Note: Descriptions are shown in the official language in which they were submitted.
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GENE AND SEQUENCE VARIATION ASSOCIATED WITH SENSING
CARBOHYDRATE COMPOUNDS AND OTHER SWEETENERS
FIELD OF THE INVENTION
The present invention relates generally to the field of mouse and human
genetics and sensing of extracellular carbohydrates. Specifically, the present
invention relates to the discovery of a gene and its sequence variation
associated
with a differential preference for sweet compounds in laboratory strains of
mice.
BACKGROUND OF THE INVENTION
The ability to sense extra-cellular carbohydrates, transduce this sensory
information, and relay it to the brain, is carried out by membrane bound
receptors
in taste papillae. Many approaches to identify the sweet receptor or receptors
have
been tried, but the problem has proved, until recently, to be difficult.
Mammals vary in their ad libitum consumption of sweeteners. To
investigate the genetic contribution to this complex behavior, behavioral,
electrophysiological, and genetic studies were conducted using two strains of
mice
that differ markedly in their preference for sucrose and saccharin (Bachmanov
et al., Behavior Genetics, 1996;26:563-573).
Recently published data indicates that the ability to sense carbohydrates is
linked to obesity. These studies demonstrated that sensation of simple
carbohydrates is suppressible by the adipose hormone, leptin.
These studies demonstrated that a locus on the telomere of mouse
chromosome 4 accounts for ~40% of the genetic variability in sucrose and
saccharin intake, and that the effect of this locus is to enhance or retard
the
gustatory neural response to sucrose.
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SUMMARY OF THE INVENTION
The present invention provides a gene and its sequence variation
associated with a preference for carbohydrate compounds, other sweeteners, or
alcohol.
The present invention provides a gene and its sequence variation
associated a differential response by the pancreas and/or muscle in response
to
dietary carbohydrates.
The present invention also relates to sequence variation and its use in the
diagnosis and prognosis of predisposition to diabetes, other obesity-related
disorders, or alcohol consumption.
The present invention also relates to the study of taste to identify
molecules responsible for signal transduction, other receptors and genes and
relationships that contribute to taste preference.
The present invention also relates to the study of diabetes to identify
molecules responsible for sensing extra-cellular carbohydrate, other receptors
and
genes and relationships that contribute to a diabetic state.
The present invention also relates to a sequence variation and its use in the
identification of specific alleles altered in their specificity for
carbohydrate
compounds.
The present invention also relates to a recombinant construct comprising
SACl (also referred to as Sac) polynucleotide suitable for expression in a
transformed host cell.
The present invention also provides primers and probes specific for the
detection and analysis of the SACl locus.
The present invention also relates to kits for detecting a polynucleotide
comprising a portion of the SAC 1 locus.
The present invention also relates to transgenic animals, which carry an
altered SAC1 allele, such as a knockout mouse.
The present invention also relates to methods for screening drugs for
inhibition or restoration of SACl function as a taste receptor.
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The present invention also relates to identification of sweeteners or
alcohols using the SACl gene and its sequence variations.
The present invention also relates to methods for screening drugs for
inhibition or restoration of SAC1 function in homeostatic regulation of
glucose
levels.
The present invention also relates to methods for screening drugs for
modification of SAC1 function in the consumption of alcohol.
Finally, the present invention provides therapies directed to diabetic or
obesity disorders. Therapies of diabetes and obesity include gene therapy,
protein
replacement, protein mimetics, and inhibitors.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1A shows genetic mapping of the SAC1 locus, using 632 F2 mice
from a cross between the B6 (high preference) and 129 (low preference)
strains.
Mapping results were obtained with MAPMAKERIQTL Version 1.1, using an
unconstrained model. A black triangle at the bottom indicates peak LOD score
at
M134G01 marker. Horizontal line at the bottom shows a 1-LOD confidence
interval.
Fig. 1B shows SAC1-containing chromosomal region defined by a donor
fragment of the 129.B6-Sacb partially congenic mice. The partially congenic
strains were constructed by identifying several founder F2 mice with small
fragments of the telomeric region of mouse chromosome 4 from the B6 strain and
successive backcrossing to the 129 strain. Presence and size of donor fragment
were determined by genotyping polymorphic markers in mice from the N4, N6,
N7, N4F4, and N3F5 generations.
Fig. 1 C shows average daily saccharin consumption by N6, N7, N4F4, and
N3F5 segregating partially congenic 129.B6-Sac mice in 4-days two-bottle tests
with water (means ~ SE). The open bar indicates intakes of mice that did not
inherit the donor fragment. The black bar indicates intakes of mice with one
or
two copies of the donor fragment, which is flanked by 280612-T7 proximally and
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D4Mon1 distally. The complete donor fragment is represented by overlapping
sequences of the BAC RPCI-23-118E21 and a genomic clone (Accession
AF 185591 ), as indicated at the bottom. The size of the SAC 1-containing
donor
fragment is 194, 478 kb.
Fig. 1D shows BAC contig of distal chromosome 4 in the SACl region.
Using 32P radioactively labeled probes from the nonrecombinant interval, a
mouse BAC library (RPCI-23) was screened; positive clones were confirmed by
PCR analysis and only clones positive by hybridization and by PCR are included
in the contig. BAC ends were sequenced and PCR primers designed. The STS
content of each BAC, using all BAC ends was determined. BAC size was
determined by digesting the BAC with Notl, and the insert size determined
using
pulse field gel electrophoresis.
Fig. 1E shows genes contained within the SACl nonrecombinant interval.
Arrows indicate predicted direction of transcription. See Table 1 for a
description
of gene prediction, and details concerning function.
Fig. 2A shows the mouse SAC1 gene (mSac; Accession AF311386), its
human ortholog (hSac), and the previously described gene TlRl, now Gpr70, are
aligned above. Residues shaded in black are identical between at least two
identical residues; residues in gray indicate conservative changes. The human
ortholog was identified by sequence homology search within the htgs database
(Accession AC026283). The amino acid sequence of the human ortholog was
predicted using GENSCAN. The amino acid sequence of mouse Gpr70 was
obtained by constructing primers based upon the nucleotide sequence, and taste
cDNA was amplified and sequenced. This amino acid and nucleotide sequence for
Gpr70 differed slightly from the initial report; the sequence reported in this
paper
has been deposited in GenBank (AF301161, AF301162). The location of the
missense mutation is indicated by an *.
Fig. 2B shows structure of the SAC1 gene. The six exons are shown as
black boxes.
Fig. 2C shows conformation of a protein predicted from the Sac gene. To
determine the transmembrane regions, the hydrophobicity was determined using
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the computer program HMMTOP, and drawn with TOPO. The missense mutation
is denoted with an asterisk.
Fig. 3 shows saccharin and sucrose preferences by mice from inbred
strains with two different haplotypes of the Sac gene. The haplotype found in
the
B6 mice and the other high sweetener-preferring inbred strains consisted of
four
variants, two variants were 5' of the predicted translation start codon, one
variant
was a missense mutation (I1e61 Thr), and the last variant was located in the
intron
between exon 2 and 3. The strains with the B6-like haplotype of Sac strongly
preferred saccharin (82 ~ 4%) and sucrose (86 ~ 6%), whereas strains with the
129-like haplotype were indifferent to these solutions (57 ~ 2% and 54 ~ 1
respectively, p = 0.0015).
Fig. 4A shows tissue expression of the SAC 1 gene. Note that cDNA was
obtained from a commercial source for the multiple tissue panel, with the
exception of tongue cDNA, which was as isolated by the investigator, as
described
within the text. Relative band intensities may differ due to differences in
cDNA
isolation methods or concentration.
Fig. 4B shows RNA from human fungiform papillae was obtained from
biopsy material, reversed transcribed, and the resulting bands from genomic
and
cDNA were amplified using primers, described in the text. The bands were
excised from the agarose gel, purified and reamplified. The PCR product was
sequenced to confirm that the bands amplified the human otholog to Sac.
Fig. 5 shows amino acid sequence alignment of the mouse cDNA sequence
for the SAC 1 gene and the cDNA for a calcium sensing metabotropic receptor.
Dark areas indicated regions of shared similarity.
Fig. 6 plots the hydrophobicity of the SACl amino acid sequence as
predicted by the computer program Top Pred. Note the seven transmembrane
domains characteristic of G-protein coupled receptors.
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DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
The present invention employs the following definitions:
As used herein, the terms "polynucleotide" and "nucleic acid" refer to
naturally occurring polynucleotides, e.g., DNA or RNA. These terms do not
refer
to a specific length. Thus, these terms include oligonucleotide, primer,
probe, etc.
These terms also refer to analogs of naturally occurring polynucleotides. The
polynucleotide may be double stranded or single stranded. The polynucleotides
may be labeled with radiolabels, fluorescent labels, enzymatic labels,
proteins,
haptens, antibodies, sequence tags.
For example, these terms include RNA, cDNA, genomic DNA, synthetic
forms, and mixed polymers, both sense and antisense strands, and may be
chemically or biochemically modified or may contain non-natural or derivatized
nucleotide bases, as will be readily appreciated by those skilled in the art.
Such
modifications include, for example, labels, methylation, substitution of one
or
more of the naturally occurring nucleotides with an analog, internucleotide
modifications such as uncharged linkages (e.g., methyl phosphonates,
phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g.,
polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators,
alkylators,
and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also
included are
synthetic molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical interactions. Such
molecules are known in the art and include, for example, those in which
peptide
linkages substitute for phosphate linkages in the backbone of the molecule.
As used herein, the term "polynucleotide amplification" refers to a broad
range of techniques for increasing the number of copies of specific
polynucleotide
sequences. Typically, amplification of either or both strand of the target
nucleic
acid comprises the use of one or more nucleic acid-modifying enzymes, such as
a
DNA polymerase, a ligase, an RNA polymerase, or an RNA-dependent reverse
transcriptase. Examples of polynucleotide amplification reaction include, but
not
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limited to, polymerase chain reaction (PCR), nucleic acid sequence based
amplification (NASB), self sustained sequence replication (3SR), strand
displacement activation (SDA), ligase chain reaction (LCR), Q[3 replicase
system,
and the like.
As used herein, the term "primer" refers to a nucleic acid, e.g., synthetic
polynucleotide, which is capable of annealing to a complementary template
nucleic acid (e.g., the SAC1 locus) and serving as a point of initiation for
template-directed nucleic acid synthesis. A primer need not reflect the exact
sequence of the template but must be sufficiently complementary to hybridize
with a template. Typically, a primer will include a free hydroxyl group at the
3' end. The appropriate length of a primer depends on the intended use of the
primer but typically ranges from 12 to 30 nucleotides. The term primer pair
means
a set of primers including a 5' upstream primer that hybridizes with the
5° end of
the target sequence to be amplified and a 3' downstream primer that hybridizes
with the complement of the 3' end of the target sequence to be amplified.
The present invention includes all novel primers having at least
eight nucleotides derived from the SAC1 locus for amplifying the SACl gene,
its
complement or functionally equivalent nucleic acid sequences. The present
invention does not include primers which exist in the prior art. That is, the
present
invention includes all primers having at least 8 nucleotides with the proviso
that it
does not include primers existing in the prior art.
"Target polynucleotide" refers to a single- or double-stranded
polynucleotide which is suspected of containing a target sequence, and which
may
be present in a variety of types of samples, including biological samples.
"Antibody" refers to polyclonal and/or monoclonal antibody and
fragments thereof, and.immunologic binding equivalents thereof, which are
capable of specifically binding to the SAC 1 polypeptides and fragments
thereof or
to polynucleotide sequences from the SAC 1 region, particularly from the
SAC1 locus or a portion thereof. Antibody may be a homogeneous molecular
entity, or a mixture such as a serum product made up of a plurality of
different
molecular entities.
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Antibodies may be produced by in vitro or in vivo techniques well-known
in the art. For example, for production of polyclonal antibodies, an
appropriate
target immune system, typically mouse or rabbit, is selected. Substantially
purified antigen is presented to the immune system. Typical sites for
injection are
in footpads, intramuscularly, intraperitoneally, or intradermally. Polyclonal
antibodies may then be purified and tested for immunological response, e.g.,
using
an immunoassay.
For production of monoclonal antibodies, protein, polypeptide, fusion
protein, or fragments thereof may be injected into mice. After the appropriate
period of time, the spleens may be excised and individual spleen cells fused,
typically, to immortalized myeloma cells under appropriate selection
conditions.
Thereafter, the cells are clonally separated and the supernatants of each
clone
tested for their production of an appropriate antibody specific for the
desired
region of the antigen. Affinities of monoclonal antibodies are typically 10-g
M-1
or preferably 10-9 to 10-10 M-1 or stronger.
Other suitable techniques involve in vitro exposure of lymphocytes to the
° antigenic polypeptides, or alternatively, to selection of libraries
of antibodies in
phage or similar vectors.
Frequently, antibodies are labeled by joining, either covalently or non-
covalently, a substance which provides for a detectable signal. A wide variety
of
labels and conjugation techniques are known. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents,
chemiluminescent agents, magnetic particles, and the like. Also, recombinant
immunoglobulins may be produced.
"Binding partner" refers to a molecule capable of binding another
molecule with specificity, as for example, an antigen and an antigen-specific
antibody or an enzyme and its inhibitor. Binding partners are known in the art
and
include, for example, biotin and avidin or streptavidin, IgG and protein A,
receptor-ligand couples, and complementary polynucleotide strands. In the case
of
complementary polynucleotide binding partners, the partners are normally at
least
about 15, 20, 25, 30, 40 bases in length.
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A "biological sample" refers to a sample of tissue or fluid suspected of
containing an analyte (e.g., polynucleotide, polypeptide) including, but not
limited
to, e.g., plasma, serum, spinal fluid, lymph fluid, the external sections of
the skin,
respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells,
organs,
tissue and samples of in vitro cell culture constituents. A biological sample
is
typically from human or other animal.
"Encode." A polynucleotide is said to "encode" a polypeptide if, in its
native state or when manipulated by methods well-known to those skilled in the
art, it can be transcribed and/or translated to produce the mRNA and/or the
polypeptide .or a fragment thereof. The antisense strand is the complement of
such
a nucleic acid, and the encoding sequence can be deduced therefrom.
"Isolated" or "substantially pure" polynucleotide or polypeptide (e.g., an
RNA, DNA, protein) is one which is substantially separated from other cellular
components which naturally accompany a native human nucleic acid or protein,
e.g., ribosomes, polymerases, many other human genome sequences and proteins.
The term embraces a nucleic acid or peptide sequence which has been removed
from its naturally occurring environment, and includes recombinant or cloned
DNA isolates and chemically synthesized analogs or analogs biologically
synthesized by heterologous systems.
"SAC 1 Allele" refers to normal alleles of the SAC 1 locus as well as alleles
carrying variations that predispose individuals to develop obesity, diabetes,
or for
alcohol consumption or alcoholism.
"SAC1 Locus" refers to polynucleotides, which are in the SAC1 region,
that are likely to be expressed in normal individual, certain alleles of which
predispose an individual to develop obesity, diabetes, or alcohol consumption
or
alcoholism. The SACl locus includes coding sequences, intervening sequences
and regulatory elements controlling transcription and/or translation. The
SAC1 locus includes all allelic variations of the DNA sequence.
The DNA sequences used in this invention will usually comprise at least
about S codons (15 nucleotides), 7, 10, 15, 20, or 30 codons, and most
preferably,
at least about 35 codons. One or more introns may also be present. This number
of
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nucleotides is usually about the minimal length required for a successful
probe
that would hybridize specifically with a SAC 1 locus.
"SAC1 Region" refers to a portion of mouse chromosome 4 bounded by
the markers 280612-T7 and D4Monl GenBank Accession number is YG7772
(SEQ ID NO: 652) and is
GCAGTGAGCTGCAGAGTTTGCAGAATGAGGGCACTCTAAACTCATCAA
GTGAGGAGGCCCTTCCCTCACACTCCAGATGGCTGATAGGTGGCATTA
CATGGTC(CA)nCGCGCGCACGCGCTCAGATGCAATCTCCACATTCATA
ACCAGATGTCCTTGGGTAGGCCT. The CA sequence in the middle is
variable in length. In the B6 mouse, n =19, while in the 129 mouse, n =16.
This
region contains the SAC1 locus, including the SAC1 gene. GenBank accession
number for the SAC1 gene is AF311386.
As used herein, a "portion" or "fragment" of the SACl gene, locus, region,
or allele is defined as having a minimal size of at least about 15
nucleotides, or
preferably at least about 20, or more preferably at least about 25
nucleotides, and
may have a minimal size of at least about 40 nucleotides.
As used herein, the term "polypeptide" refers to a polymer of amino acids
without referring to a specific length. This term includes to naturally
occurring
protein. The term also refers to modifications, analogues and functional
mimetics
thereof. For example, modifications of the polypeptide may include
glycosylations, acetylations, phosphorylations, and the like. Analogues of
polypeptide include unnatural amino acid, substituted linkage; etc. Also
included
are polypeptides encoded by DNA which hybridize under high or low stringency
conditions, to the nucleic acids of interest.
Modification of polypeptides includes those substantially homologous to
primary structural sequence, e.g., in vivo or in vitro chemical and
biochemical
modifications or incorporation unusual amino acids. Such modifications
include,
for example, acetylation, carboxylation, phosphorylation, glycosylation,
ubiquitination, labeling, e.g., with radionuclides, and various enzymatic
modifications, as will be readily appreciated by those well-skilled in the
art. A
variety of methods for labeling polypeptides and of substituents or labels
useful
for such purposes are well-known in the art, and include radioactive isotopes
such
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as 32P, ligands which bind to labeled antiligands (e.g., antibodies),
fluorophores,
chemiluminescent agents, enzymes, and antiligands which can serve as specific
' binding pair members for a labeled ligand. The choice of label depends on
the
sensitivity required, ease of conjugation with the primer, stability
requirements,
and available instrumentation. Methods of labeling polypeptides are well-known
in the art (see Sambrook et al., 1989 or Ausubel et al., 1992).
Besides substantially full-length polypeptides, the present invention
provides for biologically active fragments of the polypeptides. Significant
biological activities include ligand-binding, immunological activity, and
other
biological activities characteristic of SAC1 polypeptides. Immunological
activities
include both immunogenic function in a target immune system, as well as
sharing
of immunological epitopes for binding, serving as either a competitor or
substitute
antigen for an epitope of the SAC1 protein. As used herein, "epitope" refers
to an
antigenic determinant of a polypeptide. An epitope could comprise three amino
acids in a spatial conformation that is unique to the epitope. Generally, an
epitope
consists of at least five such amino acids, and more usually consists of at
least
8 to 10 such amino acids. Methods of determining the spatial conformation of
such amino acids are known in the art.
For immunological purposes, tandem-repeat polypeptide segments may be
used as immunogens, thereby producing highly antigenic proteins.
Alternatively,
such polypeptides will serve as highly efficient competitors for specific
binding.
Fusion proteins comprise SACl polypeptides and fragments. Homologous
polypeptides may be fusions between two or more SAC 1 polypeptide sequences
or between the sequences of SACl and a related protein. Likewise, heterologous
fusions may be constructed which would exhibit a combination of properties or
activities of the derivative proteins. For example, ligand-binding or other
domains
may be "swapped" between different new fusion polypeptides or fragments. Such
homologous or heterologous fusion polypeptides may display, for example,
altered strength or specificity of binding. Fusion partners include
immunoglobulins, bacterial ~i-galactosidase, trpE, protein A, (3-lactamase,
oc-amylase, alcohol dehydrogenase, and yeast a mating factor.
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Fusion proteins will typically be made by either recombinant nucleic acid
methods or may be chemically synthesized. Techniques for the synthesis of
polypeptides are known in the art.
Functional mimetics of a native polypeptide may be obtained using known
methods in the art. For example, polypeptides may be least about 50%
homologous to the native amino acid sequence, preferably in excess of about
70%,
and more preferably at least about 90% homologous. Substitutions typically
contain the exchange of one amino acid for another at one or more sites within
the
polypeptide, and may be designed to modulate one or more properties of the
polypeptide, such as stability against proteolytic cleavage, without the loss
of
other functions or properties. Amino acid substitutions may be made on the
basis
of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or
the amphipathic nature of the residues involved. Preferred substitutions are
ones
which are conservative, that is, one amino acid is replaced with one of
similar
shape and charge. Conservative substitutions are well-known in the art and
typically include substitutions within the following groups: glycine, alanine;
valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine,
glutamine;
serine, threonine; lysine, arginine; and tyrosine, phenylalanine.
Certain amino acids may be substituted for other amino acids in a
polypeptide structure without appreciable loss of interactive binding capacity
with
structures such as, for example, antigen-binding regions of antibodies or
binding
sites on substrate molecules or binding sites on proteins interacting with a
polypeptide. Since it is the interactive capacity and nature of a polypeptide
which
defines that polypeptide's biological functional activity, certain amino acid
substitutions can be made in a protein sequence, and its underlying DNA coding
sequence, and nevertheless obtain a protein with like properties. In making
such
changes, the hydropathic index of amino acids may be considered. The
importance of the hydrophobic amino acid index in confernng interactive
biological function on a protein is generally understood in the art.
Alternatively,
the substitution of like amino acids can be made effectively on the basis of
hydrophilicity.
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A peptide mimetic may be a peptide-containing molecule that mimics
elements of protein secondary structure. The underlying rationale behind the
use
of peptide mimetics is that the peptide backbone of proteins exists chiefly to
orient
amino acid side chains in such a way as to facilitate molecular interactions,
such
as those of antibody and antigen, enzyme and substrate or scaffolding
proteins. A
peptide mimetic is designed to permit molecular interactions similar to the
natural
molecule. A mimetic may not be a peptide at all, but it will retain the
essential
biological activity of a natural polypeptide.
Polypeptides may be produced by expression in a prokaryotic cell or
produced synthetically. These polypeptides typically lack native post-
translational
processing, such as glycosylation. Polypeptides may be labeled with
radiolabels,
fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence
tags.
"SACl polypeptide" refers to a protein or polypeptide encoded by the
SAC1 locus, variants, fragments or functional mimics thereof. A SAC
polypeptide
may be that derived from any of the exons described herein which may be in
isolated and/or purified form. The length of SAC1 polypeptide sequences is
generally at least about S amino acids, usually at least about 10, 15, 20,
30 residues.
"Alcohol consumption" relates to the intake andlor preference of an animal
for ethanol.
"Diabetes" refers to any disorder that exhibits phenotypic features of an
increased or decreased level of a biological substance associated with glucose
or
fatty acid metabolism. The term "carbohydrate" refers to simple mono and
disaccharides.
The terms "sequence variation" or "variant form" encompass all forms of
polymorphism and mutations. A sequence variation may range from a single
nucleotide variation to the insertion, modification, or deletion of more than
one
nucleotide. A sequence variation may be located at the exon, intron, or
regulatory
region of a gene.
Polymorphism refers to the occurrence of two or more genetically
determined alternative sequences or alleles in a population. A biallelic
polymorphism has two forms. A triallelic polymorphism has three forms. A
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polymorphic site is the locus at which sequence divergence occurs. Diploid
organisms may be homozygous or heterozygous for allelic forms. Polymorphic
sites have at least two alleles, each occurring at frequency of greater than 1
% of a
selected population. Polymorphic sites also include restriction fragment
length
polymorphisms, variable number of tandem repeats (VNTRs), hypervariable
regions, minisatellites, dinucleotide repeats, trinucleotide repeats,
tetranucleotide
repeats, simple sequence repeats, and insertion elements. The first identified
allelic form may be arbitrarily designated as the reference sequence and other
allelic forms may be designated as alternative or variant alleles. The allelic
form
occurring most frequently in a selected population is sometimes referred to as
the
wild type form or the consensus sequence.
Mutations include deletions, insertions and point mutations in the coding
and noncoding regions. Deletions may be of the entire gene or of only a
portion of
the gene. Point mutations may result in stop codons, frameshift mutations, or
amino acid substitutions.. Somatic mutations are those which occur only in
certain
tissues, such as liver, heart, etc. and are not inherited in the germline.
Germline
mutations can be found in any of a body's tissues and are inherited.
"Operably linked" refers to a juxtaposition wherein the components are in
a relationship permitting them to function in their intended manner. For
instance, a
promoter is operably linked to a coding sequence if the promoter affects its
transcription or expression.
The term "probes" refers to polynucleotide of any suitable length which
allows specific hybridization to the target region. Probes may be attached to
a
label or reporter molecule using known methods in the art. Probes may be
selected
by using homologous polynucleotides. Alternatively, polynucleotides encoding
these or similar polypeptides may be synthesized or selected by use of the
redundancy in the genetic code. Various codon substitutions may be introduced,
e.g., by silent changes (thereby producing various restriction sites) or to
optimize
expression for a particular system. Mutations may be introduced to modify the
properties of the polypeptide, perhaps to change ligand-binding affinities,
interchain affinities, or the polypeptide degradation or turnover rate.
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Probes comprising synthetic oligonucleotides or other polynucleotides of
the present invention may be derived from naturally occurring or recombinant
single- or double-stranded polynucleotides, or be chemically synthesized.
Probes
may also be labeled by nick translation, Klenow fill-in reaction, or other
methods
known in the art.
Portions of the polynucleotide sequence having at least about
8 nucleotides, usually at least about 1 S nucleotides, and fewer than about 6
kb,
usually fewer than about 1.0 kb, from a polynucleotide sequence encoding SAC1
are preferred as probes.
The terms "isolated," "substantially pure," and "substantially
homogeneous" are used interchangeably to describe a protein or polypeptide
which has been separated from components which accompany it in its natural
state. A monorneric protein is substantially pure when at least about 60% to
7S%
of a sample exhibits a single polypeptide sequence. A substantially pure
protein
1 S will typically comprise about 60% to 90% W/W of a protein sample, more
usually
about 9S%, and preferably will be over about 99% pure. Protein purity or
homogeneity may be indicated by a number of means well-known in the art, such
as polyacrylamide gel electrophoresis of a protein sample, followed by
visualizing
a single polypeptide band upon staining the gel. For certain purposes, higher
resolution may be provided by using HPLC or other means well-known in the art
which are utilized for purification.
A SACl protein is substantially free of naturally associated components
when it is separated from the native contaminants which accompany it in its
natural state. Thus, a polypeptide which is chemically synthesized or
synthesized
2S in a cellular system different from the cell from which it naturally
originates will
be substantially free from its naturally associated components. A protein may
also
be rendered substantially free of naturally associated components by
isolation,
using protein purification techniques well-known in the art.
"Recombinant nucleic acid" is a nucleic acid which is not naturally
occurnng, or which is made by the artificial combination of two otherwise
separated segments of sequence. This artificial combination is often
accomplished
by either chemical synthesis means, or by the artificial manipulation of
isolated
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segments of nucleic acids, e.g., by genetic engineering techniques. Such is
usually
done to replace a codon with a redundant codon encoding the same or a
conservative amino acid, while typically introducing or removing a sequence
recognition site. Alternatively, it is performed to join together nucleic acid
segments of desired functions to generate a desired combination of functions.
"Regulatory sequences" refers to those sequences normally within 100 kb
of the coding region of a locus, but they may also be more distant from the
coding
region, which affect the expression of the gene (including transcription of
the
gene, and translation, splicing, stability or the like of the messenger RNA).
"Substantial homology or similarity." A nucleic acid or fragment thereof is
of substantially homologous ("or substantially similar") to another if, when
optimally aligned (with appropriate nucleotide insertions or deletions) with
the
other nucleic acid (or its complementary strand), there is nucleotide sequence
identity in at least about 60% of the nucleotide bases, usually at least about
70%,
more usually at least about 80%, preferably at least about 90%, and more
preferably at least about 95-98% of the nucleotide bases.
Identity means the degree of sequence relatedness between two
polypeptide or two polynucleotides sequences as determined by the identity of
the
match between two strings of such sequences. Identity can be readily
calculated
(Lesk A.M., ed., Computational Molecular Biology, New York: Oxford
University Press, 1988; Smith D.W., ed., Biocomputing: hzformatics and Gen,
ome
Projects, New York: Academic Press, New York, 1993; Griffin A.M., and
Griffin H.G., eds., Computer Analysis of Sequence Data, Part l, New Jersey:
Humana Press, 1994; von Heinje G., Sequence Analysis in Molecular Biology,
Academic Press, 1987; and Gribskov M. and Devereux J., eds., Sequence Analysis
Primer, New York: M Stockton Press, 1991).
Alternatively, substantial homology or similarity exists when a nucleic
acid or fragment thereof will hybridize to another nucleic acid (or a
complementary strand thereof) under selective hybridization conditions, to a
strand, or to its complement. Selectivity of hybridization exists when
hybridization which is substantially more selective than total lack of
specificity
occurs. Typically, selective hybridization will occur when there is at least
about
Glu Leu Val Met Ala Leu A
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55% homology over a stretch of at least about 14 nucleotides, preferably at
least
about 65%, more preferably at least about 75%, and most preferably at least
about
90%. The length of homology comparison, as described, may be over longer
stretches, and in certain embodiments will often be over a stretch of at least
about
9 nucleotides, usually at least about 20 nucleotides, more usually at least
about
24 nucleotides, typically at least about 28 nucleotides, more typically at
least
about 32 nucleotides, and preferably at least about 36 or more nucleotides.
Nucleic acid hybridization will be affected by such conditions as salt
concentration, temperature, or organic solvents, in addition to the base
composition, length of the complementary strands, and the number of nucleotide
base mismatches between the hybridizing nucleic acids, as will be readily
appreciated by those skilled in the art. Stringent temperature conditions will
generally include temperatures in excess of 30°C, typically in excess
of 37°C, and
preferably in excess of 45°C. Stringent salt conditions will ordinarily
be less than
1000 mM, typically less than 500 mM, and preferably less than 200 mM.
However, the combination of parameters is much more important than the
measure of any single parameter.
The terms "substantial homology" or "substantial identity," when referring
to polypeptides, indicate that the polypeptide or protein in question exhibits
at
least about 30% identity with an entire naturally-occurring protein or a
portion
thereof, usually at least about 70% identity, and preferably at least about
95%
identity.
Homology, for polypeptides, is typically measured using sequence analysis
software (see, e.g., the Sequence Analysis Software Package of the Genetics
Computer Group, University of Wisconsin Biotechnology Center). Protein
analysis software matches similar sequences using measures of homology
assigned to various substitutions, deletions and other modifications.
Conservative
substitutions typically include substitutions within the following groups:
glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine,
glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
"Substantially similar function" refers to the function of a modified nucleic
acid or a modified protein, with reference to the wild-type SAC1 nucleic acid
or
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wild-type SAC1 polypeptide. The modified polypeptide will be substantially
homologous to the wild-type SAC1 polypeptide and will have substantially the
same function. The modified polypeptide may have an altered amino acid
sequence and/or may contain modified amino acids. In addition to the
similarity of
function, the modified polypeptide may have other useful properties, such as a
longer half life. The similarity of function (activity) of the modified
polypeptide
may be substantially the same as the activity of the wild-type SACl
polypeptide.
Alternatively, the similarity of function (activity) of the modified
polypeptide may
be higher than the activity of the wild-type SAC 1 polypeptide. The modified
polypeptide is synthesized using conventional techniques, or is encoded by a
modified nucleic acid and produced using conventional techniques. The modified
nucleic acid is prepared by conventional techniques. A nucleic acid with a
function substantially similar to the wild-type SAC1 gene function produces
the
modified protein described above.
A polypeptide "fragment," "portion," or "segment" is a stretch of amino
acid residues of at least about 5 to 7 contiguous amino acids, often at least
about
7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous
amino
acids and, most preferably, at least about 20 to 30 or more contiguous amino
acids.
The polypeptides of the present invention, if soluble, may be coupled to a
solid-phase support, e.g., nitrocellulose, nylon, column packing materials
(e.g.,
Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels,
cells,
or other substrates. Such supports may take the form, for example, of beads,
wells,
dipsticks, or membranes.
"Target region" refers to a region of the nucleic acid which is amplified
and/or detected. The term "target sequence" refers to a sequence with which a
probe or primer will form a stable hybrid under desired conditions.
II. Positional Cloning of Mouse SACl Gene and the Discovery of a Gene and
Its Sequence Variation Associated With Altered Sensation for
Carbohydrates
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Inbred strains of mice differ in their intake of sweeteners (Bachmanov
A.A., Reed D.R., Tordoff M.G., Price R.A., and Beauchamp G.K. Intake of
ethanol, sodium chloride, sucrose, citric acid, and quinine hydrochloride
solutions
by mice: a genetic analysis. Behavior Genetics, 1996;26:563-573; Lush LE.,
.The
S genetics of tasting in mice. VI. Saccharin, acesulfame, dulcin and sucrose.
