgtatccct 8981 caccccatgg
ggtggcatat tgaaaggcag gatgtgtgac cacgatactt ttctcctcct 9041
ggactatgtc taagagtctg ttattgggtt ctgaagatca gagtttaatt tccgactcct
9101 ctctgtgtag ctctgggatc ttggaaagcc acttaacctt tctgaagtcc
cctttcctca 9161 tctctaaaat gcatacactc atcactaaca tttactgagc
actgacatgt gccagacacc 9221 attctaagca ttttacacag actacaccat
ttgatcttcc aacaaacaga acactgaaac 9281 gcattacagg tcagaacaaa
tgatttgtgc ctaagcacca agaccgtaga gcccgtgctc 9341 cctattctac
cctatcctgt ctctcaaaat gattgtgaga atcgaatgag acactaggtg 9401
agaaaagggt tttataaata gcattttaaa aattttttaa agtccacaaa atttttaatt
9461 ttaatacaga taaaatagat ccctttgttt tataaaaagt aacaaaattt
gttatacaac 9521 aactatgtta tttattaatt ttgccttttt gtatgctgcc
aggaaagaaa cattaagaaa 9581 tcttaaattg attatggtga atcagaaggt
ctgcctggac tttttattgc tctaactgta 9641 cagctgatca tactacctca
ttttttttta tgacacttca agggtgcgct tagcttcatc 9701 actccttcgt
tgccaaaagc tttgtgacca aaaacaatta agcagattcc tgagtcacta 9761
aatgacacat aaccagagtt gagacttagg aacttttagt gccatgctaa gcccacaggg
9821 acacaacaaa tagcatttta caaaggcaaa gaattgtgac acttgagatt
tagcttgttg 9881 atccttgtaa aagttttctt tttaggcata attgagtttt
agatcatagt actcactatt 9941 acttagtaat aatttttttc tgatagaaat
acagtgtaac aggccgggcg cagtggctca 10001 tgcctgtaat cccagcactt
tgggaggccg aggcgggcgg atcacttgag gtcaggagtt 10061 tgagaccagc
ccggccaaca tggtgaaatc ccatctctac taaaaataca aaaaattagc 10121
caggtgtggt cgtggattcc tgtgatccca gctacttggg agggtgaggc aggagcatca
10181 gttgaaccca ggaggcggag gttgcagtga gccaagatgg tgccattgca
ctccagcctg 10241 ggccacaaag cgagactcca cttcagaaac aaaaaaaaaa
agagagagag agaaaagaag 10301 gaaggaagga aggaaggaag gaaagaagga
aggaaggaag gaaagaagga aggaaggaag 10361 gaaggaaaga aggaaggaag
gaaagaagga aggaaggaaa gaaggaagga aggaaggaag 10421 gaagggtaac
aagcaaagtg taacaatggc aatatctaaa aaaataggta tttttatatg 10481
tttgtcgttt tatatatatg acccccactt tagagatgag gaaactgaga gattaaggaa
10541 acgatccctg agagactctg ttctgacttc caaatcggtg agctttccat
cgcatcacgg 10601 tgcctccgaa agcatgacac ggagcttctc agactyagct
tcctaatccg ctaaacggga 10661 ttatgtgaga tgaggaacat gagaacgctg
acatgggtga gggttccttg gagtatcatt 10721 ttcatgtggc attttcaaaa
cttattttac ctaatcttcc caaagccctg cttttgactc 10781 taatgtgtct
cctgagactt ggagagcgca agatgctagc gacagagcaa gactccatct 10841
ccagataaat aaataagtaa aataaaaaag aacacaaata attttgaaaa tttttttgaa
10901 aattaggcac gtttgcactg accttcaatt gttattaatt gctggtttcc
cacccagaat 10961 taagttggaa tgcaactttc ttttacaatc agagtccgtt
cttggtcttg gaaacttctg 11021 aggctcctgt gctaatcmca ctcttgtatt
tttggcacct ctaccccgtg ccactgtcat 11081 ggaacccagg ctgatcgcac
ctattagtgg agaaatmtgt ccataatact gaagtttggg 11141 gacaaacagt
gttcccttag ggtaggagaa agagatcttt atttttraca aagggggagg 11201
agccagaaaa ctccagagac ccctgagttt gccctctctc caaggtttgg ggtaagcccc
11261 ccgtcaccct ttatctctgg ggctttcaca tattctggat tctctcctcc
tgtttcccag 11321 cagaaaagga tggagcctca cagattcttc ccatttctgg
agaaaaacat gcatggagct 11381 caaagttctt ctcaggagtt ttattgccaa
agccataata agaaagggtg gaggtgacaa 11441 gcagtgagga agtttaaaga
tgcatgaaat ctgtaaagtc tcagaacaag aattctccta 11501 aaatgcaaaa
ggggctttgc tggtctcccc ttggcttctc atgtagctca cctctttttt 11561
cttatcttga gactagtcaa acctaagctg tttctcattt tatttccaga agctattgag
11621 aacactctcc tgaattcttc aaattcagta gagggcgaca aatgtacata
taaatgatgg 11681 tagtgggtct taaataaaga ctcatgacac ctaaaggggc
agcacctgag tctgattgca 11741 cctgtttctg ttgctgtttc tgtctctctt
ctctctgtct gccatttcat tatcaatggt 11801 tactttactt ataagatcat
attagaacct gatatttgat aaatgatgca tcagatctat 11861 agtgagagaa
aaaattaatg caattaaagg tgttgtaaca gctagtcttc aagtggggag 11921
aaatcatttg agtaccttag gtcacagctt acatcaaaac aaaaaatcag agctacatta
11981 aaaagtgaaa ttttaactat atcaaacaat agaaaaaaac agaagaaaat
tgaatactta 12041 ctaaatctta gcatgaataa gaactgttta acacttagag
gcaaggactg ggcgtggtgg 12101 ctcatgcttt taatcccagg actttgggag
cccaaggcgg gcggatcacc tgaggtcagg 12161 agtttgagac tagcctggcc
aacatggtga aaccccgtct ctactaaaaa atgcaaaaat 12221 tagctgcgtg
tggtggtgca tgcctgtaat ctcagctact tgggaggcta aggcatgaga 12281
atcgcttgaa cctgggaggt ggaggctgta gtgagccgag attgtgccac tgcactacag
12341 cctgggtgac agtgtgaaat cctctctctc aaaaaaaaaa aaaaaaaaaa
gcaaactaga 12401 gcagtgaggt accattattt cctttgctca ctaaactgac
aacacacaaa tgttttttat 12461 aatacccaaa gctgatgagg gtagttaagg
tatgcccttt tatacacaca ctaatgatgt 12521 actactggtt ggcagtataa
catatgctgc catgtgggga tatgtatcag gagacttaaa 12581 aatgtgcata
ccttttggtc cagtaattta cttctgggaa tctgtcataa cagaataata 12641
atcttgggga aagctacatg cctaaggata tttaaaatat tatttaaaaa tcaaagtata
12701 atttcttaca gaatataaaa taatatttta aaatgaaaat atgctaaaag
tttgatgaaa 12761 tataaatggt caaatatata ttgattatat ccacttacta
gactagcact cactctgaga 12821 cgttaaaaat agtcattata aaaactagaa
aatgccaaag acaaaataaa ggaataaagt 12881 tttacataaa gtatgattcc
actatgttta aaaataaaca gagacattct tggagttgag 12941 tattgttttc
ttttctgtca tgtccaaaga actatataac tattattttt aatgaactat 13001
atatgtaata tatacatata gtttatatgt atatacaaaa tttatctcat atatatgata
13061 aagatgaaag atgagttgga tgtgccacgt gaagtgggta gtatagaaac
ccaggtaatg 13121 gggcatagga gtgggattcc agataccagg cccatgtttt
tggggtgaga ttgccaatca 13181 cggtctttct tccatccctc acagaggagt
aggtttgtct tcaacaaacc ttcagttgtc 13241 ctgaagacaa acctaattct
ggagacttca tataatctag aagagacaag caaactgatg 13301 aaaaatagtg
aatttttaag gtaaaataaa gtacatggac tacactttgt ttagaatcag 13361
attcttggga ttaaccacat taacccacag agggtcttag tgatgcctct aatccaggat
13421 cctaggacct atttctctct gtgagatgct ttctcccaac tccttggtga
gagtgggaag 13481 actaagacct cagcaatctg aggtggaggc ctaagatccc
cctaagatcg gaggcagaat 13541 ctgagagggg ataaaagtcc ctatacctgt
attgggccct tttctgggag ggggatatca 13601 aagaatgatt ttgagacagg
gaggcttttg actacctgtg ccacttgagc tctttgctag 13661 ggctccagaa
tacatatttc aaatacattc cccctccctc cttccttccc tcttccactc 13721
ttccttttta tcttcctttc ttcttttcct tcctccttcc cttcctttct ctggctctct
13781 catgatttct tttcctcatt ataaaagtgc ttatttagtc cctactctgc
tattagtgtg 13841 ttagtctttg tcccctggta cttgctgttt aatggagaaa
tgggtgagca aaacagaaat 13901 tacagcagag tgcaataata gagctaagcc
aggtgtataa atccattctc acactgctgt 13961 aaaaaactac tgggtaattt
ataaagaaaa gaggtttaat tgactcacag ttccacaggc 14021 tgtacaggaa
gcatggctgg ggaggcctca gaaaacttac aatcatggtg gaagaaagag 14081
cgaaggggaa gcaagcacat cacacagcag caggagagag agagagaaag agagagagag
14141 agaatatagg ggaagtgcta cacactttca accagatctt gtgagaattc
acctactatc 14201 atgagaacag caagggataa gtctgcctcc atgattcagt
cacctcctac caggcccctt 14261 ctccaacaca tgtcgacgtg ctatttgggt
ggggacacag acccaaacca tattaccagg 14321 gcactggaga aacacagagg
ggaaagaacc agccaaggag tgagatggag aacaaggagg 14381 acttcttgaa
acagatgaca tccaaactgg gtcctgaaag ctgaatagag attagacagg 14441
ggaggagggg cagctaaaga tggctcaggc aaacaaaggg ccaggggata tgttcatggg
14501 atgatgtgtc tctcgttgtc tgcttaacac aaggtgagtc tctccctccc
tctctctctc 14561 tttttctctg tgtgtgtttg tgtgtgtgca tgtgtgcaaa
tgtaatatac ccaatagtca 14621 aacatgtgcc ccaggagagg ggtagaggaa
gaaagagaat gagagagtaa gaaggaggaa 14681 tagacacaga aaatgagaga
raagggggga aagaaaaaga agaaaggagc cagaggagag 14741 aagctggtta
gcattraatg gagcaatctg tgtcatcgta cttgggaaac ccaaggatgg 14801
attcttggca agtygactct tggagctttc cctgtgcttg gtcctgtgct cagacatggg
14861 aaaattagag gagtgtcatc
tgtgcaatca ctgaattcat aatcttggtg aggaaaggag 14921 actacacaca
gggaataatg ctaagtatta cagatttcag ggcagaaaga gatcaaggtg 14981
ggctgcaata ttcagaaaag tcttcctgga aaagttgaat acttagaaag cagctcctag
15041 aagtagackc tgctgagatg gacggagtcc tttgtaggtc ccaactgggt
gtgtgtgtgg 15101 ggtctgtctc tccatggcyg acagtgcaca tgtggattcc ag gg
ctc agg atg 15154 Gly Leu Arg Met 15 ctg ttg ctg gga gct gtt cta
ctg cta tta gct ctg ccc ggk cat gac 15202 Leu Leu Leu Gly Ala Val
Leu Leu Leu Leu Ala Leu Pro Xaa His Asp 20 25 30 cag gaa acc acg
act caa ggg ccc gga gtc ctg ctt ccc ctg ccc aag 15250 Gln Glu Thr
Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro Lys 35 40 45 ggg
gcc tgc aca ggt tgg atg gcg ggc atc cca ggg cat ccg ggc cat 15298
Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly His 50
55 60 65 aat ggg gcc cca ggc cgt gat ggc aga gat ggc acc cct ggt
gag aag 15346 Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro
Gly Glu Lys 70 75 80 ggt gag aaa gga gat cca g gtaagaatgt
ttctggcctc tttcatcaca 15395 Gly Glu Lys Gly Asp Pro 85 gacctcctac
actgatataa actatatgaa gkcattcatt attaactaag gcctagacac 15455
agggagaaag caaagctttt ttatgttaac cataagcaac ctgargtgat ttggggttgg
15515 tcttccaagg atgagtgtag atggtgcctc tataaccaag actttggctt
tgctgcatct 15575 gcagctcctt ttccatcccc tttcccatct tcaccctcat
ccctattccc agtacattca 15635 tattctgatt cctctttctg tctgcttaac
ttccatttca cccastggca ttcaaccaca 15695 tttactgcac accccctgaa
aggctcagtc ctgcctttgg ggaactcttg atctaggtaa 15755 gatgtctaat
gtgcaaggct ctgttggtgg ttacyacaag aaagtctact ctaaaaatgt 15815
caaactgaat gtgaacaagt attcaaagta tggagcatag agaaaatrta ctcaccgtgg
15875 acctgatgaa gaatgaaggc ttcaaggagg aggcagagct tcagctaggc
cttgaatgat 15935 gggtaggcag aatagaggag gagagacatc ctagatggag
ggggtagaat tgcaaaacca 15995 gggttgatgg tgccagcaca taaagggctg
gcagggtgga gggtctatga tagagaccta 16055 taggagataa agatagagtt
gaaattatgg gagcctccat gtctgtggga gatatagaag 16115 gaggaggtaa
cacctctctc cttttgggag ctcttattgg tttcttgatc tataagtcaa 16175
gaaggttgtg agtgggagcc acagggatgg taatttaggc tgtaaccaac ctaggcagga
16235 gttctgttct ttgtagtcac tgaggtcttc tcattcctta g gt ctt att ggt
cct 16290 Gly Leu Ile Gly Pro 90 aag gga gac atc ggt gaa acc gga
gta ccc ggg gct gaa ggt ccc cga 16338 Lys Gly Asp Ile Gly Glu Thr
Gly Val Pro Gly Ala Glu Gly Pro Arg 95 100 105 ggc ttt ccg gga atc
caa ggc agg aaa gga gaa cct gga gaa ggt gcc 16386 Gly Phe Pro Gly
Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala 110 115 120 tat gta
tac cgc tca gca ttc agt gtg gga ttg gag act tac gtt act 16434 Tyr
Val Tyr Arg Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr 125 130
135 140 atc ccc aac atg ccc att cgc ttt acc aag atc ttc tac aat cag
caa 16482 Ile Pro Asn Met Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn
Gln Gln 145 150 155 aac cac tat gat ggc tcc act ggt aaa ttc cac tgc
aac att cct ggg 16530 Asn His Tyr Asp Gly Ser Thr Gly Lys Phe His
Cys Asn Ile Pro Gly 160 165 170 ctg tac tac ttt gcc tac cac atc aca
gtc tat atg aag gat gtg aag 16578 Leu Tyr Tyr Phe Ala Tyr His Ile
Thr Val Tyr Met Lys Asp Val Lys 175 180 185 gtc agc ctc ttc aag aag
gac aag gct atg ctc ttc acc tat gat cag 16626 Val Ser Leu Phe Lys
Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln 190 195 200 tac cag gaa
aat aat gtg gac cag gcc tcc ggc tct gtg ctc ctg cat 16674 Tyr Gln
Glu Asn Asn Val Asp Gln Ala Ser Gly Ser Val Leu Leu His 205 210 215
220 ctg gag gtg ggc gac caa gtc tgg ctc cag gtg tat ggg gaa gga gag
16722 Leu Glu Val Gly Asp Gln Val Trp Leu Gln Val Tyr Gly Glu Gly
Glu 225 230 235 cgt aat gga ctc tat gct gat aat gac aat gac tcc acc
ttc aca ggc 16770 Arg Asn Gly Leu Tyr Ala Asp Asn Asp Asn Asp Ser
Thr Phe Thr Gly 240 245 250 ttt ctt ctc tac cat gac acc aac tga tca
cca cta act cag agc ctc 16818 Phe Leu Leu Tyr His Asp Thr Asn Ser
Pro Leu Thr Gln Ser Leu 255 260 265 ctc cag gcc aaa cag ccc caa agt
caa tta aag gct ttc agt acg gtt 16866 Leu Gln Ala Lys Gln Pro Gln
Ser Gln Leu Lys Ala Phe Ser Thr Val 270 275 280 agg aag ttg att att
att tag ttg gag gcc ttt aga tat tat tca ttc 16914 Arg Lys Leu Ile
Ile Ile Leu Glu Ala Phe Arg Tyr Tyr Ser Phe 285 290 295 att tac tca
ttc att tat tca ttc att cat caa gta act tta aaa aaa 16962 Ile Tyr
Ser Phe Ile Tyr Ser Phe Ile His Gln Val Thr Leu Lys Lys 300 305 310
tca tat gct atg ttc cca gtc ctg ggg agc ttc aca aac atg acc aga
17010 Ser Tyr Ala Met Phe Pro Val Leu Gly Ser Phe Thr Asn Met Thr
Arg 315 320 325 330 taa ctg act aga aag aag tag ttg aca gtg cta ttt
tgt gcc cac tgt 17058 Leu Thr Arg Lys Lys Leu Thr Val Leu Phe Cys
Ala His Cys 335 340 ctc tcc tga tgc tca tat caa tcc tat aag gca cag
gga aca agc att 17106 Leu Ser Cys Ser Tyr Gln Ser Tyr Lys Ala Gln
Gly Thr Ser Ile 345 350 355 ctc ctg ttt tta cag att gta tcc tga ggc
tga gag agt taa gtg aat 17154 Leu Leu Phe Leu Gln Ile Val Ser Gly
Glu Ser Val Asn 360 365 370 gtc taa ggt cac aca agt att aag tga cag
tgc tag aaa tca aac cca 17202 Val Gly His Thr Ser Ile Lys Gln Cys
Lys Ser Asn Pro 375 380 385 gag ctg tgg act ttg ttc act aga ctg tgc
cct ttt ata gag gta cat 17250 Glu Leu Trp Thr Leu Phe Thr Arg Leu
Cys Pro Phe Ile Glu Val His 390 395 400 gtt ctc ttt gga gtg ttg gta
ggt gtc tgt ttc cca cct cac ctg aga 17298 Val Leu Phe Gly Val Leu
Val Gly Val Cys Phe Pro Pro His Leu Arg 405 410 415 gcc att gaa ttt
gcc ttc ctc atg aat taa aac ctc ccc caa gca gag 17346 Ala Ile Glu
Phe Ala Phe Leu Met Asn Asn Leu Pro Gln Ala Glu 420 425 430 ctt cct
cag aga aag tgg ttc tat gat gaa gtc ctg tct tgg aag gac 17394 Leu
Pro Gln Arg Lys Trp Phe Tyr Asp Glu Val Leu Ser Trp Lys Asp 435 440
445 tac tac tca atg gcc cct gca cta ctc tac ttc ctc tta cct atg tcc
17442 Tyr Tyr Ser Met Ala Pro Ala Leu Leu Tyr Phe Leu Leu Pro Met
Ser 450 455 460 ctt ctc atg cct ttc cct cca acg ggg aaa gcc aac tcc
atc tct aag 17490 Leu Leu Met Pro Phe Pro Pro Thr Gly Lys Ala Asn
Ser Ile Ser Lys 465 470 475 480 tgc tga act cat ccc tgt tcc tca agg
cca cct ggc cag gag ctt ctc 17538 Cys Thr His Pro Cys Ser Ser Arg
Pro Pro Gly Gln Glu Leu Leu 485 490 495 tga tgt gat atc cac ttt ttt
ttt ttt ttg aga tgg agt ctc act ctg 17586 Cys Asp Ile His Phe Phe
Phe Phe Leu Arg Trp Ser Leu Thr Leu 500 505 510 tca ccc agg ctg gag
tac agt gac acg acc tcg gct cac tgc agc ctc 17634 Ser Pro Arg Leu
Glu Tyr Ser Asp Thr Thr Ser Ala His Cys Ser Leu 515 520 525 ctt ctc
ctg ggt cca agc aat tat tgt gcc tca gcc tcc cga gta gct 17682 Leu
Leu Leu Gly Pro Ser Asn Tyr Cys Ala Ser Ala Ser Arg Val Ala 530 535
540 gag act tca ggt gca ttc cac cac aca tgg cta att ttt gta ttt tta
17730 Glu Thr Ser Gly Ala Phe His His Thr Trp Leu Ile Phe Val Phe
Leu 545 550 555 gta gaa atg ggg ttt cgt cat gtt ggc cag gct ggt ctc
gaa ctc ctg 17778 Val Glu Met Gly Phe Arg His Val Gly Gln Ala Gly
Leu Glu Leu Leu 560 565 570 gcc tag gtg atc cac ccg cct cga cct ccc
aaa gtg ctg gga tta cag 17826 Ala Val Ile His Pro Pro Arg Pro Pro
Lys Val Leu Gly Leu Gln 575 580 585 gcr tga gcc acc atg ccc agt cga
tat ctc act ttt tat ttt gcc atg 17874 Ala Ala Thr Met Pro Ser Arg
Tyr Leu Thr Phe Tyr Phe Ala Met 590 595 600 gat gag agt cct ggg tgt
gag gaa cac ctc cca cca ggc tag agg caa 17922 Asp Glu Ser Pro Gly