Genet
Res, 1989;53:95-99; Lush I. The genetics of bitterness, sweetness, and
saltiness in
strains of mice. In Genetics of Perception and Communication, Vol. 3, eds.
Wysocki C. and Kare M., New York: Marcel Dekker, 1991:227-235;
Capretta P.J. Saccharin and saccharin-glucose ingestion in two inbred strains
of
Mus musculus. Psychon. Sci., 1970;21:133-135; Nachman M. The inheritance of
saccharin preference. Journal of Comp Physiol Psychol, 1959;52:451-457).
Breeding and linkage experiments suggest that a single gene, the Sac locus
(for
saccharin intake), accounts for a large proportion of the genetic variance
(Fuller J.L. Single-locus control of saccharin preference in mice. Journal of
Heredity, 1974;65:33-36; Capeless C.G. and Whitney G. The genetic basis of
preference for sweet substances among inbred strains of mice: preference ratio
phenotypes and the alleles of the Sac and dpa loci. Chem Senses, 1995;20:291=
298; Bachmanov A.A. et al. Sucrose consumption in mice: major influence of two
genetic loci affecting peripheral sensory responses. Mammalian Genome,
1997;8:545-548; Belknap J.K. et al. Single-locus control of saccharin intake
in
BXD/Ty recombinant inbred (RI) mice: some methodological implications for RI
strain analysis. Behav Genet, 1992;22:81-100; Blizard D.A., Kotlus B., and
Frank M.E. Quantitative trait loci associated with short-term intake of
sucrose,
saccharin and quinine solutions in laboratory mice. Chem Senses, 1999;24:373-
85). Using genetic and physical mapping methods, an interval of 194 kb was
identified at the telomeric end of mouse chromosome 4 that contains the Sac
locus. BAC sequencing within this interval led to the identification of a gene
that
has a 30% amino acid homology with other putative taste receptors (Noon M.A.
et al. Putative mammalian taste receptors: a class of taste-specific GPCRs
with
distinct topographic selectivity. Cell, 1999;96:541-551). This gene is
expressed in
mouse tongue. Mutation detection on this gene revealed a missense mutation
(Ile6lThr) with four other sequence variants define a haplotype found in mice
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with low sweetener preference (129, Balb/c, AKR, and DBA2). An alternative
five variant haplotype is found in mice with a high preference for sweet
fluids
(B6, SWR, IS, ST, and SEA). A human ortholog of this gene exists, and is
expressed in human taste papillae. We therefore suggest that this gene is a
sweet
taste receptor, and variation within this gene is responsible for the
phenotype of
the Sac locus.
To identify this locus, mice from the high sweetener preference
(C57BLl6ByJ; B6) and the low sweetener preference (129P3/J; formerly 129/J,
abbreviated here as 129) were used as parental strains to produce an F2
generation. The F2 mice were phenotyped for sweetener preference using 96-hour
two-bottle taste tests and genotyped with markers polymorphic between the
B6 and 129 strains (Fig. 1A). The results of this analysis indicated peak
linkage
near marker D18346 with the B6 allele having a dominant mode of inheritance.
Using recombinant mice from the F2 generation, 129.B6-Sac partially congenic
mice were created, using genotypic (B6 allele at D18346; Fig. 1B) and
phenotypic
(high saccharin intake; Fig. 1 C) characteristics as selection criteria for
each
generation. Genotyping of partially congenic mice with polymorphic markers
defined the Sac nonrecombinant interval. Radiation hybrid mapping was
conducted with additional markers (R74924, D18402, D18346, Agrin, V2r2 and
D4Ertd296e). These markers were amplified using DNA and mouse and hamster
control DNA in the T31 mouse radiation hybrid panel, scored for the presence
or
absence of an appropriately sized band, and the data analyzed by the Jackson
Laboratory. All markers were within the SAC1 confidence interval suggested by
the initial linkage analysis, and were used in subsequent analyses.
A BAC library was screened with markers within the nonrecombinant
interval, and a contig was developed (Fig. 1D). A BAC clone was selected for
sequencing (RPCI-23-118E21, 246 kb). Within this BAC, a gene with a 30%
homology to T1R1 (a putative taste receptor) was discovered (Fig. 2A), along
with other ESTs and known genes (Table 1). The human orthalog to this gene was
identified from a BAC available in the public htgs database, and the predicted
protein sequence was aligned with SACl and TlRl. SACl is 858 amino acids in
length and contains six exons; the intron and exon boundaries were determined
by
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sequencing of the mouse tongue cDNA (Fig. 2B). The secondary structure of this
protein with regards to transmembrane domains was predicted (Fig. 2C).
To determine whether this gene might contain functional polymorphisms
that could account for the behavioral differences between the two strains,
11.8 kb
of sequence, including the SACl gene and several kb up and downstream were
amplified with PCR primers and then sequenced using DNA from the high and
low prefernng strains (Lush LE., The genetics of tasting in mice. VI.
Saccharin,
acesulfame, dulcin and sucrose. Genet Res1989;53:95-99; Lush I. The genetics
of
bitterness, sweetness, and saltiness in strains of mice. In Genetics of
Perception
and Communication, Vol. 3, eds. Wysocki C. and Kare M., New York: Marcel
Dekker, 1991:227-235). Many variants existed between these strains, and of
these,
five variants were found in the low preferring strains but not in the high
preferring
strain. One of these variants results in a missense mutation (Ile6lThr; Fig.
2). The
other four variants were in non-coding regions (T>A -2383 nt; A>G -183 nt;
A>G +134 nt; 'I>C +651 nt, between exon 2 and 3). These five variants will be
referred to as the 129-like or B6-like haplotypes. Additional inbred strains
of mice
with known saccharin and sucrose preferences (Lush LE., The genetics of
tasting
in mice. VI. Saccharin, acesulfame, dulcin and sucrose. Genet Res,
1989;53:95-99; Lush I. The genetics of bitterness, sweetness, and saltiness in
strains of mice. In Genetics ofPerception and Communication, Vol. 3, eds.
Wysocki C. and Kare M., New York: Marcel Dekker, 1991:227-235; Lush LE.
and Holland G. The genetics of tasting in mice. V. Glycine and cyclohexamide.
Genet Res, 1988;52:207-212) were also sequenced. The 129-like haplotype was
found in mice with lower sweetener preference and the B6-like haplotype was
found in mice with higher sweetener preference (Fig. 3).
B6 mice have higher maximal gustatory neural firing in response to
sweeteners compared with 129 mice, as do the 129.B6-Sac partially congenic
strains (Bachmanov A.A. et al. Sucrose consumption in mice: major influence of
two genetic loci affecting peripheral sensory responses. Mammalian Genome,
1997;8:545-548). Thus, the SAC1 gene is likely to be expressed in tongue. To
test
this hypothesis, RNA from mouse and human tongue was extracted, reversed
transcribed into cDNA and primers, chosen to span an intron, were used in a
PCR
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reaction. Genomic and cDNA yielded bands of different sizes, which were
purified and sequenced (Figure 4AB). Sequencing results confirmed that the
bands were derived from this gene with the appropriate intron/exon boundaries.
Further analysis of expression in cDNA in mouse tissue, using commercially
available mouse cDNA, indicated this gene is also expressed is widely
expressed.
The broad range of tissue expression of this gene may indicate that other
tissues
use this receptor to sense extra cellular sugars (Fig. 4A).
Hoon et al. identified a gene, Gpr70 (formerly TRl or T1R1) as a putative
sweet receptor based mainly on its expression in anterior tongue taste cells.
Since
it also mapped to distal chromosome 4, it was a logical candidate for SAC 1.
However, we have shown that Gpr70 is at least 4 cM proximal to SAC1 (Li X.
et al. The saccharin preference locus (Sac) and the putative sweet taste
receptor
(Gpr70) gene have distinct locations on mouse chromosome 4. Mammalian
Genome, 2001;12:13-16). Nevertheless, Gpr70 could be an additional sweet
receptor and there could be others. It has been argued based upon human
psychophysical studies and studies of sweet taste transduction mechanisms that
there must be more than one sweet receptor. Other lines of evidence, however,
are
more consistent with the existence of one or a very few receptors (Bartoshuk
L.M.
Is sweetness unitary? An evaluation of the evidence for multiple sweeteners.
In
Sweetness, ed. bobbing, J., London: Springer-Verlag, 197:33-46). At present no
evidence has been found of a family of Sac-like receptors resembling the large
family of bitter receptors recently reported (Matsunami H., Montmayeur J.P.,
and
Buck L.B. A family of candidate taste receptors in human and mouse [see
comments]. Nature, 2000;404:601-604; Adler E. et al. A novel family of
mammalian taste receptors [see comments]. Cell, 2000;100:693-702). The sweet
substances that exist in nature, which presumably shaped the evolution of
sweet
receptor(s), are likely much more similar amongst themselves, mostly simple
sugars, than are the vast array of structurally diverse bitter tasting
compounds.
A receptor for the sugar trehalose has recently been identified in the fruit
fly, Drosophila melanogaster. Surprisingly, the trehalose and other fly taste
receptors, have no homology with SAC 1. The specialization of flies for the
sugar
trehalose may account for this divergence.
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There may be multiple sweet receptors; evidence from across species
comparisons, psychophysical cross adaptation, and sweetness competitors has
been reviewed (Bartoshuk L.M. Is sweetness unitary? An evaluation of the
evidence for multiple sweeteners. In Sweetness, ed. bobbing, J., London:
Springer-Verlag, 1987:33-46). The SAC 1 gene accounts for ~40% of the genetic
differences in sweet perception between these two particular strains of mice,
but
other receptors, and other alleles of these receptors may exist.
Because sucrose is perceived to be bad for human health, considerable
resources are directed toward the discovery of high potency, low caloric
sweeteners. Most of the most widely known high potency sweeteners were
discovered serendipitously, i.e., the sweetener was synthesized for a
different
purpose and someone in the laboratory accidentally tasted it and discovered it
was
sweet (Waiters E.D. The rational discovery of sweeteners. In Sweeteners.
Discovery, molecular design, and chemoreception, eds. Welters D.E.,
Orthoefer F.T., and DuBois G.E., American Chemical Society, USA, 1991:1-11).
More direct methods, however, have been employed to identify new sweet
compounds, and the sweet receptor has been extensively modeled to predict
which
ligands will be sweet.
It is not known how or why different alleles of SAC1 arose in inbred
strains of mice but their existence, in addition to providing us with a tool
to
identify a sweet receptor, raises the question of whether they might also
characterize human populations. There appear to exist reliable individual
differences in human sensitivity and preference for sweet sugars but whether
these
are genetically influenced remains to be determined. The identification of SAC
1
should facilitate research in this area. Also, the observation that SAC 1 is
expressed in several tissues in addition to tongue raises the interesting
possibility
that it could be involved in other aspects of sugar recognition and that
allelic
variants in this gene could be related to diseases or conditions such as
diabetes and
obesity.
Alleles of the gene described in this application are likely to account for
the SAC 1 behavioral and neurological phenotype for four reasons. First, the
SAC 1
nonrecombinant region is small, less than 194 kb; this gene lies within this
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nonrecombinant interval and the peak of LOD score corresponds closely with the
location of the gene. Second, of the genes contained within this region, no
others
are viable candidates for SAC 1. Third, this gene has sequence homology to
other
putative taste receptors, and is expressed in the tongue. Finally, a haplotype
with a
missense mutation is found in mice with low sweetener preference but not in
mice
with high sweetener preference. These data strongly suggest that mutations of
this
gene account for differences in the acceptance and preference for sweeteners
attributed to the SAC1 locus.
Among the multiple mechanisms involved in regulation of ethanol intake,
one of the least appreciated factors is the perception of its flavor (Nachman
M.,
Larne C., Le Magnen J. The role of olfactory and orosensory factors in the
alcohol
preference of inbred strains of mice. Physiology Behavior, 1971;6:53-95).
Although individual variability in the perception of ethanol flavor by adults
and
children was described over 60 years ago (Richter C.P. Alcohol as a food.
Quart.
J. Studies Alcohol, 1941;1:650-62), the hypothesis that individual differences
in
alcohol chemosensory perception can affect alcohol intake did not receive due
attention. As a result, the relationship between alcohol chemosensation and
intake
is not well-understood. Humans perceive ethanol flavor as a combination of
components, including sweetness, bitterness, odor and irritation (burning
sensation), which depend on ethanol concentration (Green B.G. The sensitivity
of
the tongue to ethanol. Ann. NY. Acad. Sci., 1987;510:315-7; Bartoshuk L.M.,
Conner E., Grubin D., Karrer T., Kochenbach K., Palsco M., et al. PROP
supertasters and the perception of ethyl alcohol. Chem. Se~tses, 1993.). Rats
detect
sweet (sucrose-like) and bitter (quinine-like) sensory components in ethanol
(Kiefer S.W., Lawrence G.J. The sweet-bitter taste of alcohol: aversion
generalization to various sweet-quinine mixtures in the rat. Chem. Senses,
1988;13:633-41; Kiefer S.W., Mahadevan R.S. The taste of alcohol for rats as
revealed by aversion generalization tests. Chem. Senses, 1993;18:509-22) and
probably perceive the other components detected by humans as well.
The relationship between ethanol and sweetener perception and
consumption .has been studied the most and is supported by several lines of
evidence:
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(a) Electrophysiological recordings from gustatory nerves indicate that
lingual
application of ethanol activates sweetener-responsive neural fibers
(Hellekant G., Danilova V., Roberts T., Ninomiya Y. The taste of ethanol
in a primate model: I. Chorda tympani nerve response in Macaca mulatta.
Alcohol, 1997;14:473-84; Sako N., Yamamoto T. Electrophysiological and
behavioral studies on taste effectiveness of alcohols in rats. Am. J.
Physiol., 1999;276:8388-96).
(b) Conditioned taste aversions generalize between ethanol and sucrose
(Kiefer S.W., Lawrence G.J. The sweet-bitter taste of alcohol: aversion
generalization to various sweet-quinine mixtures in the rat. Chem. Senses,
1988;13:633-41; Kiefer S.W., Mahadevan R.S. The taste of alcohol for
rats as revealed by aversion generalization tests. Chem. Sepses,
1993;18:509-22; Lawrence G.J., Kiefer S.W. Generalization of specific
taste aversions to alcohol in the rat. Chem. Senses, 1987;12:591-9; Blizard
D.A., McClearn G.E. Association between ethanol and sucrose intake in
the laboratory mouse: exploration via congenic strains and conditioned
taste aversion. Alcohol. Clin. Exp. Res., 2000;24:253-8.), suggesting that
ethanol and sucrose share the same taste property, most likely sweetness.
(c) Genetic associations between preferences for ethanol and sweeteners were
found among some rat and mouse strains and within their segregating
crosses (Overstreet D.H., Kampov-Polevoy A.B., Rezvani A.H., Murelle
L., Halikas J.A., Janowsky D.S. Saccharin intake predicts ethanol intake in
genetically heterogeneous rats as well as different rat strains. Alcohol.
Clin. Exp. Res., 1993;17:366-9; Sinclair J.D., Kampov-Polevoy A.,
Stewart R., Li T-K. Taste preferences in rat lines selected for low and high
alcohol consumption. Alcohol, 1992;9:155-60; Stewart R.B., Russell R.N.,
Lumeng L., Li T-K., Murphy J.M. Consumptions of sweet, salty, sour, and
bitter solutions by selectively bred alcohol-preferring and alcohol-
nonpreferring lines of rats. Alcohol. Clih. Exp. Res., 1994;18:375-81;
Belknap J.K., Crabbe J.C., Young E.R. Voluntary consumption of alcohol
in 15 inbred mouse strains. Psychopharmacol., 1993;112:503-10;
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Bachmanov A.A., Reed D.R., Tordoff M.G., Price R.A., Beauchamp G.K.
Intake of ethanol, sodium chloride, sucrose, citric acid, and quinine
hydrochloride solutions by mice: a genetic analysis. Behav. Genet.,
1996;26:563-73; Bachmanov A.A., Tordoff M.G., Beauchamp G.K.
Ethanol consumption and taste preferences in C57BL/6ByJ and 1291)
mice. Alcohol. Clin. Exp. Res., 1996;20:201-6), reviewed in (Kampov-
Polevoy A.B., Garbutt J.C., Janowsky D.S. Association between
preference for sweets and excessive alcohol intake: a review of animal and
human studies. Alcohol. Alcohol., 1999;34:386-95; Overstreet D.H.,
Rezvani A.H., Parsian A. Behavioural features of alcohol-preferring rats:
focus on inbred strains. Alcohol. Alcohol., 1999;34:378-85); with some
exceptions (Phillips T.J., Crabbe J.C., Metten P., Belknap J.K.
Localization of genes affecting alcohol drinking in mice. Alcohol. Clin.
Exp. Res., 1994;18:931-941; Parsian A., Overstreet D.H., Rezvani A.H.
Independent segregation of alcohol and saccharin intakes in F2 progeny
from FH/ACI intercross (Abstract). Alcohol. Clin. Exp. Res.,
2000;24(Supplement):5 8A)).
(d) Human studies show that alcoholics have a stronger liking of concentrated
sucrose compared with nonalcoholics (Kampov-Polevoy A.B., Garbutt
J.C., Davis C.E., Janowsky D.S. Preference for higher sugar
concentrations and Tridimensional Personality Questionnaire scores in
alcoholic and nonalcoholic men. Alcohol. Clin. Exp. Res., 1998;22:610-4;
Kampov-Polevoy A.B., Garbutt J.C., Janowsky D. Evidence of preference
for a higher concentration sucrose solution in alcoholic men. American
Journal of Psychiatry, 1997;154:269-70).
There are several possible mechanisms that could underlie the association
between sweetener and ethanol responses:
(a) Common peripheral taste mechanisms, which may involve the interaction
of ethanol with a peripheral sweet taste transduction. At least one such
common peripheral mechanism is mediated by the Gpr98 gene (SAC1
locus) encoding a sweet taste receptor (as described below).
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(b) Common brain mechanisms. The regulation of ingestive responses to
ethanol and sweeteners may involve common opioidergic, serotonergic
and dopaminergic brain neurotransmitter systems (Gosnell B.A.,
Majchrzak M.J. Centrally administered opioid peptides stimulate saccharin
intake in nondeprived rats. Pharm. Biochem. Behav., 1989;33:805-10;
George S.R., Roldan L., Lui A., Naranjo C.A. Endogenous opioids are
involved in the genetically determined high preference for ethanol
consumption. Alcohol. Clin. Exp. Res., 1991;15:668-72; Hubell C.L.,
Marglin S.H., Spitalnic S.J., Abelson M.L., Wild K.D., Reid L.D.
Opioidergic, serotoninergic, and dopaminergic manipulations and rats'
intake of a sweetened alcoholic beverage. Alcohol, 1991;8:355-67;
Pucilowski O., Rezvani A.H., Janowsky D.S. Suppression of alcohol and
saccharin preference in rats by a novel Ca2+ channel inhibitor, Goe 5438.
Psychopharmacol., 1992;107:447-52). These mechanisms could be
responsible for the emotional response to the pleasantness of ethanol or
sweeteners, or the motivational mechanisms driving their intakes.
(c) Common signals related to the caloric value of ethanol and sugars (Gentry
R.T., Dole V.P. Why does a sucrose choice reduce the consumption of
alcohol in C57BL/6J mice? Life Sci., 1987;40:2191-4). Ethanol is
metabolized in the body through some of the same pathways as
carbohydrates and provides comparable energy. Thus, energy derived from
carbohydrates and ethanol may have similar rewarding effects through the
same hunger and satiety mechanisms.
(d) Incidental genetic linkage. Different genes affecting responses to ethanol
and sweeteners may reside nearby on the same chromosome.
Ethanol consumption is a complex trait, depending on multiple
mechanisms of its regulation and determined by multiple genes. A body of
evidence suggests that ethanol consumption may depend on perception of its
flavor, and that there is an association between perception and consumption of
ethanol and sweet-tasting compounds. However, only a few genes have been
identified as candidates affecting ethanol consumption.
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The present invention provides that a gene, SAC1, is associated with the
detection of a sensing of carbohydrates, other sweet compounds, and alcohols
including ethanol. The sequence of the mouse SAC1 cDNA (SEQ ID NO: 1) is:
ATGCCAGCTTTGGCTATCATGGGTCTCAGCCTGGCTGCTTTCCTGGAGC
TTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCAAGGCACAAG
GGGACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGG
CCACTCTCAACCAGAGAACACAACCCAACAGCATCCCGTGCAACAGGT
TCTCACCCCTTGGTTTGTTCCTGGCCATGGCTATGAAGATGGCTGTGGA
GGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTGCGGCTGGGCTA
TGACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAG
TCTCATGTTCCTGGCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTG
CAACTACACACAGTACCAACCCCGTGTGCTGGCTGTCATCGGCCCCCA
CTCATCAGAGCTTGCCCTCATTACAGGCAAGTTCTTCAGCTTCTTCCTC
ATGCCACAGGTCAGCTATAGTGCCAGCATGGATCGGCTAAGTGACCGG
GAAACGTTTCCATCCTTCTTCCGCACAGTGCCCAGTGACCGGGTGCAG
CTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGAACTGGGTG
GCCGCCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATC
TTTTCTAGTCTGGCCAATGCACGAGGTATCTGCATCGCACATGAGGGC
CTGGTGCCACAACATGACACTAGTGGCCAACAGTTGGGCAAGGTGCTG
GATGTACTACGCCAAGTGAACCAAAGTAAAGTACAAGTGGTGGTGCTG
TTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGCATCCATCA
TGGCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATC
TGACCTGGTCATGACACTTCCCAATATTGCCCGTGTGGGCACTGTGCTT
GGGTTTTTGCAGCGGGGTGCCCTACTGCCTGAATTTTCCCATTATGTGG
AGACTCACCTTGCCCTGGCCGCTGACCCAGCATTCTGTGCCTCACTGAA
TGCGGAGTTGGATCTGGAGGAACATGTGATGGGGCAACGCTGTCCACG
GTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGAA
CCTATCAGCTGGGCAATTGCACCACCAAATATTTGCAACCTATGCAGC
TGTGTACAGTGTGGCTCAAGCCCTTCACAACACCCTACAGTGCAATGT
CTCACATTGCCACGTATCAGAACATGTTCTACCCTGGCAGCTCCTGGA
GAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTACAGTT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-29-
TGATGCTGAAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGT
GTGGCAGAGCCCTACACCTGTATTACATACTGTGGGCACCTTCAACGG
CACCCTTCAGCTGCAGCAGTCTAAAATGTACTGGCCAGGCAACCAGGT
GCCAGTCTCCCAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCG
AGTAAAGGGCTTTCATTCCTGCTGCTATGACTGCGTGGACTGCAAGGC
GGGCAGCTACCGGAAGCATCCAGATGACTTCACCTGTACTCCATGTAA
CCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCTGCTTACCTCGCAG
GCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCTG
CTGCTTTGCCTGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTG
TCCACCACTGGGACAGCCCTCTTGTCCAGGCCTCAGGTGGCTCACAGT
TCTGCTTTGGCCTGATCTGCCTAGGCCTCT'TCTGCCTCAGTGTCCTTCTG
TTCCCAGGGCGGCCAAGCTCTGCCAGCTGCCTTGCACAACAACCAATG
GCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCAAGCAG
CTGAGACCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGC
TATGCAGCTACCTTCGGGGACTCTGGGCCTGGCTAGTGGTACTGTTGG
CCACTTTTGTGGAGGCAGCACTATGTGCCTGGTATTTGATCGCTTTCCC
ACCAGAGGTGGTGACAGACTGGTCAGTGCTGCCCACAGAGGTACTGG
AGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGCACATCA
CCAATGCAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACA
GAGCCAGCCTGGCCGCTACAACCGTGCCCGTGGTCTCACCTTCGCCAT
GCTAGCTTATTTCATCACCTGGGTCTCTTTTGTGCCCCTCCTGGCCAAT
GTGCAGGTGGCCTACCAGCCAGCTGTGCAGATGGGTGCTATCCTAGTC
TGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTGCTATGTGC
TTCTTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGA
ATGCCAAGAAAGCAGCAGATGAGAACAGTGGCGGTGGTGAGGCAGCT
CAGGGACACAATGAATGA
The geonomic DNA sequence of the mouse SAC 1 gene (SEQ ID NO: 2)
is:
ATCTGAGCCTTAGACACAGCACTGGTGCCAGGCAAACACTCCTGGGCC
TACATGCTTGGG
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-3 0-
GCCTCTTCATATTCCAAAAGCTGTCTTTGGGTAAGATGAAGTTCCTCTG
GCAGTGGCATG
AGTGCTGAAGGCTCTTTCCCTGCCCTTCACCTGCTTTCTTGATAGTCTCT
CTGCATACCA
AACAGGCCCTTGTCTCCTGGGAAATGGAAACTATGAAATCAATAGCTG
AGGCTTCTCTAG
GAAAGCCTGCCCTGGTCAGTACAACCTGTTTCACAGCTTCTATAGAAT
AGTTACATCAGC
CTTCTGAAGATGGCCTCTTAGAGCACATGCACCCCCAAGATTCTAAGA
TGTCAATACTAA
CTGACCAAACCATACCTCTCTAGCCAGCCCTGCTGCTCCTGTTGTCTGG
TACCCAGGTGA
CTGAGGACATGACTGGTGGAAGGAAACTAGGCCCCTTTGTCTGTCAGA
TGGCCATACCCA
GCATGGCTGATGCCCAGTGTATAAGACCCTACGCTTTTCCACTGGTCTT
AATGTTAAACC
CTAGGACAGTGTCCTCAGCATAGCTGGTGTGTGTGAATGCAAACTTTG
GGGCATATCTCT
TCCATTAAGCACTGTGATATATGTAGTATTTCCAACAAATAAATTATAC
CTACATGATTG
GGTATAGCATTCTGGGATGGGTCACAGGTGTGTCAGGTGCCTAATTAT
GTGGGGGAAGAA
CATAGAAATATATAGGTGGGGAGGGAGCTAACCCTAGGAATAAGGCT
AAAGCATGTGTCT
CCAGTCCTGAAGACTCAAAGGGCAACGTGAATCATGAGACATGTTCAG
GACTGAAGGAGT
TGCCATGTATCTGTCCTTGATGTATCTTAATCATACATACACTATGAGA
TCTGTGTTACC
TCCATTTTGCAGGTGAGAAAAGAAACACCTGAATGGCCTACCTTAAAG
GGCTAAGTGGGA
AAATAGGTCTGAAGATAACCCAGGCACTGTGTGACAAAGCGGGAAGA
AAACTAGAGATGC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-31-
TTTCTTCATGGCAACAACCTAGAGGGTACAACCTAGTGGTTTCTTCTTG
GTACTCCACTG
TATACACCCCATCTGCTTGGGCTGTACATTGTCTGACCATGCTTATAAC
AAAAGTCACAT
ACTACTAGCCAAGACTGAGAACTTAGAGCGACTGGCCAGAAAGTAAA
GATACAACAGTTG
ATATGTGTGCCACACACAGATCCATGTGTACATGTCTATTAATTATGTG
AACGTGCTTTG
TGGACATCCTCACAAAGCAGCAGGGAAATGCAAAGGTCATTTCCATAA
CACCTGCTGGAC
ACCATATGACATTGAGATTACCGGGGTGCCCATTCCAACAAGAGTTAA
TAGCTCCCCCTA
TGTTTGGGTGCCAGAAACCTGATTTGTTAGCAATAGCTCCCTCACATCC
AGATTAAGAGG
GGGATGGCTTAGCTAGGGTTACTATGATGAAACTATGACCAAAGCAAC
TTGTGGGTAAAA
GGGTGTATTTGGCTTACACTTCCATATCACTTCATCAAAGTGAGGACA
GGAACTCAAATA
GAGTAGGAATTTGGTGACAAGAGCTGATGTAGAGGCAATGCAGTGGT
GCCACTTAGTGGC
GCGCTCAGTCTGCTCCCTTTCTTAATAGAATGCAAGACCACCAGCCCAT
GGGTGGCACCA
CAATGGGACCGGGCCCTTCCCCATCGGTCACTAAGAAAATGCCCTACA
GCCAGATCTTAT
GGAGACATTTTCTCAACGGAGGCTCACTCCTTTCAGATAACTCTATATC
AAATTGACATA
AACCAGAACAGAGGAGGAGGCTAAGAAGGAAACTGCCAATTGCATAC
ATGCACACACCTG
GCCCTAGCAGCTGCAGGAAGCTATTTGTTTATGGCCTTTTCTCATTTTC
ATGGACCAGCA
TGAGCACTCTGCAGAGAGAGATGCCTGCATGCCTGCCAAGGCAGGAGT
GCTTACACTGAA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-32-
GGTCAACAGGATGGCAGGGGGGCTGCAGAGCTTCCAAGTGTCAGAAC
CCCAGCAGAAGAG
CTGAGACCCTTGCCCGAGGACTCAGGCGGGTTGGGAAGGCCAGGAAA
TTCAGCCAGAGCT
CTTCTTCAGATGGGGTACCATCTGAAGGTTAGACCAGCTAGCCAGCTG
TTGTTGAGGGAC
CACCTCTGCAGCCCCTACCTTTGGAAGATAGAAAGTGTCTCTGTGACA
AGTATGGCCATT
GTGCCCCCTTATTCCACAGTCAACAGAAACCCTGGAATCCTGAACACT
TCTGCAGCTTCT
TTTTTACAGTCTGCCAGGTTGCTCTAGGAATGAAGGGTGCCGAGAGGC
TTGGGCGTAGGC
AGGTGACAAGACCACAGTTAGTGGTCACAGCTGGCTTACTGGATCACT
CTTGGACAGAGT
TTGTTAGATATGGAGTGGAGTATACACAAGGCATCAGGCGGGGGATAT
TGAATGTATCAC
CGGAGCTCCTTGGGGCTTGGCAGCCAAGCACAGCAGTGGTTTTGCTAA
ACAAATCCACGG
TTCCCTCCCCTTGACGCAGTACATCTGTGGCTCCAACCCCACACACCCA
CCCATTGTTAG
TGCTGGAGACTTCTACCTACCATGCCAGCTTTGGCTATCATGGGTCTCA
GCCTGGCTGCT
TTCCTGGAGCTTGGGATGGGGGCCTCTTTGTGTCTGTCACAGCAATTCA
AGGCACAAGGG
GACTACATACTGGGCGGGCTATTTCCCCTGGGCTCAACCGAGGAGGCC
ACTCTCAACCAG
AGAACACAACCCAACAGCATCCCGTGCAACAGGTATGGAGGCTAGTA
GCTGGGGTGGGAG
TGAACCGAAGCTTGGCAGCTTTGGCTCCGTGGTACTACCAATCTGGGA
AGAGGTGGTGAT
CAGTTTCCATGTGGCCTCAGGTTCTCACCCCTTGGTTTGTTCCTGGCCA
TGGCTATGAAG
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-33-
ATGGCTGTGGAGGAGATCAACAATGGATCTGCCTTGCTCCCTGGGCTG
CGGCTGGGCTAT
GACCTATTTGACACATGCTCCGAGCCAGTGGTCACCATGAAATCCAGT
CTCATGTTCCTG
GCCAAGGTGGGCAGTCAAAGCATTGCTGCCTACTGCAACTACACACAG
TACCAACCCCGT
GTGCTGGCTGTCATCGGCCCCCACTCATCAGAGCTTGCCCTCATTACAG
GCAAGTTCTTC a
AGCTTCTTCCTCATGCCACAGGTGAGCCCACTTCCTTTGTGTTCTCAAC
CGATTGCACCC
ATTGAGCTCTCATATCAGAAAGTGCTTCTTGATCACCACAGGTCAGCT
ATAGTGCCAGCA
TGGATCGGCTAAGTGACCGGGAAACGTTTCCATCCTTCTTCCGCACAG
TGCCCAGTGACC
l~ GGGTGCAGCTGCAGGCAGTTGTGACTCTGTTGCAGAACTTCAGCTGGA
ACTGGGTGGCCG
CCTTAGGGAGTGATGATGACTATGGCCGGGAAGGTCTGAGCATCTTTT
CTAGTCTGGCCA
ATGCACGAGGTATCTGCATCGCACATGAGGGCCTGGTGCCACAACATG
ACACTAGTGGCC
AACAGTTGGGCAAGGTGCTGGATGTACTACGCCAAGTGAACCAAAGT
AAAGTACAAGTGG
TGGTGCTGTTTGCCTCTGCCCGTGCTGTCTACTCCCTTTTTAGTTACAGC
ATCCATCATG
GCCTCTCACCCAAGGTATGGGTGGCCAGTGAGTCTTGGCTGACATCTG
ACCTGGTCATGA
CACTTCCCAATATTGCCCGTGTGGGCACTGTGCTTGGGTTTTTGCAGCG
GGGTGCCCTAC
TGCCTGAATTTTCCCATTATGTGGAGACTCACCTTGCCGTGGCCGCTGA
CCCAGCATTCT
GTGCCTCACTGAATGCGGAGTTGGATCTGGAGGAACATGTGATGGGGC
AACGCTGTCCAC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-34-
GGTGTGACGACATCATGCTGCAGAACCTATCATCTGGGCTGTTGCAGA
ACCTATCAGCTG
GGCAATTGCACCACCAAATATTTGCAACCTATGCAGCTGTGTACAGTG
TGGCTCAAGCCC
TTCACAACACCCTACAGTGCAATGTCTCACATTGCCACGTATCAGAAC
ATGTTCTACCCT
GGCAGGTAAGGGTAGGGTTTTTTGCTGGGTTTTGCCTGCTCCTGCAGG
AACACTGAACCA
GGCAGAGCCAAATCTTGTTGTGACTGGAGAGGCCTTACCCTGACTCCA
CTCCACAGCTCC
TGGAGAACATGTACAATATGAGTTTCCATGCTCGAGACTTGACACTAC
AGTTTGATGCTG
AAGGGAATGTAGACATGGAATATGACCTGAAGATGTGGGTGTGGCAG
AGCCCTACACCTG
TATTACATACTGTGGGCACCTTCAACGGCACCCTTCAGCTGCAGCAGT
CTAAAATGTACT
GGCCAGGCAACCAGGTAAGGACAAGACAGGCAAAAAGGATGGTGGGT
AGAAGCTTGTCGG
TCTTGGGCCAGTGCTAGCCAAGGGGAGGCCTAACCCAAGGCTCCATGT
ACAGGTGCCAGT
CTCCGAGTGTTCCCGCCAGTGCAAAGATGGCCAGGTTCGCCGAGTAAA
GGGCTTTCATTC
CTGCTGCTATGACTGCGTGGACTGCAAGGCGGGCAGCTACCGGAAGCA
TCCAGGTGAACC
GTCTTCCCTAGACAGTCTGCACAGCCGGGCTAGGGGGCAGAAGCATTC
AAGTCTGGCAAG
CGCCCTCCCGCGGGGCTAATGTGGAGACAGTTACTGTGGGGGCTGGCT
GGGGAGGTCGGT
CTCCCATCAGCAGACCCCACATTACTTTTCTTCCTTCCATCACTACAGA
TGACTTCACCT
GTACTCCATGTAACCAGGACCAGTGGTCCCCAGAGAAAAGCACAGCCT
GCTTACCTCGCA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-35-
GGCCCAAGTTTCTGGCTTGGGGGGAGCCAGTTGTGCTGTCACTCCTCCT
GCTGCTTTGCC
TGGTGCTGGGTCTAGCACTGGCTGCTCTGGGGCTCTCTGTCCACCACTG
GGACAGCCCTC
TTGTCCAGGCCTCAGGTGGCTCACAGTTCTGCTTTGGCC.TGATCTGCCT
AGGCCTCTTCT
GCCTCAGTGTCCTTCTGTTCCCAGGGCGGCCAAGCTCTGCCAGCTGCCT
TGCACAACAAC
CAATGGCTCACCTCCCTCTCACAGGCTGCCTGAGCACACTCTTCCTGCA
AGCAGCTGAGA
CCTTTGTGGAGTCTGAGCTGCCACTGAGCTGGGCAAACTGGCTATGCA
GCTACCTTCGGG
GACTCTGGGCCTGGCTAGTGGTACTGTTGGCCACTTTTGTGGAGGCAG
CACTATGTGCCT
GGTATTTGATCGCTTTCCCACCAGAGGTGGTGACAGACTGGTCAGTGC
TGCCCACAGAGG
TACTGGAGCACTGCCACGTGCGTTCCTGGGTCAGCCTGGGCTTGGTGC
ACATCACCAATG
CAATGTTAGCTTTCCTCTGCTTTCTGGGCACTTTCCTGGTACAGAGCCA
GCCTGGCCGCT
ACAACCGTGCCCGTGGTCTCACCTTCGCCATGCTAGCTTATTTCATCAC
CTGGGTCTCTT
TTGTGCCCCTCCTGGCCAATGTGCAGGTGGCCTACCAGCCAGCTGTGC
AGATGGGTGCTA
TCCTAGTCTGTGCCCTGGGCATCCTGGTCACCTTCCACCTGCCCAAGTG
CTATGTGCTTC
TTTGGCTGCCAAAGCTCAACACCCAGGAGTTCTTCCTGGGAAGGAATG
CCAAGAAAGCAG
CAGATGAGAACAGTGGCGGTGGTGAGGCAGCTCAGGGACACAATGAA
TGACCACTGACCC
GTGACCTTCCCTTTAGGGAACCTAGCCCTACCAGAAATCTCCTAAGCC
AACAAGCCCCGA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-36-
ATAGTACCTCAGCCTGAGACGTGAGACACTTAACTATAGACTTGGACT
CCACTGACCTTA
GCCTCACAGTGACCCCTTCCCCAAACCCCCAAGGCCTGCAGTGCACAA
GATGGACCCTAT
GAGCCCACCTATCCTTTCAAAGCAAGATTATCCTTGATCCTATTATGCC
CACCTAAGGCC
TGCCCAGGTGACCCACAAA.AGGTTCTTTGGGACTTCATAGCCATACTTT
GAATTCAGAAA
TTCCCCAGGCAGACCATGGGAGACCAGAAGGTACTGCTTGCCTGAACA
TGCCCAGCCCTG
AGCCCTCACTCAGCACCCTGTCCAGGCGTCCCAGGAATAGAAGGCTGG
GCATGTATGTGT
GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTACGTATG
TATGTATGTAT
CAGGACAGAACAAGAAAGACATCAGGCAGAGGACACTCAGGAGGTAG
GCAACATCCAGCC
TTCTCCATCCCTAGCTGAGCCCTAGCCTGTAGGAGAGAACCAGGTCGC
CGCCAGCACCTT
GGACAGATCACACACAGGGTGCGGGTCAGCACCACGGCCAGCGCCAG
CCACGCGGGACCC
CTGGAATCAGCTTCTAGTACCAAGGACAGAAAAGTTGCCGCAAGGCCC
CTTACTGGCCAG
CACCAGGGACAGAGCCACATGCCTAAGCGGCAAGGGACAAGAGCATC
GTCCATCTGCAGG
CAGGATCAGACCCGGGTCAGTTCTGGACTGGCCCCCACACCTGAATCC
CGGAGCAGCTCA
GCTGGAGAAAAGAGAAACAAGCCACACATCAGTCCCATAAAATTAAA
CGCTTTTTTTAGT
GTTTAAAATAGCATTTACACAGAAGCAGCATTTACACAGAAGCAGCTC
TATGTCAACTAC
CCAGTCACTCAGACTTTGACACAGTGTCTAGTGTAGATGTGTGGGGCC
GCTGTGCCGGGA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-37-
TGGCAGTGGCACATGATGATGGGCAGCCACCAGAACAGAAACAGAAC
AGGGCCCAGCTCT
GCAGCTCTTGTGTTCACTGTCACCCACCACTGAGACTGAGACAGTGGC
TAGGTGCCAGGT
CTCTCTCCTGTCTCTCCTACTAGCTACCCTTCACATACCTTCAGTACAA
ACTGTGTTGTC
ATGTGCCAAGTAGCAGGTGGGGAAAGGGGCATGCAAACTGCCCCTTTG
GGTAACTAGCTG
CCACCCTTAGAGCAGGCAGGCTAGCAATAAATAAATAAGTTAGACCCC
ACCTGGGCAGCC
AGAGAGGTTTGAAGGCTCTGTCTAACCCCTCAAAAATCCCACCTTGGC
CTGACAGGTGAG
GCCCATGAACTTAGCGACAGTCAGCCTGTGTCCCTGTGCACAGTTCTGT
GAGGCTTTGGG
GCAAGGGGTACCAAGAGCCCAAGAGAGCCTTTCTTGTTCTAAATGGAG
GTCACTTCCAAA
GAAGGGAACCAGGAGGTGGTCCCTGAGACTTGTGCTGAGGACTTAAA
GTCAGAGATGTCT
CCTTACAAGACTCTATAGATACTTGAGCTGTACCACCATCAGCAGCCC
CAAGAGCAGACA
AAATGTCAAGCCAATATCCTGGTGGTATGGCTGCCCTCAGGCCCTCCT
CTGTAGCCTGCT
CCCTCTGCCCTGGCCCAGAGCCCACAGCTGATCTATCCTGGCTGGCCA
CCACCACGGCCA
GCGCAGAGCTCCTGGCACAGCAGGAGCACAGACTCAGCCACAGGCAG
CGCTGAAGACATT
GGTTGATCATCACATGATGTCCACAAAGAACTCACAGGGGTTTCCCAT
GGCCTTTTGGAA
GGACTGGCGGCTACCTGTAAGTTCTGGAGGGACAGCAGCCAGCTCCCG
GACGGGTGGCCC
TCCAGGTGGCCCACCCACTACTGCATAGGCCTTTGTAAGGGGGTGCAG
TGGGGGGAGCCC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-3 8-
TGGGGCAACAGCTGAAGCCTGACTTCGAGGGCTACTGCCACGGCTAAG
CTGGCTGACAGG
CCGCTCCCACCAGCCGGTGCTACCAGACCCACTTGGTACTGTGTGGTCT
GATTCACTGCC
ACTACCCCCAGCTCCAGTTGCCCGGCGCTCCTCTCGGCCTGGGGTCCG
ATGGCTGCTCCG
TGTGGACCCACTGCTCTTGCTCCCTAGGGGGAGGGAAGGGGACAACAG
AGTCAGCACGAG
GCCTGGCCACTTCCAGGGCCACCAGCTGCTCCCAGACAGTCAGGGCAG
GACCTGGTAAGC
CTGGAGATGGTAGGGGAATGGCAGCCATGCAGATACCAGGAACAGCT
GAGAGGCGAGAAG
CTAGGGGCAGTGGCAGACAGCAGGGACAACAGGGGCCAGCCTGGCAC
CCCACACCTAACC
CCAATGCTTGAACCAAGGGTTAATGTTACAGCTGAGAAACTAAAAACC
AGCGAAGGCCCT
GTGTGCCCAGCATTCCCATTAGCCATCCTGGGTTCACCACCCAAAGAC
CCAACCAGGGTC
CACCCAACCCCAGGACCCTGGTCATCTAATTTGCTTAGCCCCTGTCCTG
AAAGTAGTGGG
AACCTGAAAACACGTGCTGGCTGGGGACATGCTGAGAGGGACACAGG
GGGACCTGGCTTA
CCGGCCCGAGAGTCCACTCTGCTAGTCCTTCAGTCTAAGGCTTGCTCAG
CACAAAGCAAG
GGATAGCACAAGTCACACACCAGTCCAGTGCTCACCAATGGCTAATAG
GACGATTTTGGG
CCAAGCTGAGCCTGGGTACATGCAAGGGCCTGTCCATGGTCAGGATTC
ACTCGATAGCTT
CCCCTTGGGCTTTGCCACCCTCTGGCCCAACCTCTCCTGAGTCTTTCTCT
GGACCTTGTA
GCACAAGTGTGCCCCACTCTGCCTAAGACCTCCACATCAGTCCATCTCC
TCCTGAGGGAC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-39-
ACCCACCCTTCAAGATCTTCAATATCCCTGGGATATGCTTTAACACTGA
TATGCTTTAAC
AGTGTTGCTTGATACTCTTATCTGGCACTCTGTTGGGATGCAGGCTCCA
TAACTGATAAA
GCCCATTCTCCCCCTAGCTTGGGGCCTAGAGAGTGCCCCTACCTGCTAT
CAGTGGTTACT
TTCATTCTTGCCATATCATCTCCTGGCCTCTTGCCTCTGCCACCTAGCAC
ACCAGGCTGT
CTTCCTATTCTCTAACGGCTTCTACCCACATCAGCCCCTCCCTGTCCCA
CACACTGACTC
TTGAGATGGAACCCACCGGGACTCAAACACACAGCAGGAGCACAGAG
GGAAGCGTCGGGG
CCAGGCAGAGCGTGGGAGTGGGAGGGAGTGGGAGGAGGGGTGGCAC
GCCTCTCACCTTCA
CTCTGCTGGCTCCCAGCACTGCCGCTGCCGCAGCTGAAGCCAGGGTCC
TGGTAAGCAGGC
GGGAAGCAGGGCGGGGGTCCTGGGTACTGGTAGGGGTAGCCTTGACC
CAAGGGCCAGGGT
ACTGATGGGTGGGGCAGTGGGGCCAGTGTGTCCTGATCTGAGGCTCCA
CTGGAGCCACTG
TTGAGGTTCAGGGATGCGAGGTCTGGCAGGGAGGGAGGGAGGGAGGG
GTAAGTGAAGGCA
AATGAATGAGGCCACAGCAACCCTACCCAACCGCACCCCTACTCACTA
CTGCACAGGTCG
CCAAAGACATAGTAGCACTGCTCAGAAAAGGTGATCTTGTTCACGGTG
TGCCTCAGGAAA
CCGTGCTTCAGCATACTGCTGGCATACTTTCTTGCCTCCCTTCGCTCCTT
GAAGCCCTCC
ACGTGTGTGTACAGCCAGTCCACCACATCCGCCCCTGGCCACAGGTCC
ATCAAAGTCAGG
GTAGCTGAGCCCTGGGAAGCTACGCCAGAATGAGGAACAGACGGGGC
CCTTCCCACACAG
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-40-
CCAGGGACTCACCAATGACAGCATTGGCAATGGTGATCTTAAGCCACA
TGCGGTCCCGGA
TCTCCAGTCCTGAGTCTGGCAACTGCATGACGCGGACAATGGCACTCA
TGTCACTCTTCA
CAGTCAGCGGTGCCTCCTCAAGCTCTGCAGAGCACACTTCCCTGAGGC
GAGGCTCACAGC
GTGAACCTCCATGGGGTTGAGAGCAGGGGCCAGGGTCAAACCTCTTAT
CTCCCATCCTTG
GGAGATGCCCCTCATCGAAACTTGAGCTAAGACCGGGAGATTCTTCCC
GGTCCCACAGTG
CAAGTCCACGTAGGCAAGGCAGCCCCCCTCCCCTCCCCGGAGAGAACA
AGCTGTTAGCTA
TGTTAGGTAGCAGAA.AAGCAAAGCAGAGGCTGCCATGTCGTCCCAATT
CCCCCCTCCGCA
CAGGCCTGGCAGGACCCTCAATTCATGCAGATGACCAGTATGGCCAGG
CCTGGAGGGATA
TGTACATGTATCTTTGTGTACACATTTGTGAAGGTGTTGGAAGCAAAC
AAAACCTTCATA
TGTAATGGGCCCCTGTAATAGCTCTGATGAGCACCAAAGCTCAAAGCT
AGAACTGACCAT
TGTCCTTCAACCTGAGTTTCCTTGGGTGGGGGGGGGTCCTGTGAGCTGC
CACTTACGTGG
GGCGCCAGGCACTGAGCTGGTTAGTGAGGAAGAGCTGGTGCGTGTGAT
GGCGCTGGAGCA .
GGGACTCGTACCATAGCGGGGCAGGGCACCCGTCAGTGCTGCTGTGTG
GGACAGCCAGGC
AGCCGGGTCGATGGGTCGCACTGGGTCAGCTGCATAGTTTCCAGAGCA
ACGGATTACAGG
TGGTAAGTAGGGGGGCAGCACAGAGGCAGACAAGAAAGACCCCCAGA
CTGAACACAGAAA
CCCCACGCTACCCCACCTTTCCATGGGGTAACTCACCCCTTGGGATGGT
GAAGTAGCTCC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-41-
GAGGGGTTGGGTCCCAGCACTTGGCCACTGTGAGACTGATGGGCCTAC
AGAGTTGAGCAG
ACCATGTTGTAAGTGAGGCCCGCACAGCCCCTCCCATCCTGTGCCACT
CCCACCCCCACT
TGGCTCCCACCTCACCCTGTCTGGGACACGATCTCCCGAAGCACCCGT
ACAGCGTCGTCA
TTGCTCATGTTCTCAAAGTTGACATCGTTCACCTACGGGGTTTGTGGGG
TCAGGGGTTGG
TGGTGGGATGTGGGTGCCTCTTGTCCCCACAGTCCCCACATGGCTCCCA
CCTGCAGCAAC
ATGTCGCCCGGCTCAATGCGGCCATCAGCAGCCACGGCCCCGCCCTTC
ATGATGGATCCA
ATGTAGATGCCGCCATCACCCCGGTCGTTGCTCTGGCCCACGATGCTG
ATGCCCAGGAAG
TGGTGCCTCTCTGCAGGAGGGGCCGTGAGCAGGCCCCCAAAGCTCCCG
AGGCTGTACCCA
CCCCCAGCAGGCACCCACAGCCCACAAGGCCTCACCCATGTTGAGAGT
GACGGTGATGAT
GTTCAGGGACATGGTGGAGTCTGTGATGCTGCTGAAGGAGGATGCCTG
CGGAGGGACCCA
GTGAGGGGCTGTGTGGGCACCATTCAGAGCAGACACCCCACCCACCTG
CTGCCTACCCGG
TCTGTCTGCCTCAAGCGCTGCTTCCGACGACGGCATTTGTGCTTCCGAA
CTAGCCGAGAG
GAGGTGCTCTGCTCTGTGGAGCTGCTCAGCCTGAGGCAGGAGTCAGAA
AAGCACAAACAT
GTATAACCAGCTCGGACGCTCAACTACAAATCTCCAGCACGTACTGAC
ATGTGCACACGT
CACCCACCGGCTCGTATTGTCCTCCTCATCTGAGTCAATAAAGCTGCTA
GATTCAAGCTC
ACTGCTCAGTACAGTGGATGCACTGTCTGGAGGTAGTCCCAGGTCCCG
CCGCCGATCCCC
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-42-
TCTCGGGTGCCCATTGGTCCGGGCAGCTGTGGGGACAGTAGGGTGGGT
ACGACTGTGGGA
CTTCAGTCCTAACAGAATGCGGGTGGCCTGTGCATTTCAAAGTTTATGC
AGTAACTCTGG
GGCCACAGGGGCTAGGAGTACCAGGCTGGGACCTCTACCCAAGGATC
ACTGCTTGGAAGA
ATATGTGGAATACTTCCAGGCTTGGAGTATACCAAAGGGATACCAAG
GG
The polypeptide sequence of mouse SAC1 (SEQ ID NO: 3) is:
MPALAIMGLSLAAFLELGMGASLCLSQQFKAQGDYILGGLFPLGSTEEAT
LNQRTQPNSIPCNRFSPLGLFLAMAMKMAVEEINNGSALLPGLRLGYDLF
DTCSEPVVTMKSSLMFLAKVGSQSIAAYCNYTQYQPRVLAVIGPHSSELA
LITGKFFSFFLMPQVSYSASMDRLSDRETFPSFFRTVPSDRVQLQAVVTLL
QNFSWNWVAALGSDDDYGREGLSIFSSLANARGICIAHEGLVPQHDTSGQ
QLGKVLDVLRQVNQSKVQVVVLFASARAVYSLFSYSIHHGLSPKVWVAS
ESWLTSDLVMTLPNIARVGTVLGFLQRGALLPEFSHYVETHLALAADPAF
CASLNAELDLEEHVMGQRCPRCDDIMLQNLS S GLLQNLSAGQLHHQIFAT
YAAVYSVAQALHNTLQCNVSHCHVSEHVLPWQLLENMYNMSFHARDLT
LQFDAEGNVDMEYDLKMWVWQSPTPVLHTVGTFNGTLQLQQSKMYWP
GNQVPVSQCSRQCKDGQVRRVKGFHSCCYDCVDCKAGSYRKHPDDFTC
TPCNQDQWSPEKSTACLPRRPKFLAWGEPVVLSLLLLLCLVLGLALAALG
LSVHHWDSPLVQASGGSQFCFGLICLGLFCLSVLLFPGRPSSASCLAQQPM
AHLPLTGCLSTLFLQAAETFVESELPLSWANWLCSYLRGLWAWLVVLLA
TFVEAALCAWYLIAFPPEVVTDWSVLPTEVLEHCHVRSWVSLGLVHITNA
MLAFLCFLGTFLVQSQPGRYNRARGLTFAMLAYFITWVSFVPLLANVQV
AYQPAV QMGAILV CALGILV TFHLPKCYVLLWLPKLNTQEFFLGRNAKK
AADENSGGGEAAQGHNE
The cDNA of human SAC 1 (SEQ ID NO: 4) is:
ATGCTGGGCCCTGCTGTCCTGGGCCTCAGCCTCTGGGCTCTCCTGCACC
CTGGGACGGGGGCCCCATTGTGCCTGTCACAGCAACTTAGGATGAAGG
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GGGACTACGTGCTGGGGGGGCTGTTCCCCCTGGGCGAGGCCGAGGAG
GCTGGCCTCCGCAGCCGGACACGGCCCAGCAGCCCTGTGTGCACCAGG
TTCTCCTCAAACGGCCTGCTCTGGGCACTGGCCATGAAAATGGCCGTG
GAGGAGATCAACAACAAGTCGGATCTGCTGCCCGGGCTGCGCCTGGGC
TACGACCTCTTTGATACGTGCTCGGAGCCTGTGGTGGCCATGAAGCCC
AGCCTCATGTTCCTGGCCAAGGCAGGCAGCCGCGACATCGCCGCCTAC
TGCAACTACACGCAGTACCAGCCCCGTGTGCTGGCTGTCATCGGGCCC
CACTCGTCAGAGCTCGCCATGGTCACCGGCAAGTTCTTCAGCTTCTTCC
TCATGCCCCAGGTCAGCTACGGTGCTAGCATGGAGCTGCTGAGCGCCC
GGGAGACCTTCCCCTCCTTCTTCCGCACCGTGCCCAGCGACCGTGTGCA
GCTGACGGCCGCCGCGGAGCTGCTGCAGGAGTTCGGCTGGAAGTGGGT
GGCCGCCCTGGGCAGCGACGACGAGTACGGCCGGCAGGGCCTGAGCA
TCTTCTCGGCCCTGGCCTCGGCACGCGGCATCTGCATCGCGCACGAGG
GCCTGGTGCCGCTGCCCCGTGCCGATGACTCGCGGCTGGGGAAGGTGC
AGGACGTCCTGCACCAGGTGAACCAGAGCAGCGTGCAGGTGGTGCTG
CTGTTCGCCTCCGTGCACGCCGCCCACGCCCTCTTCAACTACAGCATCA
GCAGCAGGCTCTCGCCCAAGGTGTGGGTGGCCAGCGAGGCCTGGCTGA
CCTCTGACCTGGTCATGGGGCTGCCCGGCATGGCCCAGATGGGCACGG
TGCTTGGCTTCCTCCAGAGGGGTGCCCAGCTGCACGAGTTCCCCCAGT
ACGTGAAGACGCACCTGGCCCTGGCCACCGACCCGGCCTTCTGCTCTG
CCCTGGGCGAGAGGGAGCAGGGTCTGGAGGAGGACGTGGTGGGCCAG
CGCTGCCCGCAGTGTGACTGCATCACGCTGCAGAACGTGAGCGCAGGG
CTAAATCACCACCAGACGTTCTCTGTCTACGCAGCTGTGTATAGCGTG
GCCCAGGCCCTGCACAACACTCTTCAGTGCAACGCCTCAGGCTGCCCC
GCGCAGGACCCCGTGAAGCCCTGGCAGCTCCTGGAGAACATGTACAAC
CTGACCTTCCACGTGGGCGGGCTGCCGCTGCGGTTCGACAGCAGCGGA
AACGTGGACATGGAGTACGACCTGAAGCTGTGGGTGTGGCAGGGCTC
AGTGCCCAGGCTCCACGACGTGGGCAGGTTCAACGGCAGCCTCAGGAC
AGAGCGCCTGAAGATCCGCTGGCACACGTCTGACAACCAGAAGCCCGT
GTCCCGGTGCTCGCGGCAGTGCCAGGAGGGCCAGGTGCGCCGGGTCA
AGGGGTTCCACTCCTGCTGCTACGACTGTGTGGACTGCGAGGCGGGCA
GCTACCGGCAAAACCCAGACGACATCGCCTGCACCTTTTGTGGCCAGG
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ATGAGTGGTCCCCGGAGCGAAGCACACGCTGCTTCCGCCGCAGGTCTC
GGTTCCTGGCATGGGGCGAGCCGGCTGTGCTGCTGCTGCTCCTGCTGCT
GAGCCTGGCGCTGGGCCTTGTGCTGGCTGCTTTGGGGCTGTTCGTTCAC
CATCGGGACAGCCCACTGGTTCAGGCCTCGGGGGGGCCCCTGGCCTGC
TTTGGCCTGGTGTGCCTGGGCCTGGTCTGCCTCAGCGTCCTCCTGTTCC
CTGGCCAGCCCAGCCCTGCCCGATGCCTGGCCCAGCAGCCCTTGTCCC
ACCTCCCGCTCACGGGCTGCCTGAGCACACTCTTCCTGCAGGCGGCCG
AGATCTTCGTGGAGTCAGAACTGCCTCTGAGCTGGGCAGACCGGCTGA
GTGGCTGCCTGCGGGGGCCCTGGGCCTGGCTGGTGGTGCTGCTGGCCA
TGCTGGTGGAGGTCGCACTGTGCACCTGGTACCTGGTGGCCTTCCCGC
CGGAGGTGGTGACGGACTGGCACATGCTGCCCACGGAGGCGCTGGTG
CACTGCCGCACACGCTCCTGGGTCAGCTTCGGCCTAGCGCACGCCACC
AATGCCACGCTGGCCTTTCTCTGCTTCCTGGGCACTTTCCTGGTGCGGA
GCCAGCCGGGCCGCTACAACCGTGCCCGTGGCCTCACCTTTGCCATGC
TGGCCTACTTCATCACCTGGGTCTCCTTTGTGCCCCTCCTGGCCAATGT
GCAGGTGGTCCTCAGGCCCGCCGTGCAGATGGGCGCCCTCCTGCTCTG
TGTCCTGGGCATCCTGGCTGCCTTCCACCTGCCCAGGTGTTACCTGCTC
ATGCGGCAGCCAGGGCTCAACACCCCCGAGTTCTTCCTGGGAGGGGGC
CCTGGGGATGCCCAAGGCCAGAATGACGGGAACACAGGAAATCAGGG
GAAACATGAGTGA
The polypeptide sequence of human SAC1 substantially from the
translated region of the human cDNA (SEQ ID NO: 5) is:
MLGPAVLGLSLWALLHPGTGAPLCLSQQLRMKGDYVLGGLFPLGEAEEA
GLRSRTRPSSPVCTRFSSNGLLWALAMKMAVEEINNKSDLLPGLRLGYDL
FDTCSEPWAMKPSLMFLAKAGSRDIAAYCNYTQYQPRVLAVIGPHSSEL
AMVTGKFFSFFLMPQVSYGASMELLSARETFPSFFRTVPSDRVQLTAAAE
LLQEFGWNWVAALGSDDEYGRQGLSIFSALASARGICIAHEGLVPLPRAD
DSRLGKVQDVLHQVNQSSVQVVLLFASVHAAHALFNYSISSRLSPKVWV
ASEAWLTSDLVMGLPGMAQMGTVLGFLQRGAQLHEFPQYVKTHLALAT
DPAFCSALGEREQGLEEDVVGQRCPQCDCITLQNVSAGLNHHQTFSVYAA
VYSVAQALHNTLQCNASGCPAQDPVKPWQLLENMYNLTFHVGGLPLRF
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DSSGNVDMEYDLKLWVWQGSVPRLHDVGRFNGSLRTERLKIRWHTSDN
QKPVSRCSRQCQEGQVRRVKGFHSCCYDCVDCEAGSYRQNPDDIACTFC
GQDEWSPERSTRCFRRRSRFLAWGEPAVLLLLLLLSLALGLVLAALGLFV
HHRDSPLVQASGGPLACFGLVCLGLVCLSVLLFPGQPSPARCLAQQPLSHL
PLTGCLSTLFLQAAEIFVESELPLSWADRLSGCLRGPWAWLVVLLAMLVE
VALCT WYLVAFPPEV V TDWHMLPTEALVHCRTRS W V SFGLAHATNATL
AFLCFLGTFLVRSQPGRYNRARGLTFAMLAYFITWVSFVPLLANVQVVLR
PAVQMGALLLCVLGILAAFHLPRCYLLMRQPGLNTPEFFLGGGPGDAQG
QNDGNTGNQGKHE
III. SAC.l Is a G-Protein Coupled Receptor
The evidence that SAC is a G-protein coupled receptor (GPCR) comes
from its sequence homology to other GPCR and the structure predicted for the
amino acid sequence.
GPCRs (also known as 7-transmembrane receptors) bind extracellular
ligands and transduce signals into the cell by coupling to intracellular G-
proteins.
GPCRs can be subdivided into more than 30 families on the basis of their
ligands.
Sac is most closely allied by sequence homology with the Cad-sensing,
metabotropic receptors.
Proteins often contain several modules or domains, each with a distinct
evolutionary origin and function. When the Sac cDNA sequence is queried
against
the Conserved Domain Database at NCBI, the following results are obtained:
Sequences producing Score E
significant alignments:
(bits)Value
Gnl ~ Pfam ~ pfam01094ANF receptor, Receptor family145 73-36
ligand
binding region
Gnl ~ Pfam ~ pfam000037tm 3, 7-transmembrane receptor87.0 3e-18
(metabotropic glutamate
family)
Note the ANF receptor family contains the metabotropic and calcium-sensing
families of GCPs.
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The closest sequence homology of the mouse SAC gene is to the Cad
sensing receptors, all of which are GCPRs. An alignment between a calcium
sensing GPCR (BAA09453) is shown in Fig. 5.
As described above, all GPCRs have a characteristic 7-transmembrane
domain. Figure 6 is a plot of the transmembrane domains of SAC 1.
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0
r N
p ,G' V ' ~ ~ bA bD p
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p ~ 'cY ~ V1 M VN1 p Ov ~ b V ~ 3 by p a
d0'~~ N ~~'dM'~~+~+ b~' N V~~ V ~ O .~ ~ O ~ a
~o~;~z~~ o
cw o ~~ N -o ,b .. v~
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,'~-. .-~ N M 'd' N \O l~ 00 O\ ° .~-, .N-as H , ~ 3 v W b~U Q. w ~~ N
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IV. The Sac Locus and the Gpr98 Sweet Taste Receptor Gene
A substantial effort has been devoted to positional cloning of a locus on
distal Chr 4 with a major effect on sweetener intake. This locus has been
previously described as the Sac (saccharin preference) locus, and it also
explains
~8 % of the phenotypic variance in ethanol preferences within the B6 x 129
F2 generation.
Details on positional cloning of the Sac locus are found above.
The effects of SAC1 (Gpr98) on ethanol intake Two lines of evidence
point to the involvement of Gpr98 in ethanol intake. First, 129.B6-Sac
congenic
mice homozygous for a 194-kb donor fragment from the B6 strain consumed more
10% ethanol solution than did congenic mice without the donor fragment
(1.50 ~ 0.15 and 1.19 ~ 0.11 mL/day, respectively; p <0.05, one-tailed t-
test).
Second, ethanol preference was related to sequence variations of Gpr98.
Analysis
of Gpr98 sequences from genealogically remote or unrelated mouse strains
indicated the presence of two haplotypes of single nucleotide polymorphisms
within the Gpr98 locus. One, 'B6-like' haplotype, was found in mouse strains
with
elevated sweetener preference and the other, ' 129-like' haplotype, was found
in
strains relatively indifferent to sweeteners as described above. Preferences
for
10% ethanol for the same mouse strains were studied as described in Abstr. of
the
23th RSA Meeting (June 2000, Denver, Colorado). We found that strains with the
'B6-like' haplotype had higher preferences for 10% ethanol (20 ~ 4%, n =14,
strains C57BL/6J, C57L/J, CAST, FVB/NJ, KK/HIJ, NOD/LtJ, NZB/B1NJ, P/J,
RBF/DnJ, RF/J, SEA/GnJ, SJL/J, SPRET/Ei and SWR/J) compared with strains
having the ' 129-like' haplotype ( 12 ~ 2%, n =10, p <0.05, one-tailed t-test,
strains
129P3/J, AKR/J, BALB/c, BUB/BnJ, C3H/HeJ, CBA/J, DBAl2J, LP/J, PLIJ and
RIIIS/J).
V. Preparation of Recombinant or Chemicall~ynthesized Nucleic Acids,
Vectors, Transformation, Host-Cells
Large amounts of the polynucleotides of the present invention may be
produced by replication in a suitable host cell. Natural or synthetic
polynucleotide
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fragments coding for a desired fragment will be incorporated into recombinant
polynucleotide constructs, usually DNA constructs, capable of introduction
into
and replication in a prokaryotic or eukaryotic cell. Usually the
polynucleotide
constructs will be suitable for replication in a unicellular host, such as
yeast or
bacteria, but may also be intended for introduction to (with and without
integration within the genome) cultured mammalian or plant or other eukaryotic
cell lines. The purification of nucleic acids produced by the methods of the
present
invention is described, e.g., in Ausubel et al., Current Protocols i~
Molecular
Biology, Vol. 1-2, John Wiley & Sons, 1992 and Sambrook et al., Molecular
CloningA Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press,
1989.
The polynucleotides of the present invention may also be produced by
chemical synthesis, e.g., by the phosphoramidite method or the triester
method,
and may be performed on commercial, automated oligonucleotide synthesizers. A
double-stranded fragment may be obtained from the single-stranded product of
chemical synthesis either by synthesizing the complementary strand and
annealing
the strands together under appropriate conditions or by adding the
complementary
strand using DNA polymerase with an appropriate primer sequence.
Polynucleotide constructs prepared for introduction into a prokaryotic or
eukaryotic host may comprise a replication system recognized by the host,
including the intended polynucleotide fragment encoding the desired
polypeptide,
and will preferably also include transcription and translational initiation
regulatory
sequences operably linked to the polypeptide encoding segment. Expression
vectors may include, for example, an origin of replication or autonomously
replicating sequence (ARS) and expression control sequences, a promoter, an
enhancer and necessary processing information sites, such as ribosome-binding
sites, RNA splice sites, polyadenylation sites, transcriptional terminator
sequences, and mRNA stabilizing sequences. Secretion signals may also be
included where appropriate, whether from a native SAC 1 protein or from other
receptors or from secreted polypeptides of the same or related species, which
allow the protein to cross andlor lodge in cell membranes, and thus attain its
functional topology, or be secreted from the cell. Such vectors may be
prepared by
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means of standard recombinant techniques well-known in the art and discussed,
for example, in Sambrook et al., 1989 or Ausubel et al., 1992.
An appropriate promoter and other necessary vector sequences will be
selected so as to be functional in the host, and may include, when
appropriate,
those naturally associated with SAC1 genes. Examples of workable combinations
of cell lines and expression vectors are described in Sambrook et al., 1989 or
Ausubel et al., 1992. Many useful vectors are known in the art and may be
obtained from commercial vendors. Promoters such as the trp, lac and phage
promoters, TRNA promoters and glycolytic enzyme promoters may be used in
prokaryotic hosts. Useful yeast promoters include promoter regions for
metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as
enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for
maltose and galactose utilization, and others. In addition, the construct may
be
joined to an amplifiable gene so that multiple copies of the gene may be made.
For
appropriate enhancer and other expression control sequences, see also
Enhancers
and Euka~yotic Geue Expression, New York: Cold Spring Harbor Press, 1983.
See also, e.g., US Patent Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
Expression and cloning vectors will likely contain a selectable marker, a
gene encoding a protein necessary for survival or growth of a host cell
transformed with the vector. The presence of this gene ensures growth of only
those host cells which express the inserts. Typical selection genes encode
proteins
that (a) confer resistance fo antibiotics or other toxic substances, e.g.,
ampicillin,
neomycin, methotrcxate, etc.; (b) complement auxotrophic deficiencies; or
(c) supply critical nutrients not available from complex media, e.g., the gene
encoding D-alanine racemase for Bacilli. The choice of the proper selectable
marker will depend on the host cell, and appropriate markers for different
hosts
are well-known in the art.
The vectors containing the nucleic acids of interest can be transcribed
in vitro, and the resulting RNA introduced into the host cell by well-known
methods, e.g., by injection, or the vectors can be introduced directly into
host cells
by methods well-known in the art, which vary depending on the type of cellular
host, including electroporation; transfection employing calcium chloride,
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rubidium chloride, calcium phosphate, DEAF-dextran, or other substances;
microprojectile bombardment; lipofection; infection (where the vector is an
infectious agent, such as a retroviral genome); and other methods. The
introduction of the polynucleotides into the host cell by any method known in
the
S art, including, inter alia, those described above, will be referred to
herein as
"transformation." The cells into which have been introduced nucleic acids
described above are meant to also include the progeny of such cells.
Large quantities of the nucleic acids and polypeptides of the present
invention may be prepared by expressing the SAC1 nucleic acids or portions
thereof in vectors or other expression vehicles in compatible prokaryotic or
eukaryotic host cells. The most commonly used prokaryotic hosts are strains of
Eseherichia coli, although other prokaryotes, such as Bacillus subtilis or
Pseudomo~as may also be used.
Mammalian or other eukaryotic host cells, such as those of yeast,
filamentous fungi, plant, insect, or amphibian or avian species, may also be
useful
for production of the proteins of the present invention. Propagation of
mammalian
cells in culture is per se well-known. Examples of commonly used mammalian
host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells,
and
W138, BHK, and COS cell lines. An example of a commonly used insect cell line
is SF9. However, it will be appreciated by the skilled practitioner that other
cell
lines may be appropriate, e.g., to provide higher expression, desirable
glycosylation patterns, or other features.
Clones are selected by using markers depending on the mode of the vector
construction. The marker may be on the same or a different DNA molecule,
preferably the same DNA molecule. In prokaryotic hosts, the transformant may
be
selected, e.g., by resistance to ampicillin, tetracycline or other
antibiotics.
Production of a particular product based on temperature sensitivity may also
serve
as an appropriate marker.
VI. Diagnosis or Screening
Genetic analysis of obesity and diabetes and alcoholism or alcohol
consumption is often complicated by the lack of a simple diagnostic mark. For
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example, currently there is no single diagnostic marker for the diagnosis of
obesity. Sequence variation of the SACl locus may indicate a predisposition to
diabetes, obesity, and alcoholism and may provide a diagnostic mark.
In order to detect the presence of a SAC 1 allele predisposing an individual
to obesity, diabetes, or alcoholism, a biological sample may be prepared and
analyzed for the presence or absence of susceptibility alleles of SAC1.
Results of
these tests and interpretive information may be returned to the health care
professionals for communication to the tested individual. Such diagnoses may
be
performed by diagnostic laboratories. In addition, diagnostic kits may be
manufactured and available to health care providers or to private individuals
for
self diagnosis.
A basic format for sequence or expression analysis is finding sequences in
DNA or RNA extracted from affected family members which create abnormal
SAC1 gene products or abnormal levels of SACl gene product. The diagnostic or
screening method may involve amplification or molecular cloning of the
relevant
SAC1 sequences. For example, PCR based amplification may be used. Once
amplified, the resulting nucleic acid can be sequenced or used as a substrate
for
DNA probes. Primers and probes specific for the SAC 1 gene sequences may be
used to identify SAC1 alleles.
The pairs of single-stranded DNA primers can be annealed to sequences
within or surrounding the SAC1 gene in order to prime amplifying DNA synthesis
of the SAC 1 gene itself. The set of primers may allow synthesis of both
intron and
exon sequences. Allele-specific primers can also be used. Such primers anneal
only to particular SAC1 mutant alleles, and thus will only amplify a product
in the
presence of the mutant allele as a template.
In order to facilitate subsequent cloning of amplified sequences, primers
may have restriction enzyme site sequences appended to their 5' ends. Thus,
all
nucleotides of the primers are derived from SAC 1 sequences or sequences
adjacent to SACl, except for the few nucleotides necessary to form a
restriction
enzyme site. Such enzymes and sites are well-known in the art. The primers
themselves can be synthesized using techniques which are well-known in the
art.
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Generally, the primers can be made using oligonucleotide synthesizers which
are
commercially available.
The biological sample to be analyzed, such as blood, may be treated, if
desired, to extract the nucleic acids. The sample nucleic acid may be prepared
in
various ways to facilitate detection of the target sequence; e.g.,
denaturation,
restriction digestion, electrophoresis or dot blotting. The region of interest
of the
target nucleic acid is usually at least partially single-stranded to form
hybrids with
the probe. If the sequence is double-stranded, the sequence will probably need
to
be denatured. The target nucleic acid may be also be fragmented to reduce or
eliminate the formation of secondary structures. The fragmentation may be
performed using a number of methods, including enzymatic, chemical, thermal
cleavage or degradation. For example, fragmentation may be accomplished by
heat/Mg2+treatment, endonuclease (e.g., DNAase 1) treatment, restriction
enzyme digestion, shearing (e.g., by ultrasound) or NaOH treatment.
Many genotyping and expression monitoring methods have been described
previously. In general, target nucleic acid and probe are incubated under
conditions which forms hybridization complex between the probe and the target
sequence. The region of the probes which is used to bind to the target
sequence
can be made completely complementary to the targeted region of the SAC 1
locus.
Therefore, high stringency conditions may be desirable in order to prevent
false
positives. However, conditions of high stringency are typically used if the
probes
are complementary to regions of the chromosome which are unique in the
genome. The stringency of hybridization is determined by a number of factors
during hybridization and during the washing procedure, including temperature,
ionic strength, base composition, probe length, and concentration of
formamide.
Under certain circumstances, the formation of higher order hybrids, such as
triplexes, quadraplexes, etc. may be desired to provide the means of detecting
target sequences.
Detection, if any, of the resulting hybrid is usually accomplished by the
use of labeled probes. Alternatively, the probe may be unlabeled, but may be
detectable by specific binding with a ligand which is labeled, either directly
or
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indirectly. Suitable labels, and methods for labeling probes and ligands are
known
in the art, and include, for example, radioactive labels which may be
incorporated
by known methods (e.g., nick translation, random priming or kinase reaction),
biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes,
particularly
triggered dioxetanes), enzymes, antibodies and the like. Variations of this
basic
scheme are known in the art, and include those variations that facilitate
separation
of the hybrids to be detected from extraneous materials and/or that amplify
the
signal from the labeled moiety.
Two-step label amplification methodologies are known in the art. These
assays work on the principle that a small ligand (such as digoxigenin, biotin,
or
the like) is attached to a nucleic acid probe capable of specifically binding
SAC 1.
In one example, the small ligand attached to the nucleic acid probe is
specifically recognized by an antibody-enzyme conjugate. In one embodiment of
this example, digoxigenin is attached to the nucleic acid probe. Hybridization
is
detected by an antibody-alkaline phosphatase conjugate which turns over a
chemiluminescent substrate. In a second example, the small ligand is
recognized
by a second ligand-enzyme conjugate that is capable of specifically complexing
to
the first ligand. A well-known embodiment of this example is the biotin-avidin
type of interactions.
It is also contemplated within the scope of this invention that the nucleic
acid probe assays of this invention will employ a cocktail of nucleic acid
probes
capable of detecting SAG 1. Thus, in one example to detect the presence of SAC
1
in a biological sample, more than one probe complementary to SAG 1 is
employed.
Predisposition to diabetes, obesity, or alcoholism can be ascertained by
testing any fluid or tissue of a human for sequence variations of the SAC1
gene.
For example, a person who has inherited a germline SAC 1 mutation would be
prone to develop obesity, diabetes, or alcoholism. This can be determined by
testing DNA from any tissue of the person's body. Most simply, blood can be
drawn and DNA extracted from the cells of the blood. In addition, prenatal
diagnosis can be accomplished by testing fetal cells, placental cells or
amniotic
cells for mutations of the SAG 1 gene.
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The most definitive test for mutations in a candidate locus is to directly
compare genomic SAC1 sequences from obese, diabetic, or alcoholic patients,
with those from a control population. Alternatively, one could sequence
messenger RNA after amplification, e.g., by PCR, thereby eliminating the
necessity of determining the exon structure of the candidate gene.
Sequence variations from diabetic, obese, or alcoholic patients falling
outside the coding region of SAC1 can be detected by examining the non-coding
regions, such as introns and regulatory sequences near or within the SAC1
gene.
An early indication that mutations in noncoding regions are important may come
from Northern blot experiments that reveal messenger RNA molecules of
abnormal size or abundance in obese or diabetic patients as compared to
control
individuals.
Alteration of SAC 1 mRNA expression can be detected by any techniques
known in the art (see above). These include Northern blot analysis, PCR
amplification, RNase protection, and gene chip analysis. Diminished mRNA
expression indicates an alteration of the wild-type SAC1 gene.
The diabetic, obese, or alcoholic condition can also be detected on the
basis of the alteration of wild-type SACl polypeptide. For example, the
presence
of a SAC 1 gene variant, which produces a protein having a loss of function,
or
altered function, may directly correlate to an increased risk of obesity or
diabetes.
Such variation can be determined by sequence analysis in accordance with
conventional techniques. For example, antibodies (polyclonal or monoclonal)
may
be used to detect differences in, or the absence of, SAC 1 polypeptides.
Antibodies
may immunoprecipitate SAC 1 proteins from solution as well as react with SAC 1
protein on Western or immunoblots of polyacrylamide gels. Antibodies may also
detect SAC1 proteins in paraffin or frozen tissue sections, using
immunocytochemical techniques. Immunoassay include, for example, enzyme
linked immunosorbent assays (ELISA), radioimmunoassays (RIA),
immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),
sandwich assays, etc.
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Functional assays, such as protein binding determinations, can be used.
Finding a mutant SAC1 gene product indicates alteration of a wild-type SACl
gene.
VII. Drug, Sweetener, and Alcohol Preference Screening
This invention is also useful for screening compounds by using the SACl
polypeptide or binding fragment thereof in any of a variety of drug,
sweetener,
and alcohol screening techniques.
The SAC1 polypeptide or fragment employed in such a test may either be
free in solution, affixed to a solid support, or borne on a cell surface. One
method
of drug screening utilizes eukaryotic or prokaryotic host cells which are
stably
transformed with recombinant polynucleotides expressing the polypeptide or
fragment, preferably in competitive binding assays. Such cells, either in
viable or
fixed form, can be used for standard binding assays. One may measure, for
example, for the formation of complexes between a SAC1 polypeptide or
fragment and the agent being tested, or examine the degree to which the
formation
of a complex between a SAC1 polypeptide or fragment and a known ligand is
interfered with by the agent being tested.
Thus, the present invention provides methods of screening for drugs and
sweeteners comprising contacting such an agent with a SAC1 polypeptide or
fragment thereof and assaying (i) for the presence of a complex between the
agent
and the SAC 1 polypeptide or fragment, or (ii) for the presence of a complex
between the SAC 1 polypeptide or fragment and a ligand, by methods well-known
in the art. In such competitive binding assays the SACl polypeptide or
fragment is
typically labeled. Free SAC1 polypeptide or fragment is separated from that
present in a protein:protein complex, and the amount of free (i.e.,
uncomplexed)
label is a measure of the binding of the agent being tested to SAC1 or its
interference with SACl:ligand binding, respectively.
Other suitable techniques for drug, sweetener, and alcohol screening may
provide high throughput screening for compounds having suitable binding
affinity
to the SAC1 polypeptides. For example, large numbers of different small
peptide
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test compounds are synthesized on a solid substrate, such as plastic pins or
some
other surface. The peptide test compounds are reacted with SAC 1 polypeptide
and
washed. Bound SAC1 polypeptide is then detected by methods well-known in the
art.
Purified SACl can be coated directly onto plates for use in the
aforementioned drug screening techniques. However, non-neutralizing antibodies
to the polypeptide can be used to capture antibodies to immobilize the SAC1
polypeptide on the solid phase.
This invention also contemplates the use of competitive drug, sweetener,
and alcohol screening assays in which neutralizing antibodies capable of
specifically binding the SAC1 polypeptide compete with a test compound for
binding to the SAC1 polypeptide or fragments thereof. In this manner, the
antibodies can be used to detect the presence of any peptide which shares one
or
more antigenic determinants of the SAC 1 polypeptide.
A further technique for drug, sweetener, and alcohol screening involves
the use of host eukaryotic cell lines or cells which have a nonfunctional SAC
1
gene. These host cell lines or cells are defective at the SAC 1 polypeptide
level.
The host cell lines or cells are grown in the presence of the drug, sweetener,
or
alcohol compound. The rate of growth of the host cells is measured to
determine if
the compound is capable of regulating the growth of SAC1 defective cells.
Briefly, a method of screening for a substance which modulates activity of
a polypeptide may include contacting one or more test substances with the
polypeptide in a suitable reaction medium, testing the activity of the treated
polypeptide and comparing that activity with the activity of the polypeptide
in
comparable reaction medium untreated with the test substance or substances. A
difference in activity between the treated and untreated polypeptides is
indicative
of a modulating effect of the relevant test substance or substances.
Prior to or as well as being screened for modulation of activity, test
substances may be screened for ability to interact with the polypeptide, e.g.,
in a
yeast two-hybrid system. This system may be used as a coarse screen prior to
testing a substance for actual ability to modulate activity of the
polypeptide.
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Alternatively, the screen could be used to screen test substances for binding
to a
SACl specific binding partner, or to find mimetics of a SAC1 polypeptide.
VIII. Rational Drug Design
The goal of rational drug design is to produce structural analogs of
biologically active polypeptides of interest or of small molecules with which
they
interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs
which are,
for example, more active or stable forms of the polypeptide, or which, e.g.,
enhance or interfere with the function of a polypeptide in vivo. In one
approach,
one first determines the three-dimensional structure of a protein of interest
(e.g.,
SACl polypeptide) or, for example, of the SAC1-receptor or ligand complex, by
x-ray crystallography, by computer modeling or most typically, by a
combination
of approaches. Less often, useful information regarding the structure of a
polypeptide may be gained by modeling based on the structure of homologous
proteins. An example of rational drug design is the development of HIV
protease
inhibitors. In addition, peptides (e.g., SAC1 polypeptide) are analyzed by an
alanine scan. In this technique, an amino acid residue is replaced by Ala, and
its
effect on the peptide's activity is determined. Each of the amino acid
residues of
the peptide is analyzed in this manner to determine the important regions of
the
peptide.
It is also possible to isolate a target-specific antibody, selected by a
functional assay, and then to solve its crystal structure. In principle, this
approach
yields a pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating anti-
idiotypic
antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
mirror image of a mirror image, the binding site of the anti-ids would be
expected
to be an analog of the original receptor. The anti-id could then be used to
identify
and isolate peptides from banks of chemically or biologically produced banks
of
peptides. Selected peptides would then act as the pharmacore.
Thus, one may design drugs which have, e.g., improved SACl polypeptide
activity or stability or which act as inhibitors, agonists, antagonists, etc.
of SAC1
polypeptide activity. By virtue of the availability of cloned SAC1 sequences,
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sufficient amounts of the SAC1 polypeptide may be made available to perform
such analytical studies as x-ray crystallography. In addition, the knowledge
of the
SAC1 protein sequence provided herein will guide those employing computer
modeling techniques in place of, or in addition to x-ray crystallography.
Following identification of a substance which modulates or affects
polypeptide activity, the substance may be investigated further. Furthermore,
it
may be manufactured andlor used in preparation, i.e., manufacture or
formulation,
or a composition such as a medicament, pharmaceutical composition or drug.
These may be administered to individuals.
Thus, the present invention extends in various aspects not only to a
substance identified using a nucleic acid molecule as a modulator of
polypeptide
activity, in accordance with what is disclosed herein, but also a
pharmaceutical
composition, medicament, drug or other composition comprising such a
substance, a method comprising administration of such a composition comprising
such a substance, a method comprising administration of such a composition to
a
patient, e.g., for treatment of diabetes, obesity or alcohol consumption, use
of such
a substance in the manufacture of a composition for administration, e.g., for
treatment of diabetes or alcohol consumption, and a method of making a
pharmaceutical composition comprising admixing such a substance with a
pharmaceutically acceptable excipient, vehicle or carrier, and optionally
other
ingredients.
A substance identified as a modulator of polypeptide function may be
peptide or non-peptide in nature. Non-peptide "small molecules" are often
preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or
mimic
of the substance (particularly if a peptide) may be designed for
pharmaceutical
use.
The designing of mimetics to a known pharmaceutically active compound
is a known approach to the development of pharmaceuticals based on a "lead"
compound. This might be desirable where the active compound is difficult or
expensive to synthesize or where it is unsuitable for a particular method of
administration, e.g., pure peptides are unsuitable active agents for oral
compositions as they tend to be quickly degraded by proteases in the
alimentary
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canal. Mimetic design, synthesis and testing is generally used to avoid
randomly
screening large numbers of molecules for a target property.
There are several steps commonly taken in the design of a mimetic from a
compound having a given target property. First, the particular parts of the
compound that are critical and/or important in determining the target property
are
determined. In the case of a peptide, this can be done by systematically
varying
the amino acid residues in the peptide, e.g., by substituting each residue in
turn.
Alanine scans of peptide are commonly used to refine such peptide motifs.
These
parts or residues constituting the active region of the compound are known as
its
pharmacophore. .
Once the pharmacophore has been found, its structure is modeled
according to its physical properties, e.g., stereochemistry, bonding, size
and/or
charge, using data from a range of sources, e.g., spectroscopic techniques, x-
ray
diffraction data and NMR. Computational analysis, similarity mapping (which
models the charge and/or volume of a pharmacophore, rather than the bonding
between atoms) and other techniques can be used in this modeling process.
In a variant of this approach, the three-dimensional structure of the ligand
and its binding partner are modeled. This can be especially used where the
ligand
and/or binding partner change conformation on binding, allowing the model to
take account of this in the design of the mimetic.
A template molecule is then selected onto which chemical groups which
mimic the pharmacophore can be grafted. The template molecule and the chemical
groups grafted onto it can conveniently be selected so that the mimetic is
easy to
synthesize, is likely to be pharmacologically acceptable, and does not degrade
in vivo, while retaining the biological activity of the lead compound.
Alternatively, where the mimetic is peptide-based, further stability can be
achieved by cyclizing the peptide, increasing its rigidity. The mimetic(s)
found by
this approach can then be screened to see whether they have the target
property, or
to what extent they exhibit it. Further optimization or modification can then
be
carried out to arrive at one or more final mimetics for in vivo or clinical
testing.
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IX. Gene Therapy
According to the present invention, a method is also provided of supplying
wild-type SAC 1 function to a cell which carries mutant SAC 1 alleles. The
wild-
type SACl gene or a part of the gene may be introduced into the cell in a
vector
such that the gene remains extrachromosomal. In such a situation, the gene
will be
expressed by the cell from the extra chromosomal location. More preferred is
the
situation where the wild-type SAC1 gene or a part thereof is introduced into
the
mutant cell in such a way that it recombines with the endogenous mutant SAC1
gene present in the cell. Such recombination requires a double recombination
event which results in the correction of the SAC1 gene mutation. Vectors for
introduction of genes both for recombination and for extrachromosomal
maintenance are known in the art, and any suitable vector may be used. Methods
for introducing DNA into cells such as electroporation, calcium phosphate
coprecipitation and viral transduction are known in the art, and the choice of
method is within the competence of skilled practitioners.
As generally discussed above, the SAC 1 gene or fragment, where
applicable, may be employed in gene therapy methods in order to increase the
amount of the expression products of such genes in diabetic or obese cells.
Such
gene therapy is particularly appropriate, in which the level of SAC 1
polypeptide is
absent or compared to normal cells. It may also be useful to increase the
level of
expression of a given SAC 1 gene even in those situations in which the mutant
gene is expressed at a "normal" level, but the gene product is not fully
functional.
Gene therapy would be carried out according to generally accepted
methods, for example, as described by Therapy for Genetic Diseases,
T. Friedman, ed. Oxford University Press, 1991. Cells from a patient would be
first analyzed by the diagnostic methods described above, to ascertain the
production of SAC1 polypeptide in these cells. A virus or plasmid vector,
containing a copy of the SAC 1 gene linked to expression control elements and
capable of replicating inside the sample cells, is prepared. Suitable vectors
are
known, such as disclosed in PCT publications WO 93/07282 and United States
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PatentNos. 5,252,479, 5,691,198, 5,747,469, 5,436,146 and 5,753,500. The
vector
is then injected into the patient.
Gene transfer systems known in the art may be useful in the practice of the
gene therapy methods of the present invention. These include viral and
nonviral
transfer methods. A number of viruses have been used as gene transfer vectors,
including papovaviruses, e.g., SV40, adenovirus, vaccinia virus, adeno-
associated
virus, herpes viruses including HSV and EBV; lentiviruses, Sindbis and Semliki
Forest virus, and retroviruses of avian, marine, and human origin. Most human
gene therapy protocols have been based on disabled marine retroviruses.
Nonviral gene transfer methods known in the art include chemical
techniques such as calcium phosphate coprecipitation; mechanical techniques,
for
example microinjection; membrane fusion-mediated transfer via liposomes; and
direct DNA uptake and receptor-mediated DNA transfer. Viral-mediated gene
transfer can be combined with direct in vivo gene transfer using liposome
delivery, allowing one to direct the viral vectors to the affected cells and
not into
the surrounding nondividing cells. Alternatively, the retroviral vector
producer
cell line can be injected into affected cells. Injection of producer cells
would then
provide a continuous source of vector particles.
In an approach which combines biological and physical gene transfer
methods, plasmid DNA of any size is combined with a polylysine-conjugated
antibody specific to the adenovirus hexon protein, and the resulting complex
is
bound to an adenovirus vector. The trimolecular complex is then used to infect
cells. The adenovirus vector permits efficient binding, internalization, and
degradation of the endosome before the coupled DNA is damaged. For other
techniques for the delivery of adenovirus based vectors see United States
Patent
Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.
Liposome/DNA complexes have been shown to be capable of mediating
direct in vivo gene transfer. While in standard liposome preparations the gene
transfer process is nonspecific, localized in vivo uptake and expression may
be
accomplished following direct iii situ administration.
Expression vectors in the context of gene therapy are meant to include
those constructs containing sequences sufficient to express a polynucleotide
that
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has been cloned therein. In viral expression vectors, the construct contains
viral
sequences sufficient to support packaging of the construct. If the
polynucleotide
encodes SACl, expression will produce SACl. If the polynucleotide encodes an
antisense polynucleotide or a ribozyme, expression will produce the antisense
polynucleotide or ribozyme. Thus in this context, expression does not require
that
a protein product be synthesized. In addition to the polynucleotide cloned
into the
expression vector, the vector also contains a promoter functional in
eukaryotic
cells. The cloned polynucleotide sequence is under control of this promoter.
Suitable eukaryotic promoters include those described above. The expression
vector may also include sequences, such as selectable markers and other
sequences described herein.
Receptor-mediated gene transfer, for example, may be accomplished by
the conjugation of DNA (usually in the form of covalently closed supercoiled
plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis
of the
presence of the corresponding ligand receptors on the cell surface of the
target
cell/tissue type. One appropriate receptor/ligand pair may include the
estrogen
receptor and its ligand, estrogen (and estrogen analogues). These ligand-DNA
conjugates can be injected directly into the blood if desired and are directed
to the
target tissue where receptor binding and internalization of the DNA-protein
complex occurs. To overcome the problem of intracellular destruction of DNA,
coinfection with adenovirus can be included to disrupt endosome function.
X. Peptide Therapy
Peptides which have SAC 1 activity can be supplied to cells which carry
mutant or missing SAC1 alleles. Protein can be produced by expression of the
cDNA sequence in bacteria, for example, using known expression vectors.
Alternatively, SAC1 polypeptide can be extracted from SAC1-producing
mammalian cells. In addition, the techniques of synthetic chemistry can be
employed to synthesize SAC 1 protein. Any of such techniques can provide the
preparation of the present invention which comprises the SAC 1 protein.
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Preparation is substantially free of other human proteins. This is most
readily
accomplished by synthesis in a microorganism or in vitro.
Active SAC1 molecules can be introduced into cells by microinjection or
by use of liposomes, for example. Alternatively, some active molecules may be
taken up by cells, actively or by diffusion. Extra-cellular application of the
SAC 1
gene product may be sufficient. Molecules with SAC 1 activity (for example,
peptides, drugs or organic compounds) may also be used to effect such a
reversal.
Modified polypeptides having substantially similar function are also used for
peptide therapy.
XI. Transformed Hosts
Similarly, cells and animals which carry a mutant SAC1 allele can be used
as model systems to study and test for substances which have potential as
therapeutic agents. These may be isolated from individuals with SAC1
mutations,
either somatic or germline. Alternatively, the cell line can be engineered to
carry
the mutation in the SAC1 allele.
Animals for testing therapeutic agents can be selected after mutagenesis of
whole animals or after treatment of germline cells or zygotes. Such treatments
include insertion of mutant SAC1 alleles, usually from a second animal
species, as
well as insertion of disrupted homologous genes. Alternatively, the endogenous
SAC1 gene of the animals may be disrupted by insertion or deletion mutation or
other genetic alterations using conventional techniques to produce knockout or
transplacement animals. A transplacement is similar to a knockout because the
endogenous gene is replaced, but in the case of a transplacement the
replacement
is by another version of the same gene. After test substances have been
administered to the animals, the phenotype must be assessed. If the test
substance
prevents or suppresses the disease, then the test substance is a candidate
therapeutic agent for the treatment of disease. These animal models provide an
extremely important testing vehicle for potential therapeutic products.
In one embodiment of the invention, transgenic animals are produced
which contain a functional transgene encoding a functional SACl polypeptide or
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variants thereof. Transgenic animals expressing SAC1 transgenes, recombinant
cell lines derived from such animals and transgenic embryos may be useful in
methods for screening for and identifying agents that induce or repress
function of
SACl . Transgenic animals of the present invention also can be used as models
for
studying indications such as diabetes.
In one embodiment of the invention, a SAC1 transgene is introduced into a
non-human host to produce a transgenic animal expressing a human or marine
SAC 1 gene. The transgenic animal is produced by the integration of the
transgene
into the genome in a manner that permits the expression of the transgene.
Methods
for producing transgenic animals are generally described in US Patent
No. 4,873,191 and in Manipulating the Mouse Embryo; A Laboratory Manual,
2nd edition (eds., Hogan, Beddington, Costantimi and Long, New York: Cold
Spring Harbor Laboratory Press, 1994).
Tt may be desirable to replace the endogenous SAC1 by homologous
recombination between the transgene and the endogenous gene; or the endogenous
gene may be eliminated by deletion as in the preparation of "knock-out"
animals.
Typically, a SAC 1 gene flanked by genomic sequences is transferred by
microinjection into a fertilized egg. The microinjected eggs are implanted
into a
host female, and the progeny are screened for the expression of the transgene.
Transgenic animals may be produced from the fertilized eggs from a number of
animals including, but not limited to reptiles, amphibians, birds, mammals,
and
fish. Within a particularly preferred embodiment, transgenic mice are
generated
which express a mutant form of the polypeptide.
As noted above, transgenic animals and cell lines derived from such
animals may find use in certain testing experiments. In this regard,
transgenic
animals and cell lines capable of expressing wild-type or mutant SAC 1 may be
exposed to test substances. These test substances can be screened for the
ability to
reduce overexpression of wild-type SAC 1 or impair the expression or function
of
mutant SAC 1.
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XII. Pharmaceutical Compositions and Routes of Administration
The SAC1 polypeptides, antibodies, peptides and nucleic acids of the
present invention can be formulated in pharmaceutical compositions, which are
prepared according to conventional pharmaceutical compounding techniques. See,
for example, Remingtoh's Pharmaceutic. Sciehces, 18th Ed. (Euston, PA: Mack
Publishing Co., 1990). The composition may contain the active agent or
pharmaceutically acceptable salts of the active agent. These compositions may
comprise, in addition to one of the active substances, a pharmaceutically
acceptable excipient, carrier, buffer, stabilizer or other materials well-
known in
the art. Such materials should be nontoxic and should not interfere with the
efficacy of the active ingredient. The carrier may take a wide variety of
forms
depending on the form of preparation desired for administration, e.g.,
intravenous,
oral, intrathecal, epineural or parenteral.