Cys Glu Glu His Leu Pro Pro Gly Arg Gln 605 610 615 ctg ccc agg aag
gac tgt gct tcc gtc acc tct aaa tcc ctt gca gat 17970 Leu Pro Arg
Lys Asp Cys Ala Ser Val Thr Ser Lys Ser Leu Ala Asp 620 625 630 635
cct tga taa atg cct cat gaa gac caa tct ctt gaa tcc crt atc tac
18018 Pro Met Pro His Glu Asp Gln Ser Leu Glu Ser Xaa Ile Tyr 640
645 cca gaa tta act cca ttc cag tct ctg cat gta atc agt ttt atc cac
18066 Pro Glu Leu Thr Pro Phe Gln Ser Leu His Val Ile Ser Phe Ile
His 650 655 660 665 aga aac att ttc att tta gga aat ccc tgg ttt taa
gta tca atc ctt 18114 Arg Asn Ile Phe Ile Leu Gly Asn Pro Trp Phe
Val Ser Ile Leu 670 675 680 gtt cag ctg gac aat atg aat ctt ttc cac
tga agt tag gga tga ctg 18162 Val Gln Leu Asp Asn Met Asn Leu Phe
His Ser Gly Leu 685 690 tga ttt tca gaa cac gtc cag aat ttt tca tca
aga agg tag ctt gag 18210 Phe Ser Glu His Val Gln Asn Phe Ser Ser
Arg Arg Leu Glu 695 700 705 cct gaa atg caa aac cca tgg agg aat tct
gaa gcc att gtc tcc ttg 18258 Pro Glu Met Gln Asn Pro Trp Arg Asn
Ser Glu Ala Ile Val Ser Leu 710 715 720 agt acc aac agg gtc agg gaa
gac tgg gcc tcc tga att tat tat tgt 18306 Ser Thr Asn Arg Val Arg
Glu Asp Trp Ala Ser Ile Tyr Tyr Cys 725 730 735 tct tta aga att aca
ggt tga ggt agt tga tgg tgg taa aca ttc tct 18354 Ser Leu Arg Ile
Thr Gly Gly Ser Trp Trp Thr Phe Ser 740 745 750 cag gag aca ata act
cca gtg atg ttc ttc aaa gat ttt agc aaa aac 18402 Gln Glu Thr Ile
Thr Pro Val Met Phe Phe Lys Asp Phe Ser Lys Asn 755 760 765 aga gta
aat agc att ctc tat caa tat ata aat tta aaa aac tat ctt 18450 Arg
Val Asn Ser Ile Leu Tyr Gln Tyr Ile Asn Leu Lys Asn Tyr Leu 770 775
780 ttt gct tac agt ttt aaa tcc tga aca att ctc tct tay atg tgt att
18498 Phe Ala Tyr Ser Phe Lys Ser Thr Ile Leu Ser Tyr Met Cys Ile
785 790 795 gct aat cat taa ggt att att ttt tcc aca tat aaa gct ttg
tct ttt 18546 Ala Asn His Gly Ile Ile Phe Ser Thr Tyr Lys Ala Leu
Ser Phe 800 805 810 tgt tgt tgt tgt tgt ttt taa gat gga gtt tcc ctc
tgt tgc cag gct 18594 Cys Cys Cys Cys Cys Phe Asp Gly Val Ser Leu
Cys Cys Gln Ala 815 820 825 aga gtg cag tgg cat gat ctc ggc tta ctg
caa cct ttg cct ccc agg 18642 Arg Val Gln Trp His Asp Leu Gly Leu
Leu Gln Pro Leu Pro Pro Arg 830 835 840 ttc aag cga ttc ttc tgc ctc
agc ctc ccg agt agc tgg gac cac agg 18690 Phe Lys Arg Phe Phe Cys
Leu Ser Leu Pro Ser Ser Trp Asp His Arg 845 850 855 860 tgc cta cca
cca tgc cag gct aat ttt tgt att ttt agt aaa gac agg 18738 Cys Leu
Pro Pro Cys Gln Ala Asn Phe Cys Ile Phe Ser Lys Asp Arg 865 870 875
gtt tca cca tat tgg cca ggc tgg tct cga act cct gac ctt gtg atc
18786 Val Ser Pro Tyr Trp Pro Gly Trp Ser Arg Thr Pro Asp Leu Val
Ile 880 885 890 tgc cca cct cca ttt ttg ttg tta ttt ttt gag aaa gat
aga tat gag 18834 Cys Pro Pro Pro Phe Leu Leu Leu Phe Phe Glu Lys
Asp Arg Tyr Glu 895 900 905 gtt tag aga ggg atg aag agg tga gag taa
gcc ttg tgt tag tca gaa 18882 Val Arg Gly Met Lys Arg Glu Ala Leu
Cys Ser Glu 910 915 920 ctc tgt gtt gtg aat gtc att cac aac aga aaa
ccc aaa ata tta tgc 18930 Leu Cys Val Val Asn Val Ile His Asn Arg
Lys Pro Lys Ile Leu Cys 925 930 935 aaa cta ctg taa gca aga aaa ata
aag gaa aaa tgg aaa cat tta ttc 18978 Lys Leu Leu Ala Arg Lys Ile
Lys Glu Lys Trp Lys His Leu Phe 940 945 950 ctt tgc ata ata gaa att
acc aga gtt gtt ctg tct tta gat aag gtt 19026 Leu Cys Ile Ile Glu
Ile Thr Arg Val Val Leu Ser Leu Asp Lys Val 955 960 965 tga acc aaa
gct caa aac aat caa gac cct ttt ctg tat gtc ctt ctg 19074 Thr Lys
Ala Gln Asn Asn Gln Asp Pro Phe Leu Tyr Val Leu Leu 970 975 980 ttc
tgc ctt ccg cag tgt agg ctt tac cct cag gtg cta cac agt ata 19122
Phe Cys Leu Pro Gln Cys Arg Leu Tyr Pro Gln Val Leu His Ser Ile 985
990 995 gtt cta ggg ttt ccc tcc cga tat caa aaa gac tgt ggc ctg ccc
19167 Val Leu Gly Phe Pro Ser Arg Tyr Gln Lys Asp Cys Gly Leu Pro
1000 1005 1010 agc tct cgt atc ccc aag cca cac cat ctg gct aaa tgg
aca tca 19212 Ser Ser Arg Ile Pro Lys Pro His His Leu Ala Lys Trp
Thr Ser 1015 1020 1025 tgt ttt ctg gtg atg ccc aaa gag gag aga gga
agc tct ctt tcc 19257 Cys Phe Leu Val Met Pro Lys Glu Glu Arg Gly
Ser Ser Leu Ser 1030 1035 1040 cag atg ccc cag caa gtg taa cct tgc
atc tca ttg ctc tgg ctg 19302 Gln Met Pro Gln Gln Val Pro Cys Ile
Ser Leu Leu Trp Leu 1045 1050 1055 agt tgt gtg cct gtt tct gac caa
tca ctg agt cag gag gat gaa 19347 Ser Cys Val Pro Val Ser Asp Gln
Ser Leu Ser Gln Glu Asp Glu 1060 1065 1070 ata ttc ata ttg act taa
ttg cag ctt aag tta ggg gta tgt aga 19392 Ile Phe Ile Leu Thr Leu
Gln Leu Lys Leu Gly Val Cys Arg 1075 1080 1085 ggt att ttc cct aaa
gca aaa ttg gga cac tgt tat cag aaa tag 19437 Gly Ile Phe Pro Lys
Ala Lys Leu Gly His Cys Tyr Gln Lys 1090 1095 1100 gag agt gga tga
tag atg caa aat aat acc tgt cca caa caa act ctt 19485 Glu Ser Gly
Met Gln Asn Asn Thr Cys Pro Gln Gln Thr Leu 1105 1110 aat gct gtg