For oral administration, the compounds can be formulated into solid or
liquid preparations such as capsules, pills, tablets, lozenges, melts,
powders,
suspensions or emulsions. In preparing the compositions in oral dosage form,
any
of the usual pharmaceutical media may be employed, such as, for example,
water,
glycols, oils, alcohols, flavoring agents, preservatives, coloring agents,
suspending
agents, and the like in the case of oral liquid preparations (such as, for
example,
suspensions, elixirs and solutions); or carriers such as starches, sugars,
diluents,
granulating agents, lubricants, binders, disintegrating agents and the like in
the
case of oral solid preparations (such as, for example, powders, capsules and
tablets). Because of their ease in administration, tablets and capsules
represent the
most advantageous oral dosage unit form, in which case solid pharmaceutical
carriers are obviously employed. If desired, tablets may be sugar-coated or
enteric-coated by standard techniques. The active agent can be encapsulated to
make it stable to passage through the gastrointestinal tract while at the same
time
allowing for passage across the blood brain barrier. See for example,
WO 96/11698.
For parenteral administration, the compound may be dissolved in a
pharmaceutical carrier and administered as either a solution or a suspension.
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Illustrative of suitable carriers are water, saline, dextrose solutions,
fructose
solutions, ethanol, or oils of animal, vegetative or synthetic origin. The
carrier
may also contain other ingredients, for example, preservatives, suspending
agents,
solubilizing agents, buffers and the like. When the compounds are being
administered intrathecally, they may also be dissolved in cerebrospinal fluid.
The active agent is preferably administered in a therapeutically effective
amount. The actual amount administered, and the rate and time-course of
administration, will depend on the nature and severity of the condition being
treated. Prescription of treatment, e.g., decisions on dosage, timing, etc.,
is within
the responsibility of general practitioners or specialists, and Typically
takes
account of the disorder to be treated, the condition of the individual
patient, the
site of delivery, the method of administration and other factors known to
practitioners. Examples of techniques and protocols can be found in
Remington's
Pharmaceutical Sciences.
Alternatively, targeting therapies may be used to deliver the active agent
more specifically to certain types of cell, by the use of targeting systems
such as
antibodies or cell specific ligands. Targeting may be desirable for a variety
of
reasons, e.g., if the agent is unacceptably toxic, or if it would otherwise
require
too high a dosage, or if it would not otherwise be able to enter the target
cells.
Instead of administering these agents directly, they could be produced in
the target cell, e.g., in a viral vector such as described above or in a cell
based
delivery system such as described in United States Patent No. 5,550,050 and
PCT
publications WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452,
WO 96!02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97!12635,
designed for implantation in a patient. The vector could be targeted to the
specific
cells to be treated, or it could contain regulatory elements which are more
tissue
specific to the target cells. The cell based delivery system is designed to be
implanted in a patient's body at the desired target site and contains a coding
sequence for the active agent. Alternatively, the agent could be administered
in a
precursor form for conversion to the active form by an activating agent
produced
in, or targeted to, the cells to be treated. See for example, EP 425,731A and
WO 90/07936.
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EXAMPLES
The following examples further illustrate the present invention. These
examples are intended merely to be illustrative of the present invention and
are
not to be construed as being limiting.
EXAMPLE 1
Animal care and maintenance. All animal protocols used in these studies
were approved by the Monell Institutional Animal Care and Use Committee. Mice
were housed in individual cages in a temperature- controlled room at
23°C on a
12-hour light:l2-hour dark cycle. The animals had free access to deionized
water
and Teklad Rodent Diet 8604 (Harlan Teklad, Madison, WI).
EXAMPLE 2
Breeding of F2 and partially congenic mice. C57BL6/ByJ (B6) and
129P3/J (formerly named 129/J; abbreviated here as 129) mice were purchased
from The Jackson Laboratory. The B6 and 129 mice were outcrossed to produce
the first filial generation of hybrids (Fl), and these were intercrossed to
produce
the second hybrid generation (F2, n = 629).
To create the partially congenic lines, the F2 mice were genotyped with
several markers on the distal part of chromosome 4, and a few F2 mice with
recombinations in this region were used as founders of strains partially
congenic
with the 129 strain. These F2 founders were backcrossed to the 129 strain to
produce the N2 generation. Mice from this and subsequent backcross generations
were phenotyped using 96-hour two-bottle tests with saccharin solutions, and
genotyped using markers on distal chromosome 4 and on other autosomes. Mice
with high saccharin intake (with a fragment of distal chromosome 4 from the
B6 strain and homozygous for 129 alleles of markers on other chromosomes) were
selected for subsequent backcrossing. This marker-assisted selection resulted
in a
segregating 129.B6-Sac partially congenic strain. Three strains were created,
with
different overlapping fragments containing the SACl gene.
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EXAMPLE 3
Testing of sweet preference in the F2 and partially congenic mice.
Consumption of 120 mM sucrose and 17 mM saccharin (Sigma Chemical
Company, St. Louis, MO) was measured in individually caged mice using 96-hour
S two-bottle tests, with water as the second choice. The positions of the
tubes were
switched every 24 hours. Fluid intakes were expressed per 30 g of body weight
(the approximate weight of an adult mouse) per day, or as a preference score
(ratio
of average daily solution intake to total fluid intake, in percent).
EXAMPLE 4
Genotyping of F2 mice and linkage analysis. Genomic DNA was
purified from mouse tails by NaOH/Tris (Beier, personal communication;
Truett G.E. et al., Preparation of PCR-quality mouse genomic DNA with hot
sodium hydroxide and tris (HotSHOT) [In Process Citation]. Biotechniques,
2000;29:52, S4), or the phenol/chloroforzn method. All F2 mice were genotyped
1 S with all available polymorphic microsatellite markers (Research Genetics,
Huntsville, AL) known to map near the SAC 1 region with a protocol modified
slightly from that described by Dietrich W. et al., A genetic map of the mouse
suitable for typing intraspecific crosses. Genetics, 1991;131;423-447. The
maxkers
tested are as follows: D4Mit190, D4Mit42, D4Mit2S4, and D4Mit2S6. Analysis of
this framework map using MAPMAKER/QTL 1. I (Lender E. et aI.
MAPMAKER: An interactive complex package for constructing primary linkage
maps of experimental and natural populations. Genomics, 1987;1:174-181),
indicated that Sac mapped distal to D4Mit2S6, and therefore all available STS
and
EST were tested by SSCP (Orita M., Iwahana H., Kanazawa H., Hayashi K., and
2S Sekiya T. Detection of polymorphisms of human DNA by gel electrophoresis as
single-strand conformation polymorphins. Proceedings of the Natio~zal Academy
of Sciences of the USA, 1989:86) or direct sequencing, for polymorphisms
between the B6 and 129 strains. Polymorphisms between strains were found for
the following maxkers: D18346, AA410003 (K00231), V2r2, and D4Erdt296E,
and the linkage analysis conducted again using these polymorphic maiCers.
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EXAMPLE 5
Genotyping of partially congenic mice. Three partially congenic strains
of mice were genotyped with all available markers, and those markers with two
129 alleles were excluded from the SAC 1 nonrecombinant interval.
EXAMPLE 6
Radiation hybrid mapping. To generate additional markers to narrow the
Sac nonrecombinant interval, several markers were tested using the T31 RH
genome map. Primers from several sequences suggested through survey of the
public databases were constructed and DNA from the T31 panel. Results were
scored using software at the Jackson Laboratory.
EXAMPLE 7
Construction of BAC contig and marker development. To construct a
physical map of the SAC1 region, the RPCI-23 BAC library was screened with
markers within and near the SAC1 nonrecombinant interval: each marker was
tested by whole cell PCR to confirm its presence. Only those markers positive
by
both hybridization and PCR are shown. Primers for the BAC ends were
constructed from sequence obtained through TIGR (www.ti~r.org) or by direct
sequencing, when necessary. Each positive clone was tested for the presence of
each BAC end (if the BAC end contained unique sequence), and the contig
oriented using SEGMAP, Version 3.48 (Green E.D. and Green P. Sequence-
tagged site (STS) content mapping of human chromosomes: theoretical
considerations and early experiences. PCR Methods Appl., 1991;1:77-90). BAC
end sequences was amplified in B6 and 129 strains, and analyzed by SSCP or
direct sequencing. Those BAC ends polymorphic between 129 and B6 were tested
in the recombinant F2 and partially congenic mice, to further narrow the SAC 1
nonrecombinant interval.
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EXAMPLE 8
Amplification of SACl and polymorphism detection. After the SAC1
nonrecombinant interval was narrowed to less than 350 kb, a 246 kb BAC was
chosen for sequencing which spanned most of the region (RPCI-23-118E21).
Within this BAC, there was a gene with homology to other taste receptors.
Using
11.8 kb of sequence and the program GENSCAN (Barge C.B. and Karlin S.
Finding the genes in genomic DNA. Current Opinion Structural Biology,
1998;8:346-354), a 858 amino acid protein, with 6 exons, was identified.
Primers
were constructed that amplified this gene, and an additional 2600 nt upstream
and
5200 nt downstream were also amplified (primer sequence available upon
request). These PCR products were sequenced using genomic DNA from B6 and
129 mouse strains, as well as other strains with either higher (SWR/J, C57L/J,
IS,
ST/bJ, SEA/GnJ) or lower (DBA/2J, AKR/J, BALB/cByJ) saccharin preference
(Lush LE., The genetics of tasting in mice. VI. Saccharin, acesulfame, dulcin
and
sucrose. Genet Res., 1989;53:95-99; Lush I. The genetics of bitterness,
sweetness,
and saltiness in strains of mice. in Genetics of perception and communication,
Vol. 3, eds. Wysocki C. and Kare M., New York: Marcel Dekker, 1991:227-235;
Lush LE. and Holland G. The genetics of tasting in mice. V. Glycine and
cycloheximide. Genet Res., 1988;52:207-12). Sequences were aligned with
Sequencer (Gene Codes, Ann Arbor, MI) and the single nucleotide
polymorphisms, insertions and deletions identified.
EXAMPLE 9
Preparation of tongue cDNA and expression studies. Total RNA was
extracted from anterior mouse tongue from the 129 and B6 strains (TRIZOL
Reagent; GIBCOBRL). Total RNA (200 ng) was reverse transcribed using the
Life Technologies Superscript Kit. Following the reverse transcription, the
samples were amplified using Advantage cDNA PCR Kit (Clontech, Palo Alto,
CA). Primers were constructed to span exon 2 and 3, so that the genomic and
cDNA product size would differ (Primer set 3A;
Left-5'TGCATTGGCCAGACTAGAAA3';
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Right-SCGGCTGGGCTATGACCTAT'). The expected product size for primer
3A is 418 by for cDNA and 497 by for genomic DNA. Single bands of these sizes
were excised from the gel, purified and sequenced, confirming the intron-exon
boundary and expression of mRNA of this gene in mouse tongue. Primers were
then designed to cover the whole cDNA, and, the sequences obtained and
aligned,
to confirm intronlexon boundaries.
EXAMPLE 10
Human gene expression. The human ortholog of the SAC1 gene was
examined for mRNA expression in human tongue. Total RNA from human taste
papillae was obtained through biopsy, a procedure approved by the Committee on
Studies Involving Human Beings at the University of Pennsylvania. The RNA was
extracted as described above, reverse transcribed, and amplified, with human
specific primers. Two bands were obtained of the expected size for genomic and
cDNA. Sequencing of these bands co~rmed the SAC 1 gene is expressed in
human taste papillae.
EXAMPLE 11
Tissue Expression of SACl. Oligonucleotide primers specific for
different parts of the SAC1 gene were used to assay different tissues for SAC1
expression as shown in Table 2. Tissue specific cDNA pools were purchased from
OriGene Technologies Ltd. Primer pair 3A, amplifies parts of exons 2 and 3,
with
a small intron to differentiate between PCR product representing genomic DNA
versus cDNA. Primer pair 6A amplifies parts of exons 4 and 5. This part of the
protein encodes the 7TM domain, and may cross react with other GPCRs
expressed in different tissues.
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Table 2. Expression pattern of SAC1
Tissue 3A 6A
Brain - -
Heart - -
Kidney + +
Spleen + +
Thymus + +
Liver - +
Stomach - +
Sm Intestine - +
Muscle - +
Lung - +
Testis + +
Skin - -
Adrenal + -
Pancreas + +
Uterus - -
Prostrate + +
Embryo-8.5 - -
9.5 - -
12.5 - -
19 + -/+
Breast-virgin - +
Pregnant - +
Lactating + +
Involuting - -
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EXAMPLE 12
Primers for the SACl Locus (Seq. ID Nos.: 6-651) are:
Marker Forward Reverse Size, SEQ.
by ID
NO.
28.MMHAP7B4.CACTAGAGCTGCC CCCTCAGCACCA 162 6-7
seq ACCTTCC CTTTTTGT
28.MMHAP7B4.ACAAAAAGTGGTG CAGGAGACCCA 163 8-9
seq CTGAGGG AAGGATCAA
AA408705 GCTTCAGAAAATC GCATGGGCTATG 232 10-11
GAGGCAC ATAGGTGG
AA408705 TGTTGATCCCACA CAGGAAATGTCC 12-13
GCG ACTTCTGC
AA409223 TCTATCTTGCATC GTGCTGTGACTG 14-15
CAGCC TGCG
AA589460 CGCAGCATTTATT CCGACCCTTTAG 16-17
TGGAG GAGACAC
Agrin4 TGTGACTTCCTCTT TGAGCCACTCCA 156 18-19
CCCCAC GATGTCAG
Agrin4 GTGTGTCAGCATC CCAACGTGCAGT 290 20-21
ACTGCCT CAAGAAAA
Agrin4 CGAGAGACAAAG TTATGAAGGCCC 263 22-23
TGGTGCTG TCACCAAC
Agrin4 CCAGCTCCTAGAA GCAGTCTGCCGA 298 24-25
TTGCCTG AACAAGTC
Agrin4 ATAGAGGAATGG TACCAGGAGGG 299 26-27
GTGCGATG GTCAGTCAG
Agrin4 TACAAGCGAGCTG CCAATCAGCTCG 271 28-29
ACCAATG AGTTAGCC
AgrinA TGCCATTGTGGAT GAGTCCGAGGTC 575 30-31
GTTCACT GGTCAATA
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Marker Forward Reverse Size, SEQ.
by ID
NO.
AgrinB GCTGGCTTCTGTA TATGAGGGTCAA 577 32-33
GGTCAGG GGGTCAGG
AgrinC CGCTTTGGTGAGA CATGTGGAGTTG 573 34-35
ACTAGCC TGGGAGTG
AgrinD AATGGGCAGAAG TATCAGGGTCTG 507 36-37
ACAGATGG TGAAGCCC
AgrinE ATACAGGACCCTT CAGTGTTTCTAG 587 38-39
TACCCCG GTCCCCCA
Agrin GCCTCTGTCTGCC ATAATGTTACCT 594 40-41
ATCTCTC GCAGGCGG
AI115523 CTGGAAACACCCA CGGGCACATGG 200 42-43
TGTCCTC ACACTTTTA
AI225779 GAGCATGAAGTGC CGTAGGTGGCAC 266 44-45
AAGGTGA AGTTGAGA
AI225779 GCTGTTAGTGAGG CGTAGGTGGCAC 104 46-47
TCAGGGC AGTTGAGA
AI225779- GAGCATGAAGTGC TCATTTTCCTAG 126 48-49
AAGGTGA CCTCGGTG
A022703 TCTAAGAAGATGA TGTCCTTCAGGG 50-51
TGCAGACCC ATAGTGCC
Cdc212 GGCTTCAGCCTCA AAAACAACCAA 101 52-53
AGTTCTG GTTGCCCTG
Cdc212 GGCACTGAAATGA AACAATTCAAGC 265 54-55
CCTGGAT AACCTCGG
Cdc212 CTGTTCCTTCCCA TTCAGTCACGCA 225 56-57
GACTCCA AACCTGAG
Cot GCCCAGGACTTTG GGTAACCTGCAG 284 58-59
TCACTGT CTCCACTC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
Cot GGGACATGCTCTT GAACAAAGCCG 277 60-61
GGTTCAT GGTGATTTA
Cot GCCCTCAGTTCTC GGCAGAGAAGA 110 62-63
CTAGCCT CTGGTGGAG
Cot CCCAGACTTAGCG AGCAGAGACCTT 277 64-65
TCTCAGG TGGACTCG
Cot GAAGGCTGAGTGA TTGCACGAGGAG 276 66-67
GTCCCAG AAGGTTTT
Cot GATGCCAACGAGA AGAAGCCAAAA 247 68-69
CCTGAAT CCCTCACCT
Cot AAAAAGCCCTGCA ATTCAGGTCTCG 107 70-71
AGAACTT TTGGCATC
D18346 TGTCCGCAGTGTG ATGTCCAGGGTA 165 72-73
GAAACTA GAGAGCCC
D 18402 GGAGTTCTCCTAC GAGGCTCTGAGC 167 74-75
CCTGGCT AGTGTCAA
D4Bir1 GCGATGTTGTTG CAGTGTCTTTCC 76-77
CG ACATTT
D4Ertd296e AGGCATATTGTAT CCGGATGACTCT 201 78-79
AATAAATTTGTA ACTTGAC
GT
D4Hrb1 GCTGTTTATGGGG AATTTCTGAAGC 194 80-81
TCGAGAA AGGGGGAT
D4Hrb1 TCCCCCTGCTTCA AGGGGGATGATT 192 82-83
GAAATTA GTGAGTGA
D4mit313 CTTCTTTAATCAA GGGCACATATGA 196 84-85
TCTCTGTCTCTGTG ACCTCCTG
D4mit344 CCAAACTCTTAGC ACACAGAAGAC 187 86-87
TTCTTCA ACTGAAGAAC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
D4Mit51 CAGTTGTTAGAAG AGGTGCATATAC 123 88-89
CAGGATCCC CTGGGATACTC
D4Mit59 AGAGTTTGGTCTC TATCCAACACAT 108 90-91
TTCCCCTG TTATGTCTGCG
D4Mit59 GCCAGTGTGCTGA AGGGACCTGGA 119 92-93.
AAGACTG GACATCCTT
D4Nds16 CTGTAGGCTGCTT TGCCCCTTCAGC 94-95
TTATCTTTTG ACATGCCA
D4smh6b TGCAGTGTGACAT GGAAAGCCAGG 118 96-97
GTGCATAGAT CTACGCAGAA
D4smh6b CTGTAGGCTGCTT TGCCCCTTCAGC 102 98-99
TTATCTTTTG ACATGCCA
D4smh6b TAGTGTGGTTCCT CGGTCTACATAG 181 100-101
GACTAACCT TGAGTGATTC
D4smh6b AAA.AGCATCCTGC GGGTTATACAGA 83 102-103
ATCCTTCTG GAAACCCTGT
D4Xrf215 TTCCAAGCTCACA GTGCTGCTCTGC 124 104-105
CATCAGC ATTGAGTG
D4XrfZ43 GACAGTGTGGGAG CCCAAGGCATAG 203 106-107
AATCCGT GTCACAAT
D4Xrf243 ATTGTGACCTATG CGAAGGACCGTC 105 108-109
CCTTGGG ATCTGAGT
D4Xrf472 GGCTTTGATGTGA AGCTCCTCATCG 245 110-111
AAAAGGC CTCATGTT
D4Xrf472 TGGAACATCTCTG GGCTCTCATTGC 193 112-113
TCGGAAG CACCTTTA
D4X497 CCAGAGAACAGG GTGCTGGATACA 119 114-115
AGACCTGC CTGGCAGA
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Marker Forward Reverse Size, SEQ.
by ID
NO.
D4Xrf497 GCGAGACGAGTG ACACTGAAACCT 129 116-117
GGTAGTTC CGCTTGCT
D4Xrf497 AGCAAGCGAGGTT ACGGGGCTTGAT 204 118-119
TCAGTGT CCTTTTAT
Dshv4 AAGTTCATGGGCC TACTAGCTACCC 100-300120-121
TCACCACCTGTC TTCACATACC
DshvS ACCTAGCCACTGT ACAGAAGCAGC 100-300122-123
CTCAGTCT ATTTACACAG
Gnbl TGGGACAGCTTCC AATGGGAATTGT 213 124-125
TCAAGAT GCTCTTGG
Gnbl GGGCATCTGGCAA AGATAACCTGTG 281 126-127
AGATTTA TGTCCCGC
Gnbl GATGTCCGAGAAG TGTCAGCTTTGA 277 128-129
GGATGTG GTGCATCC
Gnbl ACATGCAGGCTGT TGTCAGCTTTGA 166 130-131
TTGACCT GTGCATCC
K00231 GTGCTCTGCAGAC GAGCCATTTTGA 154 132-133
AAACCAA CCCTTAAA
K00231 TTTCAGGGTCAAA TCGACAGCAACT 134-135
ATGGCTC GTGCG
K00954 GGTGAGAGTGGG CCCGGGTGAGTT 237 136-137
GAGATGAA TAAGAACC
k00954 GGTGAGAGTGGG AGGTTAGGCCCA 296 138-139
GAGATGAA ATTTCCTG
k00954 CCAGGGTTGCTGT CAGGTTAGGCCC 237 140-141
ACTGAGA AATTTCCT
K01153 GGTCAGAGTCCTT TCCAACTTCACA 124 142-143
CCTTCCC GGAAACCC
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Maxker Forward Reverse Size, SEQ.
by m
NO.
K01153 TTTCCTGTGAAGT CACCCATATGGC 213 144-145
TGGAGGG AAACATCA
K01153 GGTCAGAGTCCTT TCCAACTTCACA 125 146-147
CCTTCCC GGAAACCC
K01153 TGATGTTTGCCAT GCTTGCTGCTTC 181 148-149
ATGGGTG CGATATGT
K01599 GGAAAAGGGAGT GAGCCGCCTAAC 166 150-151
CGCCATA TCTCACAC
K01599 AGGGGATAACCTG ACAAAATTGCTC 110 152-153
CATAGG ATTTGCCC
M-05262 CCATCCCCACTAG GTCCCCTTTGTC 169 154-155
CCAGATA ACAGCAAG
M107-HO1 TGAGCACAGGATA AAAAGAACACC 217 156-157
GCTCCAC TGTTTGGGG
M111-B04 TAAACCTCGGCTG CCCTCAGTGACT 267 158-159
TGTGAG TCCTGTGA
M134-C06 CAAAACCACATGG GCCCTATTGCCA 264 160-161
TTACCGA AATGACTT
M134-GO1 GGCAGAAAGGAA CACATTAGCCAT 161 162-163
TCAGAAGC TGTCCTGG
M136-BO1 TCCTTTATGTCCA CATGGTCTGTGA 164 164-165
ACAGCCA TGTGACCA
M156-HOS ATACCCTTGGTGA GCTGTCAAATGA 139 166-167
GAGCAGG GAAAGGCA
M184-B03 TATTTCATGCTGG AGAGAAAAACA 89 168-169
GACCAAA GTGGGGGTG
Mmp23 CGGGTCCTCTCTT CTACATTTCCCT 297 170-171
CACCATA GAGCTGCC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
Mmp23 GTTGACCATGTCG CCACCTCACGGA 111 172-173
GTAACCC AACTGAAT
Mmp23 GGTGTTTGGCTCA GATGCACACACA 197 174-175
CAAACCT AAAATCCG
Mmp23 ATCACCCACCAGA ACCCTCCAGGAG 255 176-177
ACGAAAA TAGGTGCT
PCEE GATGAGACAGTGG TTGTCAATAGCA 154 178-179
GCAAGGT CCAAGCCA
PCEE GCCTTAATAGCCC GCACTCAGCATT 194 180-181
CCTTGTT GCACAGAT
PCEE GGACGGACAATTC CTATCACACCTC 142 182-183
TGGAAAA CGATGCCT
PCEE CAAGCTGGTAGAA TCTTTGGAGAAG 209 184-185
TCCCCAA CAGACCGT
Pkcz TACAGCATATGCA ATTCCTCAGGGC 294 186-187
TGCCAGG ATTACACG
Pkcz GCAATCTCTTGTG ATTCCTCAGGGC 188 188-189
TCCAGGC ATTACACG
Pkcz TACAGCATATGCA GGCCTGGACACA 127 190-191
TGCCAGG AGAGATTG
Pkcz AAGTGGGTGGACA CAGCTTCCTCCA 201 192-193
GTGAAGG TCTTCTGG
Pkcz AGAGCCTCCAGTA TCGTGGACAAGC 297 194-195
GATGGCA TCCTTCTT
Pkcz CATCGAGTATGTC TTGTCCAGTTTT 156 196-197
AATGGCG AGGTCCCG
Pkcz CAGACTGGGTTTT GTCAAAGTTGTC 132 198-199
CCGACAT CAGGCCAT
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Marker Forward Reverse Size, SEQ.
by ID
NO.
Pkcz AGGACGGACCCCA TGTCTCGCACTT 130 200-201
AGATG CCTCACAG
Pkcz CCAGAAGATGGA TCTACTGGAGGC 151 202-203
GGAAGCTG TCTTGGGA
Pkcz GAAAAACGACCA GATCTCAGCAGC 265 204-205
GATTTACG ATAGAACC
Pkcz ACACATTAAGCTG CAAACATAAGG 164 206-207
ACGGACT ACACCCAGT
Pkcz ACTGGGTGTCCTT CCTCTCTTTGGG 193 208-209
ATGTTTG ATCCTTAT
Pkcz GTCATAAAGAGGA GCTCTGTCTAGA 252 210-211
TCGACCA AGTGCCTG
Pkcz ACCAAGACCGAA GGCATTACACGC 223 212-213
GAGGGG TAACTTTTCC
874924 AGTGCCACCAACC AAGTGCCTGCAG 165 214-215
TGGTAAG GGATGC
874924 TGCTTTGGTGAGC AGGGACACCCTT 103 216-217
AATGTTT ACCAGGTT
874924 CTGATGCTTTGGT GGGACACCCTTA 218-219
GAGCAAT CCAGGTT
875150 ACAGGACAAATGC GTGGTAAAGAA 217 220-221
TGGGTTG CGCTTGGCT
875150 GGTATCTCACTTG AAGAACGCTTGG 222-223
GTAGGAACCTC CTGGC
RERl (1) GCCGATCCTGGTG ACAATGGCTCAA 224-225
ATGTACT AACCGTTC
RER1 (2) GCCTTGGGAATTT AGTACATCACCA 226-227
ACCACCT GGATCGGC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
RERl TAAAAGGCCATGC AGAGCTCTGTGG 228-229
GATAAGC GGTTCTCA
RERl GAAGGGGACAGT TCCATCAAGGAA 230-231
GTTGGAGA GGATCCAC
Tp73 GGTGGGTAATGAT TGACGTGGAGG 296-301232-233
TGGACT GAACTGCC
Tp73 TGAGATCTGGTGC GCCTGATCTAGG 222-229234-235
CCTCTCT CTGGAAAA
Txgpl AGGCAGAAAGCA CGACAGCACTTG 138 236-237
GACAAGGA TGACCACT
Txgpl CTGCAGATGTAGA CTGTGGTGGATT 269 238-239
CCAGGCA GGACAGTG
Txgpl TTGCCTAACACTC TATTAGGAGCAC 244 240-241
CCAAACC CACCAGGC
Txgpl ACCTGTCTTGTGG CTGTGGTGGATT 242-243
GTGGAAG GGACAGTG
U37351 GTGGCTTGGTGCT GGGGCTATTAAG 160 244-245
ATTGACA GCCATTTT
V2R2 CAATTGAGGAATG TGGCTTCATGTC 170 246-247
GCTACCAA CATTGTGT
V2R2 CAGAACCACAAA TCATGTTTGCTG 163 248-249
GGTAAATTGC TCCAGTTTG
TR1-likel(humaGCCACCATGCTGG TCACTCATGTTT 2520 250-251
n) GCCCTGCTGTCCT CCCCTGATTTCC
GGG
Tl-ike2(human)CTGATTTCCTGTG CATGCTGGCCTA 244 252-253
TTCCCGT CTTCATCA
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Marker Forward Reverse Size, SEQ.
by ID
NO.
Tl-like3(human)GCCTTGCAGGTCA TCACTCATGTTT 2441 254-255
GCTACGGTGCTAG CCCCTGATTTCC
CAT
T1-like4(human)AGGAAGCAGAGA TCAGAACTGCCT 274 256-257
AAGGCCAG CTGAGCTG
T1-like5(human)TCTTCACGTACTG ACTACAGCATCA 175 258-259
GGGGAAC GCAGCAGG
T1-like6(human)AAGCTGAAGAACT TGGGCTACGACC 211 260-261
TCCCGGT TCTTTGAT
h-Trllike ATCTTCAGGCGCT GTACGACCTGAA 262-263
a
CTGTCCT GCTGTGGG
h-Trllike ATCTTCAGGCGCT GTACGACCTGAA 264-265
b
CTGTCC GCTGTGGG
h-Trllike ATCTTCAGGCGCT GAGTACGACCTG 266-267
c
CTGTCC AAGCTGTGG
h-Trllike ATCTTCAGGCGCT TACGACCTGAAG 268-269
d
CTGTCCT CTGTGGG
h-Trllike ATCTTCAGGCGCT TACGACCTGAAG 270-271
a
CTGTCC CTGTGGG
h-Trllike GCTGTCCCGATGG ACCTTTTGTGGC 272-273
TGAAC CAGGATG
h-Trllike GCTGTCCCGATGG CACCTTTTGTGG 274-275
g
TGAAC CCAGGAT
h-Trllike GCTGTCCCGATGG CCTTTTGTGGCC 276-277
h
TGAAC AGGATG
h-Trllike CCTGAACCAGTGG ACCTTTTGTGGC 278-279
I
GCTGT CAGGATG
h-Trllike CCTGAACCAGTGG CACCTTTTGTGG 280-281
j
GCTGT CCAGGAT
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Marker Forward Reverse Size, SEQ.
by ID
NO.
h-Trllike TCATGTTTCCCCT CATGCTGGCCTA 282-283
k
GATTTCC CTTCATCA
h-Trllike ATGAGCAGGTAAC TCATCACCTGGG 284-285
ACCTGGG TCTCCTTT
h-Trllike ATGAGCAGGTAAC TTCATCACCTGG 286-287
m
ACCTGGG GTCTCCTT
mTrllike-lA TGGGTTGTGTTCT CCTTTTTACAGT 288-289
CTGGTTG CTGCCAGGT
mTrllike-1B TGGGTTGTGTTCT GATCCCCTTTTT 290-291
CTGGTTG ACAGTCTGC
mTrllike-2A ACGGGGTTGGTAC CACCCATTGTTA 292-293
TGTGTGT GTGCTGGA
mTrllike-2B ACGGGGTTGGTAC CACACACCCACC 294-295
TGTGTGT CATTGTTA
mTrllike-3A TGCATTGGCCAGA CGGCTGGGCTAT 296-297
CTAGAAA GACCTAT
mTrllike-3B TGCATTGGCCAGA CGGCTGGGCTAT 298-299
CTAGAAA GACCTATT
mTrllike-4A GTTCTGCAGCATG GGCAGTTGTGAC 300-301
ATGTCGT TCTGTTGC
mTrllike-4B GTTCTGCAGCATG CTGCAGGCAGTT 302-303
ATGTCGT GTGACTCT
mTrllike-SA CCATCCTTTTTGCC TCTGGAGGAACA 304-305
TGTCTT TGTGATGG
mTrllike-SB CACCATCCTTTTT GAACATGTGATG 306-307
GCCTGTC GGGCAAC
mTrllike-6A CAAAGCAGCAGG AAATGTACTGGC 308-309
AGGAGTG CAGGCAAC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
mTrllike-6B AGTGCTAGACCCA AAATGTACTGGC 310-311
GCACCAG CAGGCAAC
mTrllike-7A GCACTGACCAGTC GTCCCCAGAGAA 312-313
TGTCACC AAGCACAG
mTrllike-7B CAGTCTGTCACCA CAGTGGTCCCCA 314-315
CCTCTGG GAGAAAAG
mTrllike-8A TACTATTCGGGGC GCAGCACTATGT 316-317
TTGTTGG GCCTGGTA
mTrllike-8B TACTATTCGGGGC GCCTGGTATTTG 318-319
TTGTTGG ATCGCTTT
mTrllike-9A GCTCAGCTAGGGA CAGCTCAGGGAC 320-321
TGGAGAA ACAATGAA
mTrllike-9B TCCTACAGGCTAG CAGCTCAGGGAC 322-323
GGCTCAG ACAATGAA
mTrllike-l0AGGGACTGATGTGT AGGCGTCCCAGG 324-325
GGCTTGT AATAGAAG
mTrllike-lOBGGACTGATGTGTG AGGCGTCCCAGG 326-327
GCTTGTTT AATAGAAG
mTrllike-11ATGTTTCTGTTCTGG ATCTGCAGGCAG 328-329
TGGCTG GATCAGAC
mTrllike-11BCTCAGTGGTGGGT ATCTGCAGGCAG 330-331
GACAGTG GATCAGAC
Mutationl ACACACAGTACCA CCTGTGGTGATC 182 332-333
(mouse) ACCCCGT AAGAAGCA
Mutation2 TGCTTCTTGATCA GCAACAGAGTC 131 334-335
(mouse) CCACAGG ACAACTGCC
Mutationsl+2ACACACAGTACCA GCAACAGAGTC 293 336-337
(mouse) ACCCCGT ACAACTGCC
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Marker Forward Reverse Size, SEQ.
by ID
NO.