ttt gag ctt tca tga gtt tcc cag aga gac ata gct 19530 Asn Ala Val
Phe Glu Leu Ser Val Ser Gln Arg Asp Ile Ala 1115 1120 1125 gga aaa
ttc cta ttg att ttc tct aaa att tca aca agt agc taa 19575 Gly Lys
Phe Leu Leu Ile Phe Ser Lys Ile Ser Thr Ser Ser 1130 1135 1140 agt
ctg gct atg ctc aca gtc tca cat ctg gtt ggg gtg ggc tcc 19620 Ser
Leu Ala Met Leu Thr Val Ser His Leu Val Gly Val Gly Ser 1145 1150
1155 tta cag aac acg ctt tca cag tta ccc taa act ctc tgg ggc agg
19665 Leu Gln Asn Thr Leu Ser Gln Leu Pro Thr Leu Trp Gly Arg 1160
1165 1170 gtt att cct ttg tgg aac cag agg cac aga gag agt caa ctg
agg 19710 Val Ile Pro Leu Trp Asn Gln Arg His Arg Glu Ser Gln Leu
Arg 1175 1180 1185 cca aaa gag gcc tga gag aaa ctg agg tca aga ttt
cag gat taa 19755 Pro Lys Glu Ala Glu Lys Leu Arg Ser Arg Phe Gln
Asp 1190 1195 tgg tcc tgt gat gct ttg aag tac aat tgt gga ttt gtc
caa ttc 19800 Trp Ser Cys Asp Ala Leu Lys Tyr Asn Cys Gly Phe Val
Gln Phe 1200 1205 1210 tct tta gtt ctg tca gct ttt gct tca tat att
tta gcg ctc tat 19845 Ser Leu Val Leu Ser Ala Phe Ala Ser Tyr Ile
Leu Ala Leu Tyr 1215 1220 1225 tat tag ata tat aca tgt tta gta tta
tgt ctt att ggt gca ttt 19890 Tyr Ile Tyr Thr Cys Leu Val Leu Cys
Leu Ile Gly Ala Phe 1230 1235 1240 act ctc tta tca tta tgt aat gtc
ctt ctt tat ctg tga taa ttt 19935 Thr Leu Leu Ser Leu Cys Asn Val
Leu Leu Tyr Leu Phe 1245 1250 1255 tct gtg ttc tga agt cta ctt tgt
cta aaa ata aca tac gca ctc 19980 Ser Val Phe Ser Leu Leu Cys Leu
Lys Ile Thr Tyr Ala Leu 1260 1265 1270 aac ttc ctt ttc ttt ctt cct
tcc ttt ctt tct tcc ttc ctt tct 20025 Asn Phe Leu Phe Phe Leu Pro
Ser Phe Leu Ser Ser Phe Leu Ser 1275 1280 1285 ttc tct ctc tct ctc
ttt cct tcc ttc ctt cct cct ttt ctt tct 20070 Phe Ser Leu Ser Leu
Phe Pro Ser Phe Leu Pro Pro Phe Leu Ser 1290 1295 1300 ctc tct ctc
tct ctc tct ttt ttt gac aga ctc tcg ttc tgt ggc 20115 Leu Ser Leu
Ser Leu Ser Phe Phe Asp Arg Leu Ser Phe Cys Gly 1305 1310 1315 cct
ggc tgg agt tca gtg gtg tga tct tgg ctc act gct acc tct 20160 Pro
Gly Trp Ser Ser Val Val Ser Trp Leu Thr Ala Thr Ser 1320 1325 acc
atg agc aat tct cct gcc tca gcc tcc caa gta gct gga act 20205 Thr
Met Ser Asn Ser Pro Ala
Ser Ala Ser Gln Val Ala Gly Thr 1330 1335 1340 aca ggc tca tgc cac
tgc gcc cag cta att ttt gta ttt ttc gta 20250 Thr Gly Ser Cys His
Cys Ala Gln Leu Ile Phe Val Phe Phe Val 1345 1350 1355 gag acg ggg
ttt cac cac att cgt cag gtt ggt ttc aaa ctc ctg 20295 Glu Thr Gly
Phe His His Ile Arg Gln Val Gly Phe Lys Leu Leu 1360 1365 1370 act
ttg tga tcc acc cgc ctc ggc ctc cca aag tgc tgg gat tac 20340 Thr
Leu Ser Thr Arg Leu Gly Leu Pro Lys Cys Trp Asp Tyr 1375 1380 1385
agg cat gag cca tca cac ctg gtc aac ttt ctt ttg att agt gtt 20385
Arg His Glu Pro Ser His Leu Val Asn Phe Leu Leu Ile Ser Val 1390
1395 1400 ttt gtg gta tat ctt ttt cca tca tgt tac ttt aaa tat atc
tat 20430 Phe Val Val Tyr Leu Phe Pro Ser Cys Tyr Phe Lys Tyr Ile
Tyr 1405 1410 1415 att att gta ttt aaa atg tgt ttc tta cag act gca
tgt agt tgg 20475 Ile Ile Val Phe Lys Met Cys Phe Leu Gln Thr Ala
Cys Ser Trp 1420 1425 1430 gta taa ttt tta tcc agt cta aaa ata tct
gtc ttt taa ttg gtg 20520 Val Phe Leu Ser Ser Leu Lys Ile Ser Val
Phe Leu Val 1435 1440 1445 ttt aga caa ttt ata ttt aat aaa att gtt
gaa ttt aag atggatgact 20569 Phe Arg Gln Phe Ile Phe Asn Lys Ile
Val Glu Phe Lys 1450 1455 gttttatttg tttgctgttc accacttctg
ttttattctc tttccagaat tcttttggat 20629 tgtttaaata tttcataata
ttttatctta atttatttat tgggtatttg cctatatctc 20689 tttgtggtat
tttttagtgg ttgcttgagg gattacaatg tacttaactt ttcacagtgt 20749
gcataaagtt aatattttgc cacttgcagt aaaccgtaga aggcttataa tcatattagt
20809 acctctatcc actttctttt atgttgtagt tgtcatatat attacatcta
tatacactga 20869 aacattatag gcaatgttat gatttttgca ttcgtcagtc
atatatatat tttaaagaat 20929 ttaagaggag aaaaatacat attcagatat
tcatcat 20966 2 4811 DNA Homo sapiens 2 gctgatctgc tgcctcagcc
ttcccaaagt gctgtaattt attaggcata agccactgtg 60 cctgcctagt
gttgtacatt ctgtgggttt tgacaattgt atgcatctac atgtatgtac 120
catttatagt attcctgttt ttaattttag ccattctagt aggcatgtag tgatatctca
180 tggtgatttt aatttgcgtt tccgtaatgg ttaataatgc tgaacatctt
tgcatgtgct 240 tgtttgtcat ttgtgtttcc tacttggtga aataattgtt
catgtccttt gtccattttc 300 taattgaatt tttttttacc atttagtttt
gagatttctt tatacaatct agatccaaat 360 ctcttgtctc aaatatggtt
tgcaaataca ttcctctaat tcatatattg ccttttcctc 420 ctcttaacag
gatgtttcac agagcaaaag ttttagtttt gttgaaatct cacttttcat 480
ttttttcttt agtggattgt gcttttgttg tcatatgtaa gaactcttca ctggccctag
540 atccttgtat tggtttccta agattgccat agcaaatcac catgaactta
gtgacaaaaa 600 gacagaaatt tattttcact tcctactgtg ggcagactag
acgttaatta ttttcatgta 660 tgctcattcc tatgacatct ttctgatata
ataattatag ttattcttaa gcttcaccct 720 tttttctatt agctttgtta