34m15-T7 GGGTTTATGTGGC ACTCCATTTGCC 118 338-339
AAGCACT TTTTGTGG
34m15-SP6 CGCTACTTCGCTT ATGATGACGTAC 150 340-341
TTATCCG GACGACGA
37D20-T7 GAAAACAATCGG TGAAATTATCAC 109 342-343
GGAGAAGTC ACGCCAGG
37D20-T7(3)*AGTGAGAGGCCCA GATCTGATGCCC 247 344-345
GTCTCAA TCTTCTGC
37D20-SP6 GCTAGCCTTGAAG TGAACAGCATGC 122 346-347
CCAACAC TTACCCAG
4902-T7 TCCCTAGAGGCCT TCGTCTCGGAGC 169 348-349
GTCTGTC CTCTTCTA
4902-SP6 GATAGTCCCTTAG GCCATAGCTCCT 218 350-351
CCAGCCC CACTGCTC
73B10-T7 CAGAGTGGGCTCT TTGTGTTCAGAT 237 352-353
GGTCTTC GCTCCTGC
73B10-SP6 TTATTTCTGTGCTA ATCAAGTCAACG 267 354-355
GCCGCC TCCCCAAG
75M14 ACCTGGCCTGTGC GCACCAACCCTA 233 356-357
TAATCTC AGAAAGCA
85618 TCAGGCTAACCTC AAAGAAAAGAA 113 358-359
AAACTCACA AAGAAAAAGTC
AGACA
118E21-T7 CCCAGAACTCCAT CCCAACCTGTGG 185 360-361
CCTCAAA TCAGCTAT
118E21-SP6 GGGGCAGGTGGGT CAAAAGCCCAA 271 362-363
AATAAGT CTCCTTGAG
130A12-T7* GCTCAGTGGGTAA CTACCCTGCCGC 242 364-365
GAGCACC TAATCTCA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
_87_
Marker Forward Reverse Size, SEQ.
by ID
NO.
130A12-SP6 CAGTTAGCACCCC TCTGCACCTCTG 114 366-367
ACCCTAA TTCACCTG
138D7-T7 ACCTCTAGGGTTT CCTCAGGTAGTG 199 368-369
ACGGGGA CAAGCTCC
139J18-T7 TCAGTTACCAAGG ATAGGTTGTCAC 122 370-371
GTTTCGG AGGCCAGG
139J18-SP6 TCAGTTACCAAGG ATAGGTTGTCAC 122 372-373
GTTTCGG AGGCCAGG
147a15-T7* GTGGTTGCTGGGA CAAGCAACCAA 101 374-375
TTTGAAC ACAACCAAA
147A15-SP6 TCCGGAGGACCAT CACAGTCCCAGT 249 376-377
AAATCTG CATTCCCT
151E4-T7 GTCCCAAAAGCTA TCATGAGCCACC 240 378-379
GCACAGG ATGTGATT
151E4-SP6 GACCTTCGGAAGA AGTGTGTGTCGC 223 380-381
GCAGTTG CATATCCA
15203-T7 CCTACTCTCTCTCC GGAAAATGTTTG 142 382-383
CCGCTT GCCTTGAA
15203-SP6 CTGGAGTGAAAGG AGGCGGCACCAT 537 384-385
CAGGAAG ATGAATAA
153B21SP6 TGAGAGTGGGAAT GGATGTAATTGG 202 386-387
TCTGTTCA TGGCAAGG
153B21T7 CTGTTGGAGGAGG TGCTTGTATGTT 113 388-389
TGGCCTA TTTCCTCGT
159J19SP6 TGAGAGTGCCCTC GAACCCCTGACC 200 390-391
CTCTTTG CCAGAC
159J19T7 TGAAGTGCAGATT GTTTTGGGGTGG 213 392-393
TTTACATGG AAAAGGAT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
_88_
Marker Forward Reverse Size, SEQ.
by ID
NO.
189M12SP6* CCGTCGACATTTA GATAGTGGGGTG 189 394-395
GGTGACA GTGGGTAA
22764-SP6 CCGTCGACATTTA CGTCCCAGCTGT 219 396-397
GGTGACA GTAACTGA
22764-T7'~ GGAAGCAAATGCT TATCCCTAGCCC 243 398-399
CCACTAAA CTTGTGTG
236C12-SP6 CCGTCGACATTTA GGGTCCTGTTGG 209 400-401
GGTGACA TAGTGACC
238OST7 TATAAGCAGCCCC CAGGCCAGACA 244 402-403
TCATTGG CTGCTTACA
238OSSP6 CCTTGGGATCTGG TGGGTTTAGAGT 2S1 404-405
TGTGACT ACGGCTGG
24718-T7 ACCCATTTCCTAA ATCTCTCCAGCC 177 406-407
TCCCCTG CCTCTCAG
280612-T7* GGGCTGGGAATTG TGAATCCCTTAC 420 408-409
AACCTAT AGCCTTGC
280612-SP6 GCCCCATAAAATC GCTCCGGAAGGC 233 410-411
CACTCCT TAGAAGAT
284D21-T7 GGTTTGGGAGTGT ACTCAGTTGGCC 138 412-413
TAGGCAA TGTCCTCA
284D21-SP6 ACAGAAATCCCTC TCAGTGTGGACC lOS 414-41S
ATGCGA AGAAAGTCC
298E4 TCTGCAAGTCAGC ACTCATAAGGGT 100 416-417
TCTTGATAA CAAGCTGTCTG
298e4-T7(3)*TCTCCCCTTTTACC GCAAGGAGTCA 180 418-419
ACTCCC AAAACAGCA
307E5 GCTAGTTGGGGAA ACTGCAAATGTC 149 420-421
CAAACCA CAACTCCA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-89-
Marker ~ Forward Reverse Size, SEQ.
by ID
NO.
338N4-T7 CAGTTACACAGCT GCAAGAGCCTA 245 422-423
GGGACGA GCAATCCAC
338N4-SP6 CAGTTTAGCACCC TCTGCACCTCTG 115 424-425
CACCCTA TTCACCTG
348P19-SP6 GGGTTCCACTTGA TGGTCTGTTTCC 227 426-427
TGCTGAT TGGAGCTT
350D2-T7* TGTAGGGAATGTT ACATGGAACAG 295 428-429
TCTGCACC GATTCTGGC
350D2-SP6 GCAGGCAAACAG ATGGGGGATCCC 217 430-431
ACAGACAA TTACTGAC
360M12-T7 CGGTCAGGAGTAG CAGCAGCTGATA 123 432-433
TGTGGGT TTGAGGCA
360M12-SP6 AATGATGAAGTGT CAACAGAACTCA 100 434-435
CAGCCTCAG AAGCCTGG
382A8-SP6 AGCAGGCACAGGT AAGAACAGGAC 202 436-437
CTCTTGT AGTGGTGGG
382A8-SP6(2)CAGCGATTGGCTC GGGGCTTCCTTT 531 438-439
TTCTCTT CTGAGGTA
386N4-T7 AGCTCAGGTCCAG ATTTTCCCCTCC 107 440-441
CTTGGTA TGCTTCTC
386N4-SP6 CCAAGCCTCTGCT TGAGGGTGGAG 109 442-443
GGTTATC AATGGAAAG
387-T7 GCCCCATAAAATC TTGCCTAACACT 214 444-445
CACTCCT CCCAAACC
387-SP6 CAGTTACACAGCT GCAAGAGCCTA 245 446-447
GGGACGA GCAATCCAC
388I1 CAGCACCTTCCTC TGTCTCCAGAGG 137 448-449
TGGTCTC TTCTGCCT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-90-
Marker Forward Reverse Size, SEQ.
by ID
NO.
399I12-T7 TGGTGGTGTAATA TCTTTAATTTTT 102 450-451
CTATTCCTTTG GGCTTTTTGATA
CA
399I12-SP6 CAGCTGTGTGCAT CATCATGAAGAC 106 452-453
GTTGACC TCAGGGCA
415A22SP6 GTCCACACCTGGC CAGCACTCAGTG 199 454-455
TTTTGTT AGGTTCCA
415G24SP6 ATGTAATGGAAGG CAGCACTCAGTG 113 456-457
GCTGCTG AGGTTCCA
417B22-SP6 AAACAGGCATGA GGGTATCATTGT 116 458-459
AACTCAGGA CACCTCCA
436P10-T7 CACAGGCCAAGTT CAGGGGACCTTC 115 460-461
GTTGTTG TGAATGAT
438C18-T7 AGCTCAGGTCCAG ACCACAAAATTT 115 462-463
CTTGGTA TCCCCTCC
438C18-SP6 CGGGACCTAAAAC TGGGGACAGTTA 254 464-465
TGGACAA CCAGGAAG
457N22-T7 CCGGAGGACCATA CCTCAAAAACAA 129 466-467
AATCTGA GCCTGAGC
457N22-SP6 CCTTCAGAAATGT TCCTGAGTTCAA 252 468-469
GTTTGGACA ATCCCAGC
472018 CTTTCCATTCTCCA AGGTCCTAGGGA 260 470-471
CCCTCA GAGGTCCA
D4Mon1 AGGCCTACCCAAG GCAGTGAGCTGC 201 472-473
GACATCT AGAGTTTG
D4Mon2 AGACACCCTAGGT TGATCTTTCCAA 151 474-475
CCTGCTG ACGCATAAGA
D4Mon3 GCAAGCAACCTGA GCTTACGATGGT 188 476-477
ACATGAA CGTGAGGT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-91-
Marker Forward Reverse Size, SEQ.
by ID
NO.
D4Mon4 ACATGCCTGCCTA GGAACCTGTTTT 197 478-479
TCTTTGC CCATGGTG
D4Mon5 ACCTTGTTCCTGG TAGCTGGGACGT 200 480-481
TGTGAGC GGTATGGT
D4Mon6 CCATGGGAGACCA TGAGTGTCCTCT 206 482-483
GAAGGTA GCCTGATG
D4Mon7 GCGCTGACATCCT CCCACTATGGTC 187 484-485
CCTATGT CCAGAGAA
D4Mon8 . TTGCACGTCTTTG AAAGGGGAATA 219 486-487
TTTCGAG GACCTGAGTAG
AA
D4Mon9 CCAAGAGTCAGCC GGACAGGTAGCT 200 488-489
TTGGAGT CACCCAAC
TrllikeulcDNATGCCAGCTTTGGC TTCATTGTGTCC 490-491
mouse TATCAT CTGAGCTG
Trllikeu2cDNAAGCTTTGGCTATC ACCACCGCCACT 492-493
mouse ATGGGTCTCAG GTTCTCATCT
Trllike A1 TGTGGGGGAAGA TGATGTGTGGCT 5935 494-495
(mouse) ACATAGAA TGTTTCTCTT
Trllike A2 ATAGGTGGGGAG TGATGTGTGGCT 5903 496-49?
(mouse) GGAGCTAA TGTTTCTCTT
TRl like-2 TGTGCCTGTCACA CATGCTAGCACC 498-499
(human) GCAACTT GTAGCTGA
TRl like-3 GGAGACCTTCCCC GCTGTAGTTGAA 500-501
(human) TCCTTCT GAGGGCGT
TRllike-4 GTGCTTGGCTTCC CAGGTCGTACTC 502-503
(human) TCCAG CATGTCCA
TRl like-5 TGGAGTACGACCT ACTCATCCTGGC 504-505
(human) GAAGCTG CACAAAAG
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-92-
Marker Forward Reverse Size, SEQ.
by ID
NO.
TRllike-6 GAACAGGAGGAC CTTTTGTGGCCA 506-507
(human) GCTGAGG GGATGAGT
TRl like-7 TCACCTCACCTGG GTACGACCTGAA 508-509
(human) TTGTCAG GCTGTGGG
TRl like-8 GGCTGAGATCACA CCGTGCCTGTTG 510-511
(human) GGGTTGGGTCACT GAAGTTGCCTCT
C GCC
118e21-0 AATTCCCAGCAAC CAGACACTCCAG 585 512-513
CACTCAC AAGAGGGC
118e21-1 TGACTGCTCTTCC TTTGTGGAATAG 588 514-515
GAAGGTT CCAAAGCC
118-21-2 TCTCTCCTCTCTTC AGCAGGGTGCAT 551 516-517
TCCCCC CACCTTAT
118e21-3 TAGGAGTGCCCCA TCATTGTACCCA 518 518-519
TAGGTTG GCCAGTCA
118e21-4 AGGACTGAGCCTG CTGGGCGTTTTG 552 520-521
GATGAGA TTTTGTTT
118e21-5 CTTCCTCCTGCAG ACCCTGCTACAA 546 522-523
CTACCAC CGCAGACT
118e21-6 TCCAACCTTGACA AGCCAGGGCTAC 584 524-525
CCCATTT ACAGAGAA
139J18T7(1) CTGCTTTTCCTCA ATTCGCCGTTAG 526-527
GCAACTG AAGCTAGG
139J18T7(2) AACTGTACGTGGC ATTCGCGGTTAG 528-529
TGCTGGT AAGCTAGG
Agrin(CA)n GCCAGGTGACCCT GAGAGATGGCA 271 530-531
TATGAAA GACAGAGGC
Agrin(TG)n AGCTCTCTGTCCC TGCCAACCACTA 157 532-533
TGGTGAA GCCTCTCT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-93-
Marker Forward Reverse Size, SEQ.
by ID
NO.
repeatl CTGAACCCTCCAC AGCCAGGGCTAC 205 534-535
TCTCCTG ACAGAGAA
repeat2 AGCCAGGGCTACA ACCCTGCTACAA 153 536-537
CAGAGAA CGCAGACT
repeat3 GCAAGTTTCAGGA CCCCAGAACCAG 166 538-539
GCTAGGG AGACCATA
repeat4 CTAGGGGACTCTG CA.AGACACCCA 195 540-541
CCAAGTG GTCCCAACT
repeats TACTTCCCCTTTCC TCCTTGGTGCTT 232 542-543
CGAACT ACCCTCAC
repeat6 TGTTCCTGAGTTC ATTCCCAGCAAC 269 544-545
ACAACGC TACATGGC
repeat? ACATGTCCACTGT TGTCATGAGTTT 246 546-547
GGCAAAA GAGGCCAG
repeat8 ATCAGACAGCCCA TATGTGCCACCA 206 548-549
CAACCTC CACCTGTC
repeat9 GCTCAAGGAAGG TGCTCTTAACAT 201 550-551
ACACACCT TTTGAGCCAT
repeatl0 GCTCAGCCCCTGA GGGATCTGCCTG 111 552-553
ATCAATA TCTTACCA
repeatll GGAAGGTAGGGC GCTCCAAGATCT 277 554-555
CTGGTAAT GTGCGATT
repeatl2 TTAGCGTTAGGGT GGAGACTACGG 150 556-557
GAGGGTG ACTTGTGGC
repeatl3 CAGTTCTTCCCGA TTTCTGGGAACT 174 558-559
AAACCAC GAGATGGC
repeatl4 GTTGGGGCTGCTC GCTGTGGCTCTC 422 560-561
ATAGAAA TTGGAGTT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-94-
Marker Forward Reverse Size, SEQ.
by ID
NO.
repeatl5 CTCTGATTTCCCA AAGAGGGAGCA 152 562-563
CATGCCT CTGAGGACA
repeatl6 CAGCAGCAAATGA GAGGCAGGCAG 147 564-565
CCTTTCA ATTTCTGAG
repeatl7 GTTTCACATGTTG GGGACCTTTGGG 131 566-567
TGGTGGC ATAGCATT
repeatl8 TCAGACATCTCTG TTCACTAAGTTG 160 568-569
GCCTCCT CCCAGGCT
repeatl9 TGCCTTTTTCTCAC TTAGAAGCAGA 250 570-571
ATTGTCTC GGCAGAGGC
repeat20 GACCTTTGGAAGA TGGCAGCTCACA 296 572-573
GCAGTCG ATGTCTTT
SHANRU1 GGTGTGGTGTAGG TTTCAACTGCAA 301 574-575
GGAAGAA ACACAAACAG
SHANRU2 AGGGCCAAGGAA GCAAATATATAG 203 576-577
GGAGAAT GGTACCGAGCTG
SHANRU3 CAGATTCTCCAGC CTGTGTTTCCGC 229 578-579
TGTCAGG ACCAAGT
SHANRU4 CTGCCCGTCCTTA ACGCACGCTCAC 289 580-581
TCTTCTG' TCATACAC
SHANRUS CAGCAGAGGTGAT TTGTCACACAGT 203 582-583
GGGTTCT GGTTAAATGC
SHANRU6 TAGAACCGTGGCT CCGTAAGATAT 201 584-585
GAGGACT GAAAGAACTTG
GA
SHANRU7 TAATCCTGGCTTA TAGAAAGCACA 240 586-587
GCGCTTG GGGGACAGG
SHANRU8 CCTTCCTCGTCTG TTGGGACGTGAC 232 588-589
AGCTGTT CTGAGAAT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-95-
Marker Forward Reverse Size, SEQ.
by ID
NO.
SHANRU9 TATGTGTCTGGCC GATGTGGGTGCA 206 590-591
GTTGTTC GGTGAAG
SHANRU10 CCCCTTCTGGAGT TCTAGGCAGGGC 263 592-593
GTCTGAA TACCTTTTT
SHANRU11 GCTGAGCAGCCTC ACCATGGCTTTT 241 594-595
TAGCAA CCCAGTAA
SHANRU12 CTGTGCCTTTGGT TGTGGCACTCTA 261 596-597
GATCAGA CGGCATAA
SHANRU13 TGCATCACTATTA AAGAATTTGCAA 260 598-599
AGCCTCAACC AGACTGTGAGA
SHANRU14 AGCCAGCGCTACA CTGGACCTTTGG 199 600-601
CAGAGA AAGAGCAG
SHANRU15 GGTGGCTCAAACC GAGGGCAATGA 203 602-603
ATCCATA GCAAAATGT
SHANRU16 GGTCCTGTCTCTG TAACACCCACAT 201 604-605
GTTCAGG CAGGCAAC
SHANRU17 TTTCATTTCCTGGT AAACACAGGCG 198 606-607
GTTCCTTT GAACGATAG
SHANRU18 CTATCGTTCCGCC AAGGAAGAGGA 397 608-609
TGTGTTT TGGAGAAAGA
SHANRU19 CGGGTCTTAATGG TCCTCCCCAGTT 222 610-611
AGCAGAG ACCTAGCA
SHANRU20 CAGCAGGCAAGAT GTCCCTCACCAG 205 612-613
GACCTC CCATGTTA
SHANRU21 AGCCTGGGCTAAG TATGGGCCAATG 204 614-615
TTGTGTG TTGTTCCT
SHANRU22 ATGGTGGCTCACA TTGTCCTCTGAT 193 616-617
ACCATCT TGCAGCAT
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-96-
Marker Forward Reverse Size, SEQ.
by m
NO.
SHANRU23 CTTGGGTCATCAG AAGCTGCCCTGG 301 618-619
GCTTTGT TCTCTCTA
SHANRU24 ATGCTCAGCCTGC GCTGATAGCCCT 198 620-621
TTTGTTT GGGTTCTA
SHANRU25 TGTACGCACAAAT GAATCCACATTG 222 622-623
TGACTTGC CAAAGCCTA
SHANRU26 CACAGGCAAATGA CCAGACTTCTCC 187 624-625
AGGGAAG AGCTCTCC
SHANRU27 TCCTCGAGAGGCT TGCCTAGTCAAC 237 626-627
CTAGGTTT CACAGGAG
SHANRU28 CCTGTGGTTGACT GCCTGATAGCCT 406 628-629
AGGCAGAA GGAATACA
SHANRU29 AAAGGGATGTGTG CAAA.ACCCAACC 195 630-631
GCGTAAG TTCTCAGC
SHANRU30 TGCACTGACCGTG CGGTGTAGCTCT 200 632-633
ATAGAGG GGCTGTCT
SHANRU31 CATCTCACCAACT TTTCTGGGAACA 418 634-635
CGCACTT AAGAGGCTA
SHANRU32 GAACCCAAGTGTT TGGAAGCCCATC 222 636-637
GGGGTAA TGTCTCTT
SHANRU33 AAATGCAAGTGGG CCAGAAGAGGG 187 638-639
TGCTTCT CGTCAGAT
SHANRU34 GGTGTGCACCACC GGGAATTATCAG 201 640-641
ATATTCA CCAAAAAGC
SHANRU35 GCCCAACTGAAAG GGAAGGGGGAT 263 642-643
CTCAACT AACAATTGAA
SHANRU36 TGCTAATTTCAAG AGCTTGACACCT 369 644-645
CACAGTGAGA TGACAGCA
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
-97-
Marker Forward Reverse Size, SEQ.
by ID
NO.
SHANRU37 AACCTGCAGAGAG CTCCAAGGGGA 201 646-647
GAGACCA GGACTCATT
SHANRU38 TTCAATTGAGTTT TGCAGGACCAA 200 648-649
CTCTCCTCTGA GAAGTAGGC
SHANRU39 CGAGATCTGATGC TGCTGAGAGCAG 200 650-651
CCTCTTC AAAAGGAA
Although the foregoing invention has been described in some detail by
way of illustrating and example for purposes of clarity of understanding, it
will be
obvious that certain changes and modifications may be practiced within the
scope
of the appended claims.
All publications, patents, and web sites are herein incorporated by
reference in their entirety to the same extent as if each individual
publication,
patent, or web site was specifically and individually indicated to be
incorporated
by reference in its entirety.
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
SEQUENCE LISTING
<110> Bachmanov, Alexander A
Beauchamp, Gary K. a
Chatterjee, Aurobindo
De Jong, Pieter J.
Li, Shanru ;
Li, Xia
Ohmen, Jeffrey D
Reed, Danielle R.
Ross, David
Tordoff, Michael G.
<120> GENE AND SEQUENCE VARIATION ASSOCIATED WITH SENSING
CARBOHYDRATE COMPOUNDS AND OTHER SWEETNERS
<130> Gene & Sequence Variation......
<140>
<141>
<150> 60/200,794
<151> 2000-04-28
<160> 652
<170> PatentIn Ver. 2.1
<210> 1
<211> 2577
<212> DNA
<213> Mouse
<400> 1
atgccagctt tggctatcat gggtctcagc ctggctgctt tcctggagct tgggatgggg 60
gcctctttgt gtctgtcaca gcaattcaag gcacaagggg actacatact gggcgggcta 120
tttcccctgg gctcaaccga ggaggccact ctcaaccaga gaacacaacc caacagcatc 180
ccgtgcaaca ggttctcacc ccttggtttg ttcctggcca tggctatgaa gatggctgtg 240
gaggagatca acaatggatc tgccttgctc cctgggctgc ggctgggcta tgacctattt 300
gacacatgct ccgagccagt ggtcaccatg aaatccagtc tcatgttcct ggccaaggtg 360
ggcagtcaaa gcattgctgc ctactgcaac tacacacagt accaaccccg tgtgctggct 420
gtcatcggcc cccactcatc agagcttgcc ctcattacag gcaagttctt cagcttcttc 480
ctcatgccac aggtcagcta tagtgccagc atggatcggc taagtgaccg ggaaacgttt 540
ccatccttct tccgcacagt gcccagtgac cgggtgcagc tgcaggcagt tgtgactctg 600
ttgcagaact tcagctggaa ctgggtggcc gccttaggga gtgatgatga ctatggccgg 660
gaaggtctga gcatcttttc tagtctggcc aatgcacgag gtatctgcat cgcacatgag 720
ggcctggtgc cacaacatga cactagtggc caacagttgg gcaaggtgct ggatgtacta 780
cgccaagtga accaaagtaa agtacaagtg gtggtgctgt ttgcctctgc ccgtgctgtc 840
1
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
tactcccttt ttagttacag catccatcat ggcctctcac ccaaggtatg ggtggccagt 900
gagtcttggc tgacatctga cctggtcatg acacttccca atattgcccg tgtgggcact 960
gtgcttgggt ttttgcagcg gggtgcccta ctgcctgaat tttcccatta tgtggagact 1020
caccttgccc tggccgctga cccagcattc tgtgcctcac tgaatgcgga gttggatctg 1080
gaggaacatg tgatggggca acgctgtcca cggtgtgacg acatcatgct gcagaaccta 1140
tcatctgggc tgttgcagaa cctatcagct gggcaattgc accaccaaat atttgcaacc 1200
tatgcagctg tgtacagtgt ggctcaagcc cttcacaaca ccctacagtg caatgtctca 1260
cattgccacg tatcagaaca tgttctaccc tggcagctcc tggagaacat gtacaatatg 1320
agtttccatg ctcgagactt gacactacag tttgatgctg aagggaatgt agacatggaa 1380
tatgacctga agatgtgggt gtggcagagc cctacacctg tattacatac tgtgggcacc 1440
ttcaacggca cccttcagct gcagcagtct aaaatgtact ggccaggcaa ccaggtgcca 1500
gtctcccagt gttcccgcca gtgcaaagat ggccaggttc gccgagtaaa gggctttcat 1560
tcctgctgct atgactgcgt ggactgcaag gcgggcagct accggaagca tccagatgac 1620
ttcacctgta ctccatgtaa ccaggaccag tggtccccag agaaaagcac agcctgctta 1680
cctcgcaggc ccaagtttct ggcttggggg gagccagttg tgctgtcact cctcctgctg 1740
ctttgcctgg tgctgggtct agcactggct gctctggggc tctctgtcca ccactgggac 1800
agccctcttg tccaggcctc aggtggctca cagttctgct ttggcctgat ctgcctaggc 1860
ctcttctgcc tcagtgtcct tctgttccca gggcggccaa gctctgccag ctgccttgca 1920
caacaaccaa tggctcacct ccctctcaca ggctgcctga gcacactctt cctgcaagca 1980
gctgagacct ttgtggagtc tgagctgcca ctgagctggg caaactggct atgcagctac 2040
cttcggggac tctgggcctg gctagtggta ctgttggcca cttttgtgga ggcagcacta 2100
tgtgcctggt atttgatcgc tttcccacca gaggtggtga cagactggtc agtgctgccc 2160
acagaggtac tggagcactg ccacgtgcgt tcctgggtca gcctgggctt ggtgcacatc 2220
accaatgcaa tgttagcttt cctctgcttt ctgggcactt tcctggtaca gagccagcct 2280
ggccgctaca accgtgcccg tggtctcacc ttcgccatgc tagcttattt catcacctgg 2340
gtctcttttg tgcccctcct ggccaatgtg caggtggcct accagccagc tgtgcagatg 2400
ggtgctatcc tagtctgtgc cctgggcatc ctggtcacct tccacctgcc caagtgctat 2460
gtgcttcttt ggctgccaaa gctcaacacc caggagttct tcctgggaag gaatgccaag 2520
aaagcagcag atgagaacag tggcggtggt gaggcagctc agggacacaa tgaatga 2577
<210> 2
<211> 11809
<212> DNA
<213> Mouse
<400> 2
atctgagcct tagacacagc actggtgcca ggcaaacact cctgggccta catgcttggg 60
gcctcttcat attccaaaag ctgtctttgg gtaagatgaa gttcctctgg cagtggcatg 120
agtgctgaag gctctttccc tgcccttcac ctgctttctt gatagtctct ctgcatacca 180
aacaggccct tgtctcctgg gaaatggaaa ctatgaaatc aatagctgag gcttctctag 240
gaaagcctgc cctggtcagt acaacctgtt tcacagcttc tatagaatag ttacatcagc 300
cttctgaaga tggcctctta gagcacatgc acccccaaga ttctaagatg tcaatactaa 360
ctgaccaaac catacctctc tagccagccc tgctgctcct gttgtctggt acccaggtga 420
ctgaggacat gactggtgga aggaaactag gcccctttgt ctgtcagatg gccataccca 480
gcatggctga tgcccagtgt ataagaccct acgcttttcc actggtctta atgttaaacc 540
ctaggacagt gtcctcagca tagctggtgt gtgtgaatgc aaactttggg gcatatctct 600
tccattaagc actgtgatat atgtagtatt tccaacaaat aaattatacc tacatgattg 660
2
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
ggtatagcat tctgggatgg gtcacaagtg tgtcaggtgc ctaattatgt gggggaagaa 720
catagaaata tataggtggg gagggagcta accctaggaa taaggctaaa gcatgtgtct 780
ccagtcctga agactcaaag ggcaacgtga atcatgagac atgttcagga ctgaaggagt 840
tgccatgtat ctgtccttga tgtatcttaa tcatacatac actatgagat ctgtgttacc 900
tccattttgc aggtgagaaa agaaacacct gaatggccta ccttaaaggg ctaagtggga 960
aaataggtct gaagataacc caggcactgt gtgacaaagc gggaagaaaa ctagagatgc 1020
tttcttcatg gcaacaacct agagggtaca acctagtggt ttcttcttgg tactccactg 1080
tatacacccc atctgcttgg gctgtacatt gtctgaccat gcttataaca aaagtcacat 1140
actactagcc aagactgaga acttagagcg actggccaga aagtaaagat acaacagttg 1200
atatgtgtgc cacacacaga tccatgtgta catgtctatt aattatgtga acgtgctttg 1260
tggacatcct cacaaagcag cagggaaatg caaaggtcat ttccataaca cctgctggac 1320
accatatgac attgagatta ccggggtgcc cattccaaca agagttaata gctcccccta 1380
tgtttgggtg ccagaaacct gatttgttag caatagctcc ctcacatcca gattaagagg 1440
gggatggctt agctagggtt actatgatga aactatgacc aaagcaactt gtgggtaaaa 1500
gggtgtattt ggcttacact tccatatcac ttcatcaaag tgaggacagg aactcaaata 1560
gagtaggaat ttggtgacaa gagctgatgt agaggcaatg cagtggtgcc acttagtggc 1620
gcgctcagtc tgctcccttt cttaatagaa tgcaagacca ccagcccatg ggtggcacca 1680
caatgggacc gggcccttcc ccatcggtca ctaagaaaat gccctacagc cagatcttat 1740
ggagacattt tctcaacgga ggctcactcc tttcagataa ctctatatca aattgacata 1800
aaccagaaca gaggaggagg ctaagaagga aactgccaat tgcatacatg cacacacctg 1860
gccctagcag ctgcaggaag ctatttgttt atggcctttt ctcattttca tggaccagca 1920
tgagcactct gcagagagag atgcctgcat gcctgccaag gcaggagtgc ttacactgaa 1980
ggtcaacagg atggcagggg ggctgcagag cttccaagtg tcagaacccc agcagaagag 2040
ctgagaccct tgcccgagga ctcaggcggg ttgggaaggc caggaaattc agccagagct 2100
cttcttcaga tggggtacca tctgaaggtt agaccagcta gccagctgtt gttgagggac 2160
cacctctgca gcccctacct ttggaagata gaaagtgtct ctgtgacaag tatggccatt 2220
gtgccccctt attccacagt caacagaaac cctggaatcc tgaacacttc tgcagcttct 2280
tttttacagt ctgccaggtt gctctaggaa tgaagggtgc cgagaggctt gggcgtaggc 2340
aggtgacaag accacagtta gtggtcacag ctggcttact ggatcactct tggacagagt 2400
ttgttagata tggagtggag tatacacaag gcatcaggcg ggggatattg aatgtatcac 2460
cggagctcct tggggcttgg cagccaagca cagcagtggt tttgctaaac aaatccacgg 2520
ttccctcccc ttgacgcagt acatctgtgg ctccaacccc acacacccac ccattgttag 2580
tgctggagac4ttctacctac catgccagct ttggctatca tgggtctcag cctggctgct 2640
ttcctggagc ttgggatggg ggcctctttg tgtctgtcac agcaattcaa ggcacaaggg 2700
gactacatac tgggcgggct atttcccctg ggctcaaccg aggaggccac tctcaaccag 2760
agaacacaac ccaacagcat cccgtgcaac aggtatggag gctagtagct ggggtgggag 2820
tgaaccgaag cttggcagct ttggctccgt ggtactacca atctgggaag aggtggtgat 2880
cagtttccat gtggcctcag gttctcaccc cttggtttgt tcctggccat ggctatgaag 2940
atggctgtgg aggagatcaa caatggatct gccttgctcc ctgggctgcg gctgggctat 3000
gacctatttg acacatgctc cgagccagtg gtcaccatga aatccagtct catgttcctg 3060
gccaaggtgg gcagtcaaag cattgctgcc tactgcaact acacacagta ccaaccccgt 3120
gtgctggctg tcatcggccc ccactcatca gagcttgccc tcattacagg caagttcttc 3180
agcttcttcc tcatgccaca ggtgagccca cttcctttgt gttctcaacc gattgcaccc 3240
attgagctct catatcagaa agtgcttctt gatcaccaca ggtcagctat agtgccagca 3300
tggatcggct aagtgaccgg gaaacgtttc catccttctt ccgcacagtg cccagtgacc 3360
gggtgcagct gcaggcagtt gtgactctgt tgcagaactt cagctggaac tgggtggccg 3420
ccttagggag tgatgatgac tatggccggg aaggtctgag catcttttct agtctggcca 3480
atgcacgagg tatctgcatc gcacatgagg gcctggtgcc acaacatgac actagtggcc 3540
3
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
aacagttggg caaggtgctg gatgtactac gccaagtgaa ccaaagtaaa gtacaagtgg 3600
tggtgctgtt tgcctctgcc cgtgctgtct actccctttt tagttacagc atccatcatg 3660
gcctctcacc caaggtatgg gtggccagtg agtcttggct gacatctgac ctggtcatga 3720
cacttcccaa tattgcccgt gtgggcactg tgcttgggtt tttgcagcgg ggtgccctac 3780
tgcctgaatt ttcccattat gtggagactc accttgccct ggccgctgac ccagcattct 3840
gtgcctcact gaatgcggag ttggatctgg aggaacatgt gatggggcaa cgctgtccac 3900
ggtgtgacga catcatgctg cagaacctat catctgggct gttgcagaac ctatcagctg 3960