ccttgggtgt cactttttct tttttgacat tgtgacctat 780 gccagatcat
gtctgttagt acttagccct ccattcacct ctccataatc ccttttgtat 840
tcctggagct tgatgcctga aatgacacat cctacattcc tttgccagat gggtaccagt
900 tagcttgtgc acatgggaga caaccgtgaa aagactgaag tggggaagaa
gggaggagct 960 gttgtgtttc agtgagcgcc cttggcagtg gcggtgacag
tggctcctgt tcagtggcaa 1020 tggtggagca gctagcaaga catgcagtaa
gcgcaggctc ataggctatg gtccaggagc 1080 agtcaccgat tcctggtctt
taggcaatat catctccctt tgcttctcca gcctttctaa 1140 aattattgta
ccttgactag tacaattttt tagtattggg ggtagtccaa ggacacaggc 1200
tttaaaaagt atgaattcag ggttgcctac ctgcattgac tgcgcttgaa tcatgatggc
1260 cttctggtcg gtggcaggag gtgacagtcc aaatcatgca gtagcaaacc
agatacttaa 1320 attatcatct gagatacttc agaagtacag ccgtagccat
accttcagaa gagataaaga 1380 aatgttctcc tggccaggcg cggtggctca
cgcctgtcat tccagcactt tgggaggccg 1440 agggggtgga tcacctgagg
tcgggagttc gagaccagcc tgaccaacat ggggaaaccc 1500 tgtctctact
aaaaatacaa aattagcggg gcgtggtggc acatgcccat aatcccagct 1560
actcgggagg ctaaggcagg ataatcgctt gaacctgaga ggcagaggtt gcggtgaact
1620 gagatcatgc catagtactc cagcctgggc aacaagagtg aaactccatc
tcaaaaaaaa 1680 aaaaaagaaa aaaagataaa gaaatgttct cctttcttgc
catttctagg ggtttgggga 1740 tggcgtacat tgctgcaggg cgtgctcact
ctaccatctt gctccaatct ttatttttca 1800 aaatacagtg cttatgcttg
gttacttcag ttaagattat ttttaaaaat cataattaag 1860 caaaaatata
tggccatgct taaacatatt taagataaat taagtgattt ggcctgtttc 1920
agtatcccaa ctcacatgct aacaggggct tgacctgtag ctacggtacc ctggaggaaa
1980 tgatcgcatt tatttggtta tttcggtcta agtagtaata gttctgtcct
gggaaaaaga 2040 ctagcctcaa ggcatttctg attgaatgtt tttcaattac
agtctttaaa ccagtatgcc 2100 acagaactgg ctctttccac atgacggcct
ttgtggtggg tggcagattg ccctgaggcc 2160 tcgcaaaatg ctaggctttc
acaatgtcac tgactgacag ccaggcccag cacagtcttg 2220 gtgtgattgt
ggggctaaag ttattccacc ttgtgcaata gctacagcct tctctaacca 2280
gctgcattct tataaagtta gaagaaaata cttttttttt tttgagatgg attctcgctc
2340 tgttgcccag gctggagtgc aatggtgcga tctcggctcg ctgcaacctc
cgcctcctgg 2400 gttcaaacga ttctcctccc tcagaccccc gagtagctgg
gattgcaggt gcctgccacc 2460 acgcccggct aacttttttg tatttttagt
ggagacgggg tttcaccatc ttcgtcaggc 2520 tggtctcaga ctcctgacct
caagtgatct gcccgcctca gcctcccaaa atgctgggat 2580 tacaggcatg
agctactgtg cccggccaaa gaaaatactt tttatgccag ccctgaaact 2640
accctgaagc acatacatca accttgaggc ctcacactcc atcaagaggg gtgaagggca
2700 tgaggaatta gaaagcatag ggatttttag ttagacagat ctggttcaaa
tcctagactt 2760 gtgccttgaa caaattattt accctcattg aactctagat
tcattatttg taaaatgaaa 2820 gacaataata gttatctcca aaggaaagtt
gaatatgatc attcatttat tcattaattc 2880 aacatttatt attgcctact
ttgtgccagg ttctattcta ggaactaagg gatacaactt 2940 tgaataggca
aaatctctgc tctcctgaag tttacttttt tttttttttt ttgagacaga 3000
gtttcactct tgtcacccag gctggagcgc aatggtgctc ttggctcact gcaacctcca
3060 cctcctgggt tcaagtgatt ctcttgtctc agcctcccaa gtagctggga
ctacaggtat 3120 gtgccaccac gcccggctat ttctgcattt ttagtagaga
tggggtttca ccatgttggc 3180 cagactggtc tcaaactcct gatctcaggt
gatatgcctg tcttggcctt ccaaagtact 3240 gggattacag gcctgagcca
ctgcacctga cctgaagttt atgttctatt aaatagcaac 3300 agacagtaac
ataaaccaaa aataaatagg aaaacaccat aacaaaaatc aaacagtgat 3360
ataattgaga gttgcttcta tttctttttg ttgtcttctt ggttcaatca gcctgctaaa
3420 ctatatggaa cctcattttc atgggccact tatttaagcc gggggacctt
ggaaagtctc 3480 tcatgtctct catctcaacg gcctaatgtg acttctcttg
aaatatttgg acattagcag 3540 gaagctgagg ctttacatca gatctttact
ttaatggtgg acttgacttt actggtagat 3600 ttttaggctc tgtgtggact
gtggagatga tatctggggg gcaggcagac acttgccctg 3660 cctctgtctg
agaaaattct gttttggatg tcttgttgaa gttggtgctg gcatcctaag 3720
cccttgctgg ggtcgtaatt taattcatca gaatgtgtgg cttgcaagaa ccggctcaga
3780 tcctgcgctt caaaaacaaa acatgagcgt gccaagaaag tccaaggtgt
tgaatgttgc 3840 cacttcaagc ctaaactttc taggaacacc taagtgggtg
gcagcttcca gttctccagg 3900 ctgcttctag gccagagctg ggttccacaa
gagacagaat aggcatatat atgcttaagg 3960 aactggaaaa acaggctctc
tctctctcac aaacacacac acacacatac caaggtagct 4020 gtcaaaatgt
tatccgaaat tttggaacca aaaaatcttg aaagatggta ttccaatatc 4080
acattttatg taagttttct attatattag attcaaatta cgattcgagg ccacaagctt
4140 taagaattca gggccttttt aacttgccaa gccccacacc actccaggaa
cttccccaca 4200 ccccagttct cagaattcat gtgcaaggtc tttcctaaat
ccagggtcca ggtcagagag 4260 tggaggatgt gctctatttc ttacctgatt
gcagacccct ctgacagtgc tcccttctga 4320 agcactcact gtctgaacgt
acacagtctc agacttaatc atgcacagtg agcaagactg 4380 tggtgtgata
attggcgtcc ctgacttatt agggcaaatc tatgggaggg ggagacctcc 4440
tggaccactg agcaattaat tcatttacat taggaagttt ctccgtcaga tgcaggaaaa
4500 aaatcttgtt ttcctgctgt ggttttgact tttgccccat cttctgttgc
tgttgtagga 4560 ggcaaaataa gggtcaaggc ctggaaacac aagtgctttg
actgaagctc cacttggctt 4620 ccgaagccca agctgggttg taccaggttc
cctagggtgc aggctgtggg caactgccag 4680 ggacatgtgc ctgcccaccg
gcctctggcc ctcactgagt tggccaatgg gaaatgacaa 4740 ttgtgaggtg
gggactgcct gcccccgtga gtaccaggct gttgaggctg ggccatctcc 4800
tcctcacttc c 4811 3 407 DNA Homo sapiens 3 atggatgact gttttatttg
tttgctgttc accacttctg ttttattctc tttccagaat 60 tcttttggat
tgtttaaata tttcataata ttttatctta atttatttat tgggtatttg 120
cctatatctc tttgtggtat tttttagtgg ttgcttgagg gattacaatg tacttaactt
180 ttcacagtgt gcataaagtt aatattttgc cacttgcagt aaaccgtaga
aggcttataa 240 tcatattagt acctctatcc actttctttt atgttgtagt
tgtcatatat attacatcta 300 tatacactga aacattatag gcaatgttat
gatttttgca ttcgtcagtc atatatatat 360 tttaaagaat ttaagaggag
aaaaatacat attcagatat tcatcat 407 4 366 DNA Homo sapiens
misc_feature (267)..(267) n=a, g, c or t 4 attctgactg cagtctgtgg
ttctgattcc ataccagagg ggctcaggat gctgttgctg 60 ggagctgttc
tactgctatt agctctgccc gggcatgacc aggaaaccac gactcaaggg 120
cccggagtcc tgcttcccct gcccaagggg gcctgcacag gttggatggc gggcatccca
180 gggcatccgg gccataatgg ggccccaggc cgtgatggca gagatggcac
ccctggtgag 240 aagggtgaga aaggagatcc aggtctnatt ggtcctaagg
gagacatcgg tgaaacggag 300 tacccggggc tgaaggtccc cgaggctttc
cgggaatcca aggcaggaaa ggagaaccgg 360 agaagg 366 5 4545 DNA Homo
sapiens CDS (49)..(783) 5 attctgactg cagtctgtgg ttctgattcc
ataccagagg ggctcagg atg ctg ttg 57 Met Leu Leu 1 ctg gga gct gtt
cta ctg cta tta gct ctg ccc ggk cat gac cag gaa 105 Leu Gly Ala Val
Leu Leu Leu Leu Ala Leu Pro Xaa His Asp Gln Glu 5 10 15 acc acg act
caa ggg ccc gga gtc ctg ctt ccc ctg ccc aag ggg gcc 153 Thr Thr Thr
Gln Gly Pro Gly Val Leu Leu Pro Leu Pro Lys Gly Ala 20 25 30 35 tgc
aca ggt tgg atg gcg ggc atc cca ggg cat ccg ggc cat aat ggg 201 Cys
Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly His Asn Gly 40 45
50 gcc cca ggc cgt gat ggc aga gat ggc acc cct ggt gag aag ggt gag
249 Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu Lys Gly Glu
55 60 65 aaa gga gat cca ggt ctt att ggt cct aag gga gac atc ggt
gaa acc 297 Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile Gly
Glu Thr 70 75 80 gga gta ccc ggg gct gaa ggt ccc cga ggc ttt ccg
gga atc caa ggc 345 Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro
Gly Ile Gln Gly 85 90 95 agg aaa gga gaa cct gga gaa ggt gcc tat
gta tac cgc tca gca ttc 393 Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr
Val Tyr Arg Ser Ala Phe 100 105 110 115 agt gtg gga ttg gag act tac
gtt act atc ccc aac atg ccc att cgc 441 Ser Val Gly Leu Glu Thr Tyr
Val Thr Ile Pro Asn Met Pro Ile Arg 120 125 130 ttt acc aag atc ttc
tac aat cag caa aac cac tat gat ggc tcc act 489 Phe Thr Lys Ile Phe
Tyr Asn Gln Gln Asn His Tyr Asp Gly Ser Thr 135 140 145 ggt aaa ttc
cac tgc aac att cct ggg ctg tac tac ttt gcc tac cac 537 Gly Lys Phe
His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr His 150 155 160 atc
aca gtc tat atg aag gat gtg aag gtc agc ctc ttc aag aag gac 585 Ile
Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys Asp 165 170
175 aag gct atg ctc ttc acc tat gat cag tac cag gaa aat aat gtg gac
633 Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val Asp
180 185 190 195 cag gcc tcc ggc tct gtg ctc ctg cat ctg gag gtg ggc
gac caa gtc 681 Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly
Asp Gln Val 200 205 210 tgg ctc cag gtg tat ggg gaa gga gag cgt aat
gga ctc tat gct gat 729 Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn
Gly Leu Tyr Ala Asp 215 220 225 aat gac aat gac tcc acc ttc aca ggc
ttt ctt ctc tac cat gac acc 777 Asn Asp Asn Asp Ser Thr Phe Thr Gly
Phe Leu Leu Tyr His Asp Thr 230 235 240 aac tga tcaccactaa
ctcagagcct cctccaggcc aaacagcccc aaagtcaatt 833 Asn aaaggctttc
agtacggtta ggaagttgat tattatttag ttggaggcct ttagatatta 893
ttcattcatt tactcattca tttattcatt cattcatcaa gtaactttaa aaaaatcata
953 tgctatgttc ccagtcctgg ggagcttcac aaacatgacc agataactga
ctagaaagaa 1013 gtagttgaca gtgctatttt gtgcccactg tctctcctga
tgctcatatc aatcctataa 1073 ggcacaggga acaagcattc tcctgttttt
acagattgta tcctgaggct gagagagtta 1133 agtgaatgtc taaggtcaca
caagtattaa gtgacagtgc tagaaatcaa acccagagct 1193 gtggactttg
ttcactagac tgtgcccttt tatagaggta catgttctct ttggagtgtt 1253
ggtaggtgtc tgtttcccac ctcacctgag agccattgaa tttgccttcc tcatgaatta
1313 aaacctcccc caagcagagc ttcctcagag aaagtggttc tatgatgaag
tcctgtcttg 1373 gaaggactac tactcaatgg cccctgcact actctacttc
ctcttaccta tgtcccttct 1433 catgcctttc cctccaacgg ggaaagccaa
ctccatctct aagtgctgaa ctcatccctg 1493 ttcctcaagg ccacctggcc
aggagcttct ctgatgtgat atccactttt tttttttttt 1553 gagatggagt
ctcactctgt cacccaggct ggagtacagt gacacgacct cggctcactg 1613
cagcctcctt ctcctgggtc caagcaatta ttgtgcctca gcctcccgag tagctgagac
1673 ttcaggtgca ttccaccaca catggctaat ttttgtattt ttagtagaaa
tggggtttcg 1733 tcatgttggc caggctggtc tcgaactcct ggcctaggtg
atccacccgc ctcgacctcc 1793 caaagtgctg ggattacagg crtgagccac
catgcccagt cgatatctca ctttttattt 1853 tgccatggat gagagtcctg