ggcaattgca ccaccaaata tttgcaacct atgcagctgt gtacagtgtg gctcaagccc 4020
ttcacaacac cctacagtgc aatgtctcac attgccacgt atcagaacat gttctaccct 4080
ggcaggtaag ggtagggttt tttgctgggt tttgcctgct cctgcaggaa cactgaacca 4140
ggcagagcca aatcttgttg tgactggaga ggccttaccc tgactccact ccacagctcc 4200
tggagaacat gtacaatatg agtttccatg ctcgagactt gacactacag tttgatgctg 4260
aagggaatgt agacatggaa tatgacctga agatgtgggt gtggcagagc cctacacctg 4320
tattacatac tgtgggcacc ttcaacggca cccttcagct gcagcagtct aaaatgtact 4380
ggccaggcaa ccaggtaagg acaagacagg caaaaaggat ggtgggtaga agcttgtcgg 4440
tcttgggcca gtgctagcca aggggaggcc taacccaagg ctccatgtac aggtgccagt 4500
ctcccagtgt tcccgccagt gcaaagatgg ccaggttcgc cgagtaaagg gctttcattc 4560
ctgctgctat gactgcgtgg actgcaaggc gggcagctac cggaagcatc caggtgaacc 4620
gtcttcccta gacagtctgc acagccgggc tagggggcag aagcattcaa gtctggcaag 4680
cgccctcccg cggggctaat gtggagacag ttactgtggg ggctggctgg ggaggtcggt 4740
ctcccatcag cagaccccac attacttttc ttccttccat cactacagat gacttcacct 4800
gtactccatg taaccaggac cagtggtccc cagagaaaag cacagcctgc ttacctcgca 4860
ggcccaagtt tctggcttgg ggggagccag ttgtgctgtc actcctcctg ctgctttgcc 4920
tggtgctggg tctagcactg gctgctctgg ggctctctgt ccaccactgg gacagccctc 4980
ttgtccaggc ctcaggtggc tcacagttct gctttggcct gatctgccta ggcctcttct 5040
gcctcagtgt ccttctgttc ccagggcggc caagctctgc cagctgcctt gcacaacaac 5100
caatggctca cctccctctc acaggctgcc tgagcacact cttcctgcaa gcagctgaga 5160
cctttgtgga gtctgagctg ccactgagct gggcaaactg gctatgcagc taccttcggg 5220
gactctgggc ctggctagtg gtactgttgg ccacttttgt ggaggcagca ctatgtgcct 5280
ggtatttgat cgctttccca ccagaggtgg tgacagactg gtcagtgctg cccacagagg 5340
tactggagca ctgccacgtg cgttcctggg tcagcctggg cttggtgcac atcaccaatg 5400
caatgttagc tttcctctgc tttctgggca ctttcctggt acagagccag cctggccgct 5460
acaaccgtgc ccgtggtctc accttcgcca tgctagctta tttcatcacc tgggtctctt 5520
ttgtgcccct cctggccaat gtgcaggtgg cctaccagcc agctgtgcag atgggtgcta 5580
tcctagtctg tgccctgggc atcctggtca ccttccacct gcccaagtgc tatgtgcttc 5640
tttggctgcc aaagctcaac acccaggagt.tcttcctggg aaggaatgcc aagaaagcag 5700
cagatgagaa cagtggcggt ggtgaggcag ctcagggaca caatgaatga ccactgaccc 5760
gtgaccttcc ctttagggaa cctagcccta ccagaaatct cctaagccaa caagccccga 5820
atagtacctc agcctgagac gtgagacact taactataga cttggactcc actgacctta 5880
gcctcacagt gaccccttcc ccaaaccccc aaggcctgca gtgcacaaga tggaccctat 5940
gagcccacct atcctttcaa agcaagatta tccttgatcc tattatgccc acctaaggcc 6000
tgcccaggtg acccacaaaa ggttctttgg gacttcatag ccatactttg aattcagaaa 6060
ttccccaggc agaccatggg agaccagaag gtactgcttg cctgaacatg cccagccctg 6120
agccctcact cagcaccctg tccaggcgtc ccaggaatag aaggctgggc atgtatgtgt 6180
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtat gtacgtatgt atgtatgtat 6240
caggacagaa caagaaagac atcaggcaga ggacactcag gaggtaggca acatccagcc 6300
ttctccatcc ctagctgagc cctagcctgt aggagagaac caggtcgccg ccagcacctt 6360
ggacagatca cacacagggt gcgggtcagc accacggcca gcgccagcca cgcgggaccc 6420
4
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
ctggaatcag cttctagtac caaggacaga aaagttgccg caaggcccct tactggccag 6480
caccagggac agagccacat gcctaagcgg caagggacaa gagcatcgtc catctgcagg 6540
caggatcaga cccgggtcag ttctggactg gcccccacac ctgaatcccg gagcagctca 6600
gctggagaaa agagaaacaa gccacacatc agtcccataa aattaaacgc tttttttagt 6660
gtttaaaata gcatttacac agaagcagca tttacacaga agcagctcta tgtcaactac 6720
ccagtcactc agactttgac acagtgtcta gtgtagatgt gtggggccgc tgtgccggga 6780
tggcagtggc acatgatgat gggcagccac cagaacagaa acagaacagg gcccagctct 6840
gcagctcttg tgttcactgt cacccaccac tgagactgag acagtggcta ggtgccaggt 6900
ctctctcctg tctctcctac tagctaccct tcacatacct tcagtacaaa ctgtgttgtc 6960
atgtgccaag tagcaggtgg ggaaaggggc atgcaaactg cccctttggg taactagctg 7020
ccacccttag agcaggcagg ctagcaataa ataaataagt tagaccccac ctgggcagcc 7080
agagaggttt gaaggctctg tctaacccct caaaaatccc accttggcct gacaggtgag 7140
gcccatgaac ttagcgacag tcagcctgtg tccctgtgca cagttctgtg aggctttggg 7200
gcaaggggta ccaagagccc aagagagcct ttcttgttct aaatggaggt cacttccaaa 7260
gaagggaacc aggaggtggt ccctgagact tg~gctgagg acttaaagtc agagatgtct 7320
ccttacaaga ctctatagat acttgagctg taccaccatc agcagcccca agagcagaca 7380
aaatgtcaag ccaatatcct ggtggtatgg ctgccctcag gccctcctct gtagcctgct 7440
ccctctgccc tggcccagag cccacagctg atctatcctg gctggccacc accacggcca 7500
gcgcagagct cctggcacag caggagcaca gactcagcca caggcagcgc tgaagacatt 7560
ggttgatcat cacatgatgt ccacaaagaa ctcacagggg tttcccatgg ccttttggaa 7620
ggactggcgg ctacctgtaa gttctggagg gacagcagcc agctcccgga cgggtggccc 7680
tccaggtggc ccacccacta ctgcataggc ctttgtaagg gggtgcagtg gggggagccc 7740
tggggcaaca gctgaagcct gacttcgagg gctactgcca cggctaagct ggctgacagg 7800
ccgctcccac cagccggtgc taccagaccc acttggtact gtgtggtctg attcactgcc 7860
actaccccca gctccagttg cccggcgctc ctctcggcct ggggtccgat ggctgctccg 7920
tgtggaccca ctgctcttgc tccctagggg gagggaaggg gacaacagag tcagcacgag 7980
gcctggccac ttccagggcc accagctgct cccagacagt cagggcagga cctggtaagc 8040
ctggagatgg taggggaatg gcagccatgc agataccagg aacagctgag aggcgagaag 8100
ctaggggcag tggcagacag cagggacaac aggggccagc ctggcacccc acacctaacc 8160
ccaatgcttg aaccaagggt taatgttaca gctgagaaac taaaaaccag cgaaggccct~8220
gtgtgcccag cattcccatt agccatcctg ggttcaccac ccaaagaccc aaccagggtc 8280
cacccaaccc caggaccctg gtcatctaat ttgcttagcc cctgtcctga aagtagtggg 8340
aacctgaaaa cacgtgctgg ctggggacat gctgagaggg acacaggggg acctggctta 8400
ccggcccgag agtccactct gctagtcctt cagtctaagg cttgctcagc acaaagcaag 8460
ggatagcaca agtcacacac cagtccagtg ctcaccaatg gctaatagga cgattttggg 8520
ccaagctgag cctgggtaca tgcaagggcc tgtccatggt caggattcac tcgatagctt 8580
ccccttgggc tttgccaccc tctggcccaa cctctcctga gtctttctct ggaccttgta 8640
gcacaagtgt gccccactct gcctaagacc tccacatcag tccatctcct cctgagggac 8700
acccaccctt caagatcttc aatatccctg ggatatgctt taacactgat atgctttaac 8760
agtgttgctt gatactctta tctggcactc tgttgggatg caggctccat aactgataaa 8820
gcccattctc cccctagctt ggggcctaga gagtgcccct acctgctatc agtggttact 8880
ttcattcttg ccatatcatc tcctggcctc ttgcctctgc cacctagcac accaggctgt 8940
cttcctattc tctaacggct tctacccaca tcagcccctc cctgtcccac acactgactc 9000
ttgagatgga acccaccggg actcaaacac acagcaggag cacagaggga agcgtcgggg 9060
ccaggcagag cgtgggagtg ggagggagtg ggaggagggg tggcacgcct ctcaccttca 9120
ctctgctggc tcccagcact gccgctgccg cagctgaagc cagggtcctg gtaagcaggc 9180
gggaagcagg gcgggggtcc tgggtactgg taggggtagc cttgacccaa gggccagggt 9240
actgatgggt ggggcagtgg ggccagtgtg tcctgatctg aggctccact ggagccactg 9300
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
ttgaggttca gggatgcgag gtctggcagg gagggaggga gggaggggta agtgaaggca 9360
aatgaatgag gccacagcaa ccctacccaa ccgcacccct actcactact gcacaggtcg 9420
ccaaagacat agtagcactg ctcagaaaag gtgatcttgt tcacggtgtg cctcaggaaa 9480
ccgtgcttca gcatactgct ggcatacttt cttgcctccc ttcgctcctt gaagccctcc 9540
acgtgtgtgt acagccagtc caccacatcc gcccctggcc acaggtccat caaagtcagg 9600
gtagctgagc cctgggaagc tacgccagaa tgaggaacag acggggccct tcccacacag 9660
ccagggactc accaatgaca gcattggcaa tggtgatctt aagccacatg cggtcccgga 9720
tctccagtcc tgagtctggc aactgcatga cgcggacaat ggcactcatg tcactcttca 9780
cagtcagcgg tgcctcctca agctctgcag agcacacttc cctgagccca ggctcacagc 9840
gtgaacctcc atggggttga gagcaggggc cagggtcaaa cctcttatct cc~atccttg 9900
ggagatgccc ctcatcgaaa cttgagctaa gaccgggaga ttcttccccg tcccacagtg 9960
caagtccacg taggcaaggc agcccccctc ccctccccgg agagaacaag ctgttagcta 10020
tgttaggtag cagaaaagca aagcagaggc tgccatgtcc tcccaattcc cccctccgca 10080
caggcctggc aggaccctca attcatgcag atgaccagta tggccaggcc tggagggata 10140
tgtacatgta tctttgtgta cacatttgtg aaggtgttgg aagcaaacaa aaccttcata 10200
tgtaatgggc ccctgtaata gctctgatga gcaccaaagc tcaaagctag aactgaccat 10260
tgtccttcaa cctcagtttc cttgggtggg ggggggtcct gtgagctgcc acttacgtgg 10320
ggcgccaggc actgagctgg ttagtgagga agagctggtg cgtgtgatgg cgctggagca 10380
gggactcgta ccatagcggg gcagggcacc cgtcagtgct gctgtgtggg acagccaggc 10440
agccgggtcg atgggtcgca ctgggtcagc tgcatagttt ccacagcaac ggattacagg 10500
tggtaagtag gggggcagca cagaggcaga caagaaagac ccccagactg aacacagaaa 10560
ccccacccta ccccaccttt ccatggggta actcacccct tgggatggtg aagtagctcc 10620
gaggggttgg gtcccagcac ttggccactg tgagactgat gggcctacag agttgagcag 10680
accatgttgt aagtgaggcc cgcacagccc ctcccatcct gtgccactcc cacccccact 10740
tggctcccac ctcaccctgt ctgggacacg atctcccgaa gcacccgtac agcgtcgtca 10800
ttgctcatgt tctcaaagtt gacatcgttc acctacgggg tttgtggggt caggggttgg 10860
tggtgggatg tgggtgcctc ttgtccccac agtccccaca tggctcccac ctgcagcaac 10920
atgtcgcccg gctcaatgcg gccatcagca gccacggccc cgcccttcat gatggatcca 10980
atgtagatgc cgccatcacc ccggtcgttg ctctggccca cgatgctgat gcccaggaag 11040
tggtgcctct ctgcaggagg ggccgtgagc aggcccccaa agctcccgag gctgtaccca 11100
cccccagcag gcacccacag cccacaaggc ctcacccatg ttgagagtga cggtgatgat 11160
gttcagggac atggtggagt ctgtgatgct gctgaaggag gatgcctgcg gagggaccca 11220
gtgaggggct gtgtgggcac cattcagagc agacacccca cccacctgct gcctacccgg 11280
tctgtctgcc tcaagcgctg cttccgacga cggcatttgt gcttccgaac tagccgagag 11340
gaggtgctct gctctgtgga gctgctcagc ctgaggcagg agtcagaaaa gcacaaacat 11400
gtataaccag ctcggacgct caactacaaa tctccagcac gtactgacat gtgcacacgt 11460
cacccaccgg ctcgtattgt cctcctcatc tgagtcaata aagctgctag attcaagctc 11520
actgctcagt acagtggatg cactgtctgg aggtagtccc aggtcccgcc gccgatcccc 11580
tctcgggtgc ccattggtcc gggcagctgt ggggacagta gggtgggtac gactgtggga 11640
cttcagtcct aacagaatgc gggtggcctg tgcatttcaa agtttatgca gtaactctgg 11700
ggccacaggg gctaggagta ccaggctggg acctctaccc aaggatcact gcttggaaga 11760
atatgtggaa tacttccagg cttggagtat accaaaggga taccaaggg 1180:
<210> 3
<211> 858
<212> PRT
<213> Mouse
6
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 3
Met Pro Ala Leu Ala Ile Met Gly Leu Ser Leu Ala Ala Phe Leu Glu
1 5 10 15
Leu Gly Met Gly Ala Ser Leu Cys Leu Ser Gln Gln Phe Lys Ala Gln
20 25 30
Gly Asp Tyr Ile Leu Gly Gly Leu Phe Pro Leu Gly Ser Thr Glu Glu
35 40 45
Ala Thr Leu Asn Gln Arg Thr Gln Pro Asn Ser Ile Pro Cys Asn Arg
50 55 60
Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val
65 70 75 80
Glu Glu Ile Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly
85 90 95
Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Ser
100 105 110
Ser Leu Met Phe Leu Ala Lys Val Gly Ser Gln Ser Ile Ala Ala Tyr
115 120 125
Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro
130 135 140
His Ser Ser Glu Leu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe Phe
145 150 155 160
Leu Met Pro Gln Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp
165 170 175
Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val
180 185 190
Gln Leu Gln Ala Val Val Thr Leu Leu Gln Asn Phe Ser Trp Asn Trp
195 200 205
Val Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser
210 215 220
Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly Ile Cys Ile Ala His Glu
225 230 235 240
Gly Leu Val Pro Gln His Asp Thr Ser Gly Gln Gln Leu Gly Lys Val
7
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
245 250 255
Leu Asp Val Leu Arg Gln Val Asn Gln Ser Lys Val Gln Val Val Val
260 265 270
Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser Ile
275 280 285
His His Gly Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu
290 295 300
Thr Ser Asp Leu Val Met Thr Leu Pro Asn Ile Ala Arg Val Gly Thr
305 310 315 320
Val Leu Gly Phe Leu Gln Arg Gly Ala Leu Leu Pro Glu Phe Ser His
325 330 335
Tyr Val Glu Thr His Leu Ala Leu Ala Ala Asp Pro A1a Phe Cys Ala
340 345 350
Ser Leu Asn Ala Glu Leu Asp Leu Glu Glu His Val Met Gly Gln Arg
355 360 365
Cys Pro Arg Cys Asp Asp Ile Met Leu Gln Asn Leu Ser Ser Gly Leu
370 375 380
Leu Gln Asn Leu Ser Ala Gly Gln Leu His His Gln Ile Phe Ala Thr
385 390 395 400
Tyr Ala Ala Val Tyr Ser Val Ala Gln Ala Leu His Asn Thr Leu Gln
405 410 415
Cys Asn Val Ser His Cys His Val Ser Glu His Val Leu Pro Trp Gln
420 425 430
Leu Leu Glu Asn Met Tyr Asn Met Ser Phe His Ala Arg Asp Leu Thr
435 440 445
Leu Gln Phe Asp Ala Glu Gly Asn Val Asp Met Glu Tyr Asp Leu Lys
450 455 460
Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu His Thr Val Gly Thr
465 470 475 480
Phe Asn Gly Thr Leu Gln Leu Ghn Gln Ser Lys Met Tyr Trp Pro Gly
485 490 495
Asn Gln Val Pro Val Ser Gln Cys Ser Arg Gln Cys Lys Asp Gly Gln
8
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
500 505 510
Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp
515 520 525
Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr
530 535 540
Pro Cys Asn Gln Asp Gln Trp Ser Pro Glu Lys Ser Thr Ala Cys Leu
545 550 555 560
Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Val Val Leu Ser
565 570 575
Leu Leu Leu Leu Leu Cys Leu Val Leu Gly Leu Ala Leu Ala Ala Leu
580 585 590
Gly Leu Ser Val His His Trp Asp Ser Pro Leu Val Gln Ala Ser Gly
595 600 605
Gly Ser Gln Phe Cys Phe Gly Leu Ile Cys Leu Gly Leu Phe Cys Leu
610 615 620
Ser Val Leu Leu Phe Pro Gly Arg Pro Ser Ser Ala Ser Cys Leu Ala
625 630 635 640
Gln Gln Pro Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu
645 650 655
Phe Leu Gln Ala Ala Glu Thr Phe Val Glu Ser Glu Leu Pro Leu Ser
660 665 670
Trp Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly Leu Trp Ala Trp Leu
675 680 685
Val Val Leu Leu Ala Thr Phe Val Glu Ala Ala Leu Cys Ala Trp Tyr
690 695 700
Leu Ile Ala Phe Pro Pro Glu Val Val Thr Asp Trp Ser Val Leu Pro
705 710 715 720
Thr Glu Val Leu Glu His Cys His Val Arg Ser Trp Val Ser Leu Gly
725 730 735
Leu Val His Ile Thr Asn Ala Met Leu Ala Phe Leu Cys Phe Leu Gly
740 745 750
Thr Phe Leu Val Gln Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly
9
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
755 760 765
Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val
770 775 780
Pro Leu Leu Ala Asn Val Gln Val Ala Tyr Gln Pro A1a Val Gln Met
785 790 795 800
Gly Ala Ile Leu Val Cys Ala Leu Gly Ile Leu Val Thr Phe His Leu
805 810 815
Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Lys Leu Asn Thr Gln Glu
820 825 830
Phe Phe Leu Gly Arg Asn Ala Lys Lys Ala Ala Asp Glu Asn Ser Gly
835 840 845
Gly Gly Glu Ala Ala Gln Gly His Asn Glu
850 855
<210> 4
<211> 2559
<212> DNA
<213> Homo Sapiens
<400> 4
atgctgggcc ctgctgtcct gggcctcagc ctctgggctc tcctgcaccc tgggacgggg 60
gccccattgt gcctgtcaca gcaacttagg atgaaggggg actacgtgct gggggggctg 120
ttccccctgg gcgaggccga ggaggctggc ctccgcagcc ggacacggcc cagcagccct 180
gtgtgcacca ggttctcctc aaacggcctg ctctgggcac tggccatgaa aatggccgtg 240
gaggagatca acaacaagtc ggatctgctg cccgggctgc gcctgggcta cgacctcttt 300
gatacgtgct cggagcctgt ggtggccatg aagcccagcc tcatgttcct ggccaaggca 360
ggcagccgcg acatcgccgc ctactgcaac tacacgcagt accagccccg tgtgctggct 420
gtcatcgggc cccactcgtc agagctcgcc atggtcaccg gcaagttctt cagcttcttc 480
ctcatgcccc aggtcagcta cggtgctagc atggagctgc tgagcgcccg ggagaccttc 540
ccctccttct tccgcaccgt gcccagcgac cgtgtgcagc tgacggccgc cgcggagctg 600
ctgcaggagt tcggctggaa ctgggtggcc gccctgggca gcgacgacga gtacggccgg 660
cagggcctga gcatcttctc ggccctggcc tcggcacgcg gcatctgcat cgcgcacgag 720
ggcctggtgc cgctgccccg tgccgatgac tcgcggctgg ggaaggtgca ggacgtcctg 780
caccaggtga accagagcag cgtgcaggtg gtgctgctgt tcgcctccgt gcacgccgcc 840
cacgccctct tcaactacag catcagcagc aggctctcgc ccaaggtgtg ggtggccagc 900
gaggcctggc tgacctctga cctggtcatg gggctgcccg gcatggccca gatgggcacg 960
gtgcttggct tcctccagag gggtgcccag ctgcacgagt tcccccagta cgtgaagacg 1020
cacctggccc tggccaccga cccggccttc tgctctgccc tgggcgagag ggagcagggt 1080
ctggaggagg acgtggtggg ccagcgctgc ccgcagtgtg actgcatcac gctgcagaac 1140
gtgagcgcag ggctaaatca ccaccagacg ttctctgtct acgcagctgt gtatagcgtg 1200
gcccaggccc tgcacaacac tcttcagtgc aacgcctcag gctgccccgc gcaggacccc 1260
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
gtgaagccct ggcagctcct ggagaacatg tacaacctga ccttccacgt gggcgggctg 1320
ccgctgcggt tcgacagcag cggaaacgtg gacatggagt acgacctgaa gctgtgggtg 1380
tggcagggct cagtgcccag gctccacgac gtgggcaggt tcaacggcag cctcaggaca 1440
gagcgcctga agatccgctg gcacacgtct gacaaccaga agcccgtgtc ccggtgctcg 1500
cggcagtgcc aggagggcca ggtgcgccgg gtcaaggggt tccactcctg ctgctacgac 1560
tgtgtggact gcgaggcggg cagctaccgg caaaacccag acgacatcgc ctgcaccttt 1620
tgtggccagg atgagtggtc cccggagcga agcacacgct gcttccgccg caggtctcgg 1680
ttcctggcat ggggcgagcc ggctgtgctg ctgctgctcc tgctgctgag cctggcgctg 1740
ggccttgtgc tggctgcttt ggggctgttc gttcaccatc gggacagccc actggttcag 1800
gcctcggggg ggcccctggc ctgctttggc ctggtgtgcc tgggcctggt ctgcctcagc 1860
gtcctcctgt tccctggcca gcccagccct gcccgatgcc tggcccagca gcccttgtcc 1920
cacctcccgc tcacgggctg cctgagcaca ctcttcctgc aggcggccga gatcttcgtg 1980
gagtcagaac tgcctctgag ctgggcagac cggctgagtg gctgcctgcg ggggccctgg 2040
gcctggctgg tggtgctgct ggccatgctg gtggaggtcg cactgtgcac ctggtacctg 2100
gtggccttcc cgccggaggt ggtgacggac tggcacatgc tgcccacgga ggcgctggtg 2160
cactgccgca cacgctcctg ggtcagcttc ggcctagcgc acgccaccaa tgccacgctg 2220
gcctttctct gcttcctggg cactttcctg gtgcggagcc agccgggccg ctacaaccgt 2280
gcccgtggcc tcacctttgc catgctggcc tacttcatca cctgggtctc ctttgtgccc 2340
ctcctggcca atgtgcaggt ggtcctcagg cccgccgtgc agatgggcgc cctcctgctc 2400
tgtgtcctgg gcatcctggc tgccttccac ctgcccaggt gttacctgct catgcggcag 2460
ccagggctca acacccccga gttcttcctg ggagggggcc ctggggatgc ccaaggccag 2520
aatgacggga acacaggaaa tcaggggaaa catgagtga 2559
<210> 5
<211> 852
<212> PRT
<213> Homo sapiens
<400> 5
Met Leu Gly Pro Ala Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His
1 5 10 15
Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gln Gln Leu Arg Met Lys
20 25 30
Gly Asp Tyr Va1 Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu
35 40 45
Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg
50 55 60
Phe Ser Ser Asn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val
65 70 75 80
Glu Glu Ile Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly
85 90 95
11
CA 02406999 2002-10-18
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Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro
100 105 110
Ser Leu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr
115 120 125
Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro
130 135 140
His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe
145 150 155 160
Leu Met Pro Gln Val Ser Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala
165 170 175
Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser~Asp Arg Val
180 185 190
Gln Leu Thr Ala Ala Ala Glu Leu Leu Gln Glu Phe Gly Trp Asn Trp
195 200 205
Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gln Gly Leu Ser
210 215 220
Ile Phe Ser Ala Leu Ala Ser Ala Arg Gly Ile Cys Ile Ala His Glu
225 230 235 240
Gly Leu Va1 Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val
245 250 255
Gln Asp Val Leu His Gln Val Asn Gln Ser Ser Val Gln Val Val Leu
260 265 270
Leu Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser Ile
275 280 285
Ser Ser Arg Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ala Trp Leu
290 295 300
Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gln Met Gly Thr
305 310 315 320
Val Leu Gly Phe Leu Gln Arg Gly Ala Gln Leu His Glu Phe Pro Gln
325 330 335
Tyr Val Lys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser
340 345 350
12
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
Ala Leu Gly Glu Arg Glu Gln Gly Leu Glu G1u Asp Val Val Gly Gln
355 360 365
Arg Cys Pro Gln Cys Asp Cys Ile Thr Leu G1n Asn Val Ser Ala Gly
370 375 380
Leu Asn His His Gln Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val
385 390 395 400
Ala Gln Ala Leu His Asn Thr Leu Gln Cys Asn Ala Ser Gly Cys Pro
405 410 415
Ala Gln Asp Pro Val Lys Pro Trp Gln Leu Leu Glu Asn Met Tyr Asn
420 425 430
Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly
435 440 445
Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gln Gly Ser
450 455 460
Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg Thr
465 470 475 480
Glu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gln Lys Pro Val
485 490 495
Ser Arg Cys Ser Arg Gln Cys Gln Glu Gly Gln Val Arg Arg Val Lys
500 505 510
Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys Glu Ala Gly Ser
515 520 525
Tyr Arg Gln Asn Pro Asp Asp Ile Ala Cys Thr Phe Cys Gly Gln Asp
530 535 540
Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg
545 550 555 560
.,
Phe Leu Ala Trp Gly Glu Pro Ala Val Leu Leu Leu Leu Leu Leu Leu
565 570 575
Ser Leu Ala Leu Gly Leu Val Leu Ala Ala Leu Gly Leu Phe Val His
580 585 590
His Arg Asp Ser Pro Leu Val Gln Ala Ser Gly Gly Pro Leu Ala Cys
595 600 605
13
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
Phe Gly Leu Val Cys Leu Gly Leu Val Cys Leu Ser Val Leu Leu Phe
610 615 620
Pro Gly Gln Pro Ser Pro Ala Arg Cys Leu Ala Gln Gln Pro Leu Ser
625 630 635 640
His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gln Ala Ala
645 650 655
Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asp Arg Leu.