ggtgtgagga acacctccca ccaggctaga ggcaactgcc 1913 caggaaggac
tgtgcttccg tcacctctaa atcccttgca gatccttgat aaatgcctca 1973
tgaagaccaa tctcttgaat cccrtatcta cccagaatta actccattcc agtctctgca
2033 tgtaatcagt tttatccaca gaaacatttt cattttagga aatccctggt
tttaagtatc 2093 aatccttgtt cagctggaca atatgaatct tttccactga
agttagggat gactgtgatt 2153 ttcagaacac gtccagaatt tttcatcaag
aaggtagctt gagcctgaaa tgcaaaaccc 2213 atggaggaat tctgaagcca
ttgtctcctt gagtaccaac agggtcaggg aagactgggc 2273 ctcctgaatt
tattattgtt ctttaagaat tacaggttga ggtagttgat ggtggtaaac 2333
attctctcag gagacaataa ctccagtgat gttcttcaaa gattttagca aaaacagagt
2393 aaatagcatt ctctatcaat atataaattt aaaaaactat ctttttgctt
acagttttaa 2453 atcctgaaca attctctctt ayatgtgtat tgctaatcat
taaggtatta ttttttccac 2513 atataaagct ttgtcttttt gttgttgttg
ttgtttttaa gatggagttt ccctctgttg 2573 ccaggctaga gtgcagtggc
atgatctcgg cttactgcaa cctttgcctc ccaggttcaa 2633 gcgattcttc
tgcctcagcc tcccgagtag ctgggaccac aggtgcctac caccatgcca 2693
ggctaatttt tgtattttta gtaaagacag ggtttcacca tattggccag gctggtctcg
2753 aactcctgac cttgtgatct gcccacctcc atttttgttg ttattttttg
agaaagatag 2813 atatgaggtt tagagaggga tgaagaggtg agagtaagcc
ttgtgttagt cagaactctg 2873 tgttgtgaat gtcattcaca acagaaaacc
caaaatatta tgcaaactac tgtaagcaag 2933 aaaaataaag gaaaaatgga
aacatttatt cctttgcata atagaaatta ccagagttgt 2993 tctgtcttta
gataaggttt gaaccaaagc tcaaaacaat caagaccctt ttctgtatgt 3053
ccttctgttc tgccttccgc agtgtaggct ttaccctcag gtgctacaca gtatagttct
3113 agggtttccc tcccgatatc aaaaagactg tggcctgccc agctctcgta
tccccaagcc 3173 acaccatctg gctaaatgga catcatgttt tctggtgatg
cccaaagagg agagaggaag 3233 ctctctttcc cagatgcccc agcaagtgta
accttgcatc tcattgctct ggctgagttg 3293 tgtgcctgtt tctgaccaat
cactgagtca ggaggatgaa atattcatat tgacttaatt 3353 gcagcttaag
ttaggggtat gtagaggtat tttccctaaa gcaaaattgg gacactgtta 3413
tcagaaatag gagagtggat gatagatgca aaataatacc tgtccacaac aaactcttaa
3473 tgctgtgttt gagctttcat gagtttccca gagagacata gctggaaaat
tcctattgat 3533 tttctctaaa atttcaacaa gtagctaaag tctggctatg
ctcacagtct cacatctggt 3593 tggggtgggc tccttacaga acacgctttc
acagttaccc taaactctct ggggcagggt 3653 tattcctttg tggaaccaga
ggcacagaga gagtcaactg aggccaaaag aggcctgaga 3713 gaaactgagg
tcaagatttc aggattaatg gtcctgtgat gctttgaagt acaattgtgg 3773
atttgtccaa ttctctttag ttctgtcagc ttttgcttca tatattttag cgctctatta
3833 ttagatatat acatgtttag tattatgtct tattggtgca tttactctct
tatcattatg 3893 taatgtcctt ctttatctgt gataattttc tgtgttctga
agtctacttt gtctaaaaat 3953 aacatacgca ctcaacttcc ttttctttct
tccttccttt ctttcttcct tcctttcttt 4013 ctctctctct ctctttcctt
ccttccttcc tccttttctt tctctctctc tctctctctc 4073 tttttttgac
agactctcgt tctgtggccc tggctggagt tcagtggtgt gatcttggct 4133
cactgctacc tctaccatga gcaattctcc tgcctcagcc tcccaagtag ctggaactac
4193 aggctcatgc cactgcgccc agctaatttt tgtatttttc gtagagacgg
ggtttcacca 4253 cattcgtcag gttggtttca aactcctgac tttgtgatcc
acccgcctcg gcctcccaaa 4313 gtgctgggat tacaggcatg agccatcaca
cctggtcaac tttcttttga ttagtgtttt 4373 tgtggtatat ctttttccat
catgttactt taaatatatc tatattattg tatttaaaat 4433 gtgtttctta
cagactgcat gtagttgggt ataattttta tccagtctaa aaatatctgt 4493
cttttaattg gtgtttagac aatttatatt taataaaatt gttgaattta ag 4545 6
244 PRT Homo sapiens misc_feature (15)..(15) The 'Xaa' at location
15 stands for Gly. 6 Met Leu Leu Leu Gly Ala Val Leu Leu Leu Leu
Ala Leu Pro Xaa His 1 5 10 15 Asp Gln Glu Thr Thr Thr Gln Gly Pro
Gly Val Leu Leu Pro Leu Pro 20 25 30 Lys Gly Ala Cys Thr Gly Trp
Met Ala Gly Ile Pro Gly His Pro Gly 35 40 45 His Asn Gly Ala Pro
Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu 50 55 60 Lys Gly Glu
Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile 65 70 75 80 Gly
Glu Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly 85 90
95 Ile Gln Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg
100 105 110 Ser Ala Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro
Asn Met 115 120 125 Pro Ile Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln
Asn His Tyr Asp 130 135 140 Gly Ser Thr Gly Lys Phe His Cys Asn Ile
Pro Gly Leu Tyr Tyr Phe 145 150 155 160 Ala Tyr His Ile Thr Val Tyr
Met Lys Asp Val Lys Val Ser Leu Phe 165 170 175 Lys Lys Asp Lys Ala
Met Leu Phe Thr Tyr Asp Gln
Tyr Gln Glu Asn 180 185 190 Asn Val Asp Gln Ala Ser Gly Ser Val Leu
Leu His Leu Glu Val Gly 195 200 205 Asp Gln Val Trp Leu Gln Val Tyr
Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220 Tyr Ala Asp Asn Asp Asn
Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr 225 230 235 240 His Asp Thr
Asn 7 18 DNA Artificial Sequence sequencing oligonucleotide
PrimerPU 7 tgtaaaacga cggccagt 18 8 18 DNA Artificial Sequence
sequencing oligonucleotide PrimerRP 8 caggaaacag ctatgacc 18
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