660 665 670
Ser Gly Cys Leu Arg Gly Pro Trp Ala Trp Leu Val Val Leu Leu Ala
675 680 685
Met Leu Val Glu Val Ala Leu Cys Thr Trp Tyr Leu Val Ala Phe Pro
690 695 700
Pro Glu Val Val Thr Asp Trp His Met Leu Pro Thr Glu Ala Leu Val
705 710 715 720
His Cys Arg Thr Arg Ser Trp Val Ser Phe Gly Leu Ala His Ala Thr
725 730 ' 735
Asn Ala Thr Leu Ala Phe Leu Cys Phe Leu Gly Thr Phe Leu Val Arg
740 745 750
Ser Gln Pro Gly Arg Tyr Asn Arg Ala Arg Gly Leu Thr Phe Ala Met
755 ~ 760 765
Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val Pro Leu Leu Ala Asn
770 775 780
Val Gln Val Val Leu Arg Pro Ala Val Gln Met Gly Ala Leu Leu Leu
785 790 795 800
Cys Val Leu Gly Ile Leu Ala Ala Phe His Leu Pro Arg Cys Tyr Leu
805 810 815
Leu Met Arg Gln Pro Gly Leu Asn Thr Pro Glu Phe Phe Leu Gly Gly
820 825 830
Gly Pro Gly Asp Ala Gln Gly Gln Asn Asp Gly Asn Thr Gly Asn Gln
835 840 845
Gly Lys His Glu
850
14
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 6
<211> 20
<212> DNA
<213> Mouse
<400> 6
cactagagct gccaccttcc 20
<210> 7
<211> 20
<212> DNA
<213> Mouse
<400> 7
ccctcagcac cactttttgt 20
<210> 8
<211> 20
<212> DNA
<213> Mouse
<400> 8
acaaaaagtg gtgctgaggg 20
<210> 9
<211> 20
<212> DNA
<213> Mouse
<400> 9
caggagaccc aaaggatcaa 20
<210> 10
<211> 20
<212> DNA
<213> Mouse
<400> 10
gcttcagaaa atcgaggcac 20
<210> 11
<211> 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 11
gcatgggcta tgataggtgg 20
<210> 12
<211> 16
<212> DNA
<213> Mouse
<400> 12
tgttgatccc acagcg 16
<210> 13
<211> 20
<212> DNA
<213> Mouse ,
<400> 13
caggaaatgt ccacttctgc 20
<210> 14
<211> 18
<212> DNA
<213> Mouse
<400> 14
tctatcttgc atccagcc 18
<210> 15
<211> 16
<212> DNA
<213> Mouse
<400> 15
gtgctgtgac tgtgcg 16
<210> 16
<211> 18
<212> DNA
<213> Mouse
16
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 16
cgcagcattt atttggag 18
<210> 17
<211> 19
<212> DNA
<213> Mouse
<400> 17
ccgacccttt aggagacac 19
<210> 18
<211> 20
<212> DNA
<213> Mouse
<400> 18
tgtgacttcc tcttccccac 20
<210> 19
<211> 20
<212> DNA
<213> Mouse
<400> 19
tgagccactc cagatgtcag 20
<210>20
<211>20
<212>DNA
<213>Mouse
<400> 20 .
ccaacgtgca gtcaagaaaa 20
<210> 21
<211> 20
<212> DNA
<213> Mouse
<400> 21
ccaacgtgca gtcaagaaaa 20
17
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 22
<211> 20
<212> DNA
<213> Mouse
<400> 22
cgagagacaa agtggtgctg 20
<210> 23
<211> 20
<212> DNA
<213> Mouse
<400> 23
ttatgaaggc cctcaccaac 20
<210> 24
<211> 20
<212> DNA
<213> Mouse
<400> 24
ccagctccta gaattgcctg 20
<210> 25
<211> 20
<212> DNA
<213> Mouse
<400> 25
gcagtctccc gaaacaagtc 20
<210> 26
<211> 20
<212> DNA
<213> Mouse
<400> 26
atagaggaat gggtgcgatg 20
<210> 27
<211> 20
18
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 27
taccaggagg ggtcagtcag 20
<210> 28
<211> 20
<212> DNA
<213> Mouse
<400> 28
tacaagcgag ctgaccaatg 20
<210> 29
<211> 20
<212> DNA
<213> Mouse
<400> 29
ccaatcagct cgagttagcc 20
<210> 30
<211> 20
<212> DNA
<213> Mouse
<400> 30
tgccattgtg gatgttcact 20
<210> 31
<211> 20
<212> DNA
<213> Mouse
<400> 31
gagtccgagg tcggtcaata 20
<210> 32
<211> 20
<212> DNA
<213> Mouse
19
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 32
gctggcttct gtaggtcagg 20
<210> 33
<211> 20
<212> DNA
<213> Mouse
<400> 33
tatgagggtc aagggtcagg 20
<210> 34
<211> 20
<212> DNA
<213> Mouse
<400> 34
cgctttggtg agaactagcc 20
<210> 35
<211> 20
<212> DNA
<213> Mouse
<400> 35
catgtggagt tgtgggagtg ' 20
<210> 36
<211> 20
<212> DNA
<213> Mouse
<400> 36
aatgggcaga agacagatgg 20
<210> 37
<211> 20
<212> DNA
<213> Mouse
<400> 37
tatcagggtc tgtgaagccc 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 38
<211> 20
<212> DNA
<213> Mouse
<400> 38
atacaggacc ctttaccccg 20
<210> 39
<211> 20
<212> DNA
<213> Mouse
<400> 39
cagtgtttct aggtccccca 20
<210> 40
<211> 20
<212> DNA
<213> Mouse
<400> 40
gcctctgtct gccatctctc 20
<210> 41
<211> 20
<212> DNA
<213> Mouse
<400> 41
ataatgttac ctgcaggcgg 20
<210> 42
<211> 20
<212> DNA
<213> Mouse
<400> 42
ctggaaacac ccatgtcctc 20
<210> 43
<211> 20
21
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 43
cgggcacatg gacactttta 20
<210> 44
<211> 20
<212> DNA
<213> Mouse
<400> 44
gagcatgaag tgcaaggtga 20
<210> 45
<211> 20
<212> DNA
<213> Mouse
<400> 45
cgtaggtggc acagttgaga 20
<210> 46
<211> 20
<212> DNA
<213> Mouse
<400> 46
gctgttagtg aggtc,agggc 20
<210> 47
<211> 20
<212> DNA
<213> Mouse
<400> 47
cgtaggtggc acagttgaga 20
<210> 48
<211> 20
<212> DNA
<213> Mouse
22
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 48
gagcatgaag tgcaaggtga 20
<210> 49
<211> 20
<212> DNA
<213> Mouse
<400> 49
tcattttcct agcctcggtg 20
<210> 50
<211> 22
<212> DNA
<213> Mouse
<400> 50
tctaagaaga tgatgcagac cc 22
<210> 51
<211> 20
<212> DNA
<213> Mouse
<400> 51
tgtccttcag ggatagtgcc 20
<210> 52
<211> 20
<212> DNA
<213> Mouse
<400> 52
ggcttcagcc tcaagttctg 20
<210> 53
<211> 20
<212> DNA
<213> Mouse
<400> 53
aaaacaacca agttgccctg 20
23
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 54
<211> 20
<212> DNA
<213> Mouse
<400> 54
ggcactgaaa tgacctggat 20
<210> 55
<211> 20
<212> DNA
<213> Mouse
<400> 55
aacaattcaa gcaacctcgg 20
<210> 56
<211> 20
<212> DNA
<213> Mouse
<400> 56
ctgttccttc ccagactcca 20
<210> 57
<211> 20
<212> DNA
<213> Mouse
<400> 57
ttcagtcacg caaacctgag . 20
<210> 58
<211> 20
<212> DNA
<213> Mouse
<400> 58
gcccaggact ttgtcactgt 20
<210> 59
<211> 20
24
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 59
ggtaacctgc agctccactc 20
<210> 60
<211> 20
<212> DNA
<213> Mouse ,
<400> 60
gggacatgct cttggttcat 20
<210> 61
<211> 20
<212> DNA
<213> Mouse
<400> 61
gaacaaagcc gggtgattta 20
<210> 62
<211> 20
<212> DNA
<213> Mouse
<400> 62
gccctcagtt ctcctagcct 20
<210> 63
<211> 20
<212> DNA
<213> Mouse
<400> 63
ggcagagaag actggtggag 20
<210> 64
<211> 20
<212> DNA
<213> Mouse
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 64
cccagactta gcgtctcagg 20
<210> 65
<211> 20
<212> DNA
<213> Mouse
<400> 65
agcagagacc tttggactcg 20
<210> 66
<211> 20
<212> DNA
<213> Mouse
<400> 66
gaaggctgag tgagtcccag 20
<210> 67
<211> 20
<212> DNA
<213> Mouse
<400> 67
ttgcacgagg agaaggtttt 20
<210> 68
<211> 20
<212> DNA
<213> Mouse
<400> 68
gatgccaacg agacctgaat 20
<210> 69
<211> 20
<212> DNA
<213> Mouse
<400> 69
agaagccaaa accctcacct 20
26
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 70
<211> 20
<212> DNA
<213> Mouse
<400> 70
aaaaagccct gcaagaactt 20
<210> 71
<211> 20
<212> DNA
<213> Mouse
<400> 71
attcaggtct cgttggcatc 20
<210> 72
<211> 20
<212> DNA
<213> Mouse
<400> 72
tgtccgcagt gtggaaacta 20
<210> 73
<211> 20
<212> DNA
<213> Mouse
<400> 73
atgtccaggg tagagagccc 20
<210> 74
<211> 20
<212> DNA
<213> Mouse
<400> 74
ggagttctcc taccctggct 20
<210> 75
<211> 20
27
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 75
gaggctctga gcagtgtcaa 20
<210> 76
<211> 14
<212> DNA
<213> Mouse
<400> 76
gcgatgttgt tgcg 14
<210> 77
<211> 18
<212> DNA
<213> Mouse
<400> 77
cagtgtcttt ccacattt 18
<210> 78
<211> 27
<212> DNA
<213> Mouse
<400> 78
aggcatattg tataataaat ttgtagt 27
<210> 79
<211> 19
<212> DNA
<213> Mouse
<400> 79
ccggatgact ctacttgac 19
<210> 80
<211> 20
<212> DNA
<213> Mouse
28
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 80
gctgtttatg gggtcgagaa 20
<210> 81
<211> 20
<212> DNA
<213> Mouse
<400> 81
aatttctgaa gcagggggat 20
<210> 82
<211> 20
<212> DNA
<213> Mouse
<400> 82
tccccctgct tcagaaatta 20
<210> 83
<211> 20
<212> DNA
<213> Mouse
<400> 83
agggggatga ttgtgagtga 20
<210> 84
<211> 27
<212> DNA
<213> Mouse
<400> 84
cttctttaat caatctctgt ctctgtg 27
<210> 85
<211> 20
<212> DNA
<213> Mouse
<400> 85
gggcacatat gaacctcctg 20
29
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 86
<211> 20
<212> DNA
<213> Mouse
<400> 86
ccaaactctt agcttcttca 20
<210> 87
<211> 21
<212> DNA
<213> Mouse
<400> 87
acacagaaga cactgaagaa c 21
<210> 88
<211> 22
<212> DNA
<213> Mouse
<400> 88
cagttgttag aagcaggatc cc 22
<210> 89
<211> 23
<212> DNA
<213> Mouse
<400> 89
aggtgcatat acctgggata ctc 23
<210> 90
<211> 21
<212> DNA
<213> Mouse
<400> 90
agagtttggt ctcttcccct g 21
<210> 91
<211> 23
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 91
tatccaacac atttatgtct gcg 23
<210> 92
<211> 20
<212> DNA
<213> Mouse
<400> 92
gccagtgtgc tgaaagactg 20
<210> 93
<211> 20
<212> DNA
<213> Mouse
<400> 93
agggacctgg agacatcctt 20
<210> 94
<211> 23
<212> DNA
<213> Mouse
<400> 94
ctgtaggctg cttttatctt ttg 23
<210> 95
<211> 20
<212> DNA
<213> Mouse
<400> 95
tgccccttca gcacatgcca 20
<210> 96
<211> 23
<212> DNA
<213> Mouse
31
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 96
tgcagtgtga catgtgcata gat 23
<210> 97
<211> 21
<212> DNA
<213> Mouse
<400> 97
ggaaagccag gctacgcaga a 21
<210> 98
<211> 23
<212> DNA
<213> Mouse
<400> 98
ctgtaggctg cttttatctt ttg 23
<210> 99
<211> 20
<212> DNA
<213> Mouse
<400> 99
tgccccttca gcacatgcca 20
<210> 100
<211> 22
<212> DNA
<213> Mouse
<400> 100
tagtgtggtt cctgactaac ct 22
<210> 101
<211> 22
<212> DNA
<213> Mouse
<400> 101
cggtctacat agtgagtgat tc 22
32
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 102
<211> 22
<212> DNA
<213> Mouse
<400> 102
aaaagcatcc tgcatccttc tg 22
<210> 103
<211> 22
<212> DNA
<213> Mouse
<400> 103
gggttataca gagaaaccct gt 22
<210> 104
<211> 20
<212> DNA
<213> Mouse
<400> 104
ttccaagctc acacatcagc 20
<210> 105
<211> 20
<212> DNA
<213> Mouse
<400> 105
gtgctgctct gcattgagtg 20
<210> 106
<211> 20
<212> DNA
<213> Mouse
<400> 106
gacagtgtgg gagaatccgt 20
<210> 107
<211> 20
33
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 107
cccaaggcat aggtcacaat 20
<210> 108
<211> 20
<212> DNA
<213> Mouse
<400> 108
attgtgacct atgccttggg 20
<210> 109
<211> 20
<212> DNA
<213> Mouse
<400> 109
cgaaggaccg tcatctgagt 20
<210> 110
<211> 20
<212> DNA
<213> Mouse
<400> 110
ggctttgatg tgaaaaaggc 20
<210> 111
<211> 20
<212> DNA
<213> Mouse
<400> 111
agctcctcat cgctcatgtt 20
<210> 112
<211> 20
<212> DNA
<213> Mouse
34
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 112
tggaacatct ctgtcggaag 20
<210> 113
<211> 20
<212> DNA
<213> Mouse
<400> 113
ggctctcatt gccaccttta 20
<210> 114
<211> 20
<212> DNA
<213> Mouse
<400> 114
ccagagaaca ggagacctgc 20
<210> 115
<211> 20
<212> DNA
<213> Mouse
<400> 115
gtgctggata cactggcaga 20
<210> 116
<211> 20
<212> DNA
<213> Mouse
<400> 116
gcgagacgag tgggtagttc 20
<210> 117
<211> 20
<212> DNA
<213> Mouse
<400> 117
acactgaaac ctcgcttgct 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 118
<211> 20
<212> DNA
<213> Mouse
<400> 118
agcaagcgag gtttcagtgt 20
<210> 119
<211> 20
<212> DNA
<213> Mouse
<400> 119
acggggcttg atccttttat 20
<210> 120
<211> 25
<212> DNA
<213> Mouse
<400> 120
aagttcatgg gcctcaccac ctgtc 25
<210> 121
<211> 22
<212> DNA
<213> Mouse
<400> 121
tactagctac ccttcacata cc 22
<210> 122
<211> 21
<212> DNA
<213> Mouse
<400> 122
acctagccac tgtctcagtc t 21
<210> 123
<211> 21
36
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 123
acagaagcag catttacaca g 21
<210> 124
<211> 20
<212> DNA
<213> Mouse
<400> 124
tgggacagct tcctcaagat r 20
<210> 125
<211> 20
<212> DNA
<213> Mouse
<400> 125
aatgggaatt gtgctcttgg 20
<210> 126
<211> 20
<212> DNA
<213> Mouse
<400> 126
gggcatctgg caaagattta 20
<210> 127
<211> 20
<212> DNA
<213> Mouse
<400> 127
agataacctg tgtgtcccgc 20
<210> 128
<211> 20
<212> DNA
<213> Mouse
37
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 128
gatgtccgag aagggatgtg 20
<210> 129
<211> 20
<212> DNA
<213> Mouse
<400> 129
tgtcagcttt gagtgcatcc 20
<210> 130
<211> 20
<212> DNA
<213> Mouse
<400> 130
acatgcaggc tgtttgacct 20
<210> 131
<211> 20
<212> DNA
<213> Mouse
<400> 131
tgtcagcttt gagtgcatcc 20
<210> 132
<211> 20
<212> DNA
<213> Mouse
<400> 132
gtgctctgca gacaaaccaa 20
<210> 133
<211> 20
<212> DNA
<213> Mouse
<400> 133
gagccatttt gacccttaaa 20
38
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 134
<211> 20
<212> DNA
<213> Mouse
<400> 134
tttcagggtc aaaatggctc 20
<210> 135
<211> 17
<212> DNA
<213> Mouse
<400> 135
tcgacagcaa ctgtgcg 17
<210> 136
<211> 20
<212> DNA
<213> Mouse
<400> 136
ggtgagagtg gggagatgaa 20
<210> 137
<211> 20
<212> DNA
<213> Mouse
<400> 137
cccgggtgag tttaagaacc 20
<210> 138
<211> 20
<212> DNA
<213> Mouse
<400> 138
ggtgagagtg gggagatgaa 20
<210> 139
<211> 20
39
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 139
aggttaggcc caatttcctg 20
<210> 140
<211> 20
<212> DNA
<213> Mouse
<400> 140
ccagggttgc tgtactgaga 20
<210> 141
<211> 20
<212> DNA
<213> Mouse
<400> 141
caggttaggc ccaatttcct 20
<210> 142
<211> 20
<212> DNA
<213> Mouse
<400> 142
ggtcagagtc cttccttccc 20
<210> 143
<211> 20
<212> DNA
<213> Mouse
<400> 143
tccaacttca caggaaaccc 20
<210> 144
<211> 20
<212> DNA
<213> Mouse
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 144
tttcctgtga agttggaggg 20
<210> 145
<211> 20
<212> DNA
<213> Mouse
<400> 145
cacccatatg gcaaacatca .20
<210> 146
<211> 20
<212> DNA
<213> Mouse
<400> 146
ggtcagagtc cttccttccc 20
<210> 147
<211> 20
<212> DNA
<213> Mouse
<400> 147
tccaacttca.caggaaaccc 20
<210> 148
<211> 20
<212> DNA
<213> Mouse
<400> 148
tgatgtttgc catatgggtg 20
<210> 149
<211> 20
<212> DNA
<213> Mouse
<400> 149
gcttgctgct tccgatatgt 20
41
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 150
<211> 19
<212> DNA
<213> Mouse
<400> 150
ggaaaaggga gtcgccata 19
<210> 151
<211> 20
<212> DNA
<213> Mouse
<400> 151
gagccgccta actctcacac 20
<210> 152
<211> 19
<212> DNA
<213> Mouse
<400> 152
aggggataac ctgcatagg 19
<210> 153
<211> 20
<212> DNA
<213> Mouse
<400> 153 ,
acaaaattgc tcatttgccc 20
<210> 154
<211> 20
<212> DNA
<213> Mouse
<400> 154
ccatccccac tagccagata~ 20
<210> 155
<211> 20
42
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 155
gtcccctttg tcacagcaag 20
<210> 156
<211> 20
<212> DNA
<213> Mouse
<400> 156
tgagcacagg atagctccac 20
<210'> 157
<211> 20
<212> DNA
<213> Mouse
<400> 157
aaaagaacac ctgtttgggg 20
<210> 158
<211> 19
<212> DNA
<213> Mouse
<400> 158
taaacctcgg ctgtgtgag 19
<210> 159
<211> 20
<212> DNA
<213> Mouse
<400> 159
ccctcagtga cttcctgtga 20
<210> 160
<211> 20
<212> DNA
<213> Mouse
43
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 160
caaaaccaca tggttaccga 20
<210> 161
<211> 20
<212> DNA
<213> Mouse
<400> 161
gccctattgc caaatgactt 20
<210> 162
<211> 20
<212> DNA
<213> Mouse
<400> 162
ggcagaaagg aatcagaagc 20
<210> 163
<211> 20
<212> DNA
<213> Mouse
<400> 163
cacattagcc attgtcctgg 20
<210> 164
<211> 20
<212> DNA
<213> Mouse
<400> 164
tcctttatgt ccaacagcca 20
<210> 165
<211> 20
<212> DNA
<213> Mouse
<400> 165
catggtctgt gatgtgacca 20
44
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 166
<211> 20
<212> DNA
<213> Mouse
<400> 166
atacccttgg tgagagcagg 20
<210> 167
<211> 20
<212> DNA
<213> Mouse
<400> 167
gctgtcaaat gagaaaggca 20
<210> 168
<211> 20
<212> DNA
<213> Mouse
<400> 168
tatttcatgc tgggaccaaa 20
<210> 169
<211> 20
<212> DNA
<213> Mouse
<400> 169
agagaaaaac agtgggggtg 20
<210> 170
<211> 20
<212> DNA
<213> Mouse
<400> 170
cgggtcctct cttcaccata 20
<210> 171
<211> 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 171
ctacatttcc ctgagctgcc 20
<210> 172
<211> 20
<212> DNA
<213> Mouse
<400> 172
gttgaccatg tcggtaaccc 20
<210> 173
<211> 20
<212> DNA
<213> Mouse
<400> 173
ccacctcacg gaaactgaat 20
<210> 174
<211> 20
<212> DNA
<213> Mouse
<400> 174
ggtgtttggc tcacaaacct 20
<210> 175
<211> 20
<212> DNA
<213> Mouse
<400> 175
gatgcacaca caaaaatccg 20
<210> 176
<211> 20
<212> DNA
<213> Mouse
46
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 176
atcacccacc agaacgaaaa 20
<210> 177
<211> 20
<212> DNA
<213> Mouse
<400> 177
accctccagg agtaggtgct 20
<210>178
<211>20
<212>DNA
<213>Mouse
<400> 178
gatgagacag tgggcaaggt 20
<210> 179
<211> 20
<212> DNA
<213> Mouse
<400> 179
ttgtcaatag caccaagcca ' 20
<210> 180
<211> 20
<212> DNA
<213> Mouse
<400> 180
gccttaatag cccccttgtt 20
<210> 181
<211> 20
<212> DNA
<213> Mouse
v1
<400> 181
gcactcagca ttgcacagat 20
47
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 182
<211> 20
<212> DNA
<213> Mouse
<400> 182
ggacggacaa ttctggaaaa 20
<210> 183
<211> 20
<212> DNA
<213> Mouse
<400> 183
ctatcacacc tccgatgcct 20
<210> 184
<211> 20
<212> DNA
<213> Mouse
<400> 184
caagctggta gaatccccaa 20
<210> 185
<211> 20
<212> DNA
<213> Mouse
<400> 185
tctttggaga agcagaccgt 20
<210> 186
<211> 20
<212> DNA
<213> Mouse
<400> 186
tacagcatat gcatgccagg , 20
<210> 187
<211> 20
48
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 187
attcctcagg gcattacacg 20
<210> 188
<211> 20
<212> DNA
<213> Mouse
<400> 188
gcaatctctt gtgtccaggc 20
<210> 189
<211> 20
<212> DNA
<213> Mouse
<400> 189
attcctcagg gcattacacg 20
<210> 190
<211> 20
<212> DNA
<213> Mouse
<400> 190
tacagcatat gcatgccagg 20
<210> 191
<211> 20
<212> DNA
<213> Mouse
<400> 191
ggcctggaca caagagattg 20
<210> 192
<211> 20
<212> DNA
<213> Mouse
49
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 192
aagtgggtgg acagtgaagg 20
<210> 193
<211> 20
<212> DNA
<213> Mouse
<400> 193
cagcttcctc catcttctgg 20
<210> 194
<211> 20
<212> DNA
<213> Mouse
<400> 194
agagcctcca gtagatggca 20
<210> 195
<211> 20
<212> DNA
<213> Mouse
<400> 195
tcgtggacaa gctccttctt 20
<210> 196
<211> 20
<212> DNA
<213> Mouse
<400> 196
catcgagtat gtcaatggcg 20
<210> 197
<211> 20
<212> DNA
<213> Mouse
<400> 197
ttgtccagtt ttaggtcccg 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 198
<211> 20
<212> DNA
<213> Mouse
<400> 198
cagactgggt tttccgacat 20
<210> 199 -
<211> 20
<212> DNA
<213> Mouse
<400> 199
gtcaaagttg tccaggccat 20
<210> 200
<211> 18
<212> DNA
<213> Mouse
<400> 200
aggacggacc ccaagatg 18
<210> 201
<211> 20
<212> DNA
<213> Mouse
<400> 201
tgtctcgcac ttcctcacag 20
<210> 202
<211> 20
<212> DNA
<213> Mouse
<400> 202
ccagaagatg gaggaagctg 20
<210> 203
<211> 20
51
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 203
tctactggag gctcttggga 20
<210> 204
<211> 20
<212> DNA
<213> Mouse
<400> 204
gaaaaacgac cagatttacg 20
<210> 205
<211> 20
<212> DNA
<213> Mouse
<400> 205
gatctcagca gcatagaacc 20
<210> 206
<211> 20
<212> DNA
<213> Mouse
<400> 206
acacattaag ctgacggact 20
<210> 207
<211> 20
<212> DNA
<213> Mouse
<400> 207
caaacataag gacacccagt 20
<210> 208
<211> 20
<212> DNA
<213> Mouse
52
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 208
actgggtgtc cttatgtttg 20
<210> 209
<211> 20
<212> DNA
<213> Mouse
<400> 209
cctctctttg ggatccttat 20
<210> 210
<211> 20
<212> DNA
<213> Mouse
<400> 210
gtcataaaga ggatcgacca 20
<210> 211
<211> 20
<212> DNA
<213> Mouse
<400> 211
gctctgtcta gaagtgcctg 20
<210> 212
<211> 18
<212> DNA
<213> Mouse
<400> 212
accaagaccg aagagggg , 18
<210>213
<211>22
<212>DNA
<213>Mouse
<400> 213
ggcattacac gctaactttt cc 22
53
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 214
<211> 20
<212> DNA
<213> Mouse
<400> 214
agtgccacca acctggtaag 20
<210> 215
<211> 18
<212> DNA
<213> Mouse
<400> 215
aagtgcctgc agggatgc 18
<210> 216
<211> 20
<212> DNA
<213> Mouse
<400> 216
tgctttggtg agcaatgttt 20
<210> 217
<211> 20
<212> DNA
<213> Mouse
<400> 217
agggacaccc ttaccaggtt 20
<210> 218
<211> 20
<212> DNA
<213> Mouse
<400> 218
ctgatgcttt ggtgagcaat 20
<210> 219
<211> 19
54
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 219
gggacaccct taccaggtt 19
<210> 220
<211> 20
<212> DNA
<213> Mouse
<400> 220
acaggacaaa tgctgggttg 20
<210> 221
<211> 20
<212> DNA
<213> Mouse
<400> 221
gtggtaaaga acgcttggct 20
<210> 222
<211> 24
<212> DNA
<213> Mouse
<400> 222
ggtatctcac ttggtaggaa cctc 24 .
<210> 223
<211> 17
<212> DNA
<213> Mouse
<400> 223
aagaacgctt ggctggc 17
<210> 224
<211> 20
<212> DNA
<213> Mouse
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 224
gccgatcctg gtgatgtact 20
<210> 225
<211> 20
<212> DNA
<213> Mouse
<400> 225
acaatggctc aaaaccgttc 20
<210> 226
<211> 20
<212> DNA
<213> Mouse
<400> 226
gccttgggaa tttaccacct 20
<210> 227
<211> 20
<212> DNA
<213> Mouse
<400> 227
agtacatcac caggatcggc 20
<210> 228
<211> 20
<212> DNA
<213> Mouse
<400> 228
taaaaggcca tgcgataagc 20
<210> 229
<211> 20
<212> DNA
<213> Mouse
<400> 229
agagctctgt ggggttctca 20
56
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 230
<211> 20
<212> DNA
<213> Mouse
<400> 230
gaaggggaca gtgttggaga 20
<210> 231
<211> 20
<212> DNA
<213> Mouse
<400> 231
tccatcaagg aaggatccac~ 20
<210> 232
<211> 19
<212> DNA
<213> Mouse
<400> 232
ggtgggtaat gattggact 19
<210> 233
<211> 19
<212> DNA
<213> Mouse
<400> 233
tgacgtggag ggaactgcc 19
<210> 234
<211> 20
<212> DNA
<213> Mouse
<400> 234
tgagatctgg tgccctctct 20
<210> 235
<211> 20
57
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 235
gcctgatcta ggctggaaaa 20
<210> 236
<211> 20
<212> DNA
<213> Mouse
<400> 236
aggcagaaag cagacaagga 20
<210> 237
<211> 20
<212> DNA
<213> Mouse
<400> 237
cgacagcact tgtgaccact 20
<210> 238
<211> 20
<212> DNA
<213> Mouse
<400> 238
ctgcagatgt agaccaggca 20
<210> 239
<211> 20
<212> DNA
<213> Mouse
<400> 239
ctgtggtgga ttggacagtg 20
<210> 240
<211> 20
<212> DNA
<213> Mouse
58
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 240
ttgcctaaca ctcccaaacc 20
<210> 241
<211> 20
<212> DNA
<213> Mouse
<400> 241
tattaggagc accaccaggc 20
<210> 242
<211> 20
<212> DNA
<213> Mouse
<400> 242
acctgtcttg tgggtggaag 20
<210> 243
<211> 20
<212> DNA
<213> Mouse
<400> 243
ctgtggtgga ttggacagtg 20
<210> 244
<211> 20
<212> DNA
<213> Mouse
<400> 244
gtggcttggt gctattgaca 20
<210> 245
<211> 20
<212> DNA
<213> Mouse
<400> 245
ggggctatta aggccatttt 20
59
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 246
<211> 21
<212> DNA
<213> Mouse
<400> 246
caattgagga atggctacca a ~ 21
<210> 247
<211> 20
<212> DNA
<213> Mouse
<400> 247
tggcttcatg tccattgtgt 20
<210> 248
<211> 22
<212> DNA
<213> Mouse
<400> 248
cagaaccaca aaggtaaatt gc 22
<210> 249
<211> 21
<212> DNA
<213> Mouse
<400> 249
tcatgtttgc tgtccagttt g 21
<210> 250
<211> 29
<212> DNA
<213> Homo Sapiens
<400> 250
gccaccatgc tgggcc.ctgc tgtcctggg 29
<210> 251
<211> 24
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Homo Sapiens
<400> 251
tcactcatgt ttcccctgat ttcc 24
<210> 252
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 252
ctgatttcct gtgttcccgt 20
<210>253
<211>20
<212>DNA
<213>Homo sapiens
<400> 253
catgctggcc tacttcatca 20
<210> 254
<211> 29
<212> DNA
<213> Homo Sapiens
<400> 254
gccttgcagg tcagctacgg tgctagcat 29
<210> 255
<211> 24
<212> DNA
<213> Homo Sapiens
<400> 255
tcactcatgt ttcccctgat ttcc 24
<210> 256
<211> 20
<212> DNA
<213> Homo Sapiens
61
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 256
aggaagcaga gaaaggccag 20
<210> 257
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 257
tcagaactgc ctctgagctg 20
<210> 258
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 258
tcttcacgta ctgggggaac 20
<210> 259
<211> 20
<212> DNA
<213> Homo sapiens
<400> 259
actacagcat cagcagcagg 20
<210>260
<211>20
<212>DNA
<213>Homo sapiens
<400> 260
aagctgaaga acttcccggt 20
<210> 261
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 261
tgggctacga cctctttgat 20
62
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 262
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 262
atcttcaggc gctctgtcct 20
<210>263
<211>20
<212>DNA
<213>Homo Sapiens
<400> 263
gtacgacctg aagctgtggg 20
<210> 264
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 264
atcttcaggc gctctgtcc 19
<210> 265
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 265
gtacgacctg aagctgtggg 20
<210> 266
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 266
atcttcaggc gctctgtcc 19
<210> 267
<211> 21
63
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Homo Sapiens
<400> 267
gagtacgacc tgaagctgtg g 21
<210> 268
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 268
atcttcaggc gctctgtcc 19
<210> 269
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 269
tacgacctga agctgtggg 19
<210> 270
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 270 .
atcttcaggc gctctgtcc 19
<210> 271
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 271
tacgacctga agctgtggg 19
<210> 272
<211> 18
<212> DNA
<213> Homo Sapiens
64
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 272
gctgtcccga tggtgaac 18
<210>273
<211>19
<212>DNA
<213>Homo Sapiens
<400> 273
accttttgtg gccaggatg 19
<210>274
<211>18
<212>DNA
<213>Homo Sapiens
<400> 274
gctgtcccga tggtgaac 18
<210>275
<211>19
<212DNA
>
<213>Homo Sapiens
<400> 275
caccttttgt ggccaggat 19
<210> 276
<211> 18
<212> DNA
<213> Homo Sapiens
<400> 276
gctgtcccga tggtgaac 18
<210> 277
<211> 18
<212> DNA
<213> Homo Sapiens
<400> 277
ccttttgtgg ccaggatg 18
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 278
<211> 18
<212> DNA
<213> Homo Sapiens
<400> 278
cctgaaccag tgggctgt 18
<210> 279
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 279
accttttgtg gccaggatg 19
<210> 280
<211> 18
<212> DNA
<213> Homo Sapiens
<400> 280
cctgaaccag tgggctgt 18
<210> 281
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 281
caccttttgt ggccaggat 19
<210> 282
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 282
tcatgtttcc cctgatttcc 20
<210> 283
<211> 20
66
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Homo Sapiens
<400> 283
catgctggcc tacttcatca 20
<210> 284
<211> 20
<212> DNA
<213> Homo sapiens
<400> 284
atgagcaggt aacacctggg 20
<210> 285
<211> 20
<212> DNA
<213> Homo sapiens
<400> 285
tcatcacctg ggtctccttt 20
<210> 286
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 286
atgagcaggt aacacctggg 20
<210> 287
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 287
ttcatcacct gggtctcctt 20
<210> 288
<211> 20
<212> DNA
<213> Mouse
67
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 288
tgggttgtgt tctctggttg 20
<210> 289
<211> 21
<212> DNA
<213> Mouse
<400> 289
cctttttaca gtctgccagg t 21
<210> 290
<211> 20
<212> DNA
<213> Mouse
<400> 290
tgggttgtgt tctctggttg 20
<210> 291
<211> 21
<212> DNA
<213> Mouse
<400> 291
gatccccttt ttacagtctg c 21
<210> 292
<211> 20
<212> DNA
<213> Mouse
<400> 292
acggggttgg tactgtgtgt 20
<210> 293
<211> 20
<212> DNA
<213> Mouse
<400> 293
cacccattgt tagtgctgga 20
68
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 294
<211> 20
<212> DNA
<213> Mouse
<400> 294
acggggttgg tactgtgtgt 20
<210> 295
<211> 20
<212> DNA
<213> Mouse
<400> 295
cacacaccca cccattgtta 20
<210> 296
<211> 20
<212> DNA
<213> Mouse
<400> 296
tgcattggcc agactagaaa 20
<210> 297
<211> 19
<212> DNA
<213> Mouse
<400> 297
cggctgggct atgacctat 19
<210> 298
<211> 20
<212> DNA
<213> Mouse
<400> 298
tgcattggcc agactagaaa 20
<210> 299
<211> 20
69
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 299
cggctgggct atgacctatt 20
<210> 300
<211> 20
<212> DNA
<213> Mouse
<400> 300
gttctgcagc atgatgtcgt 20
<210> 301
<211> 20
<212> DNA
<213> Mouse
<400> 301 _
ggcagttgtg actctgttgc 20
<210> 302
<211> 20
<212> DNA
<213> Mouse
<400> 302
gttctgcagc atgatgtcgt 20
<210> 303
<211> 20
<212> DNA
<213> Mouse
<400> 303
ctgcaggcag ttgtgactct 20
<210> 304
<211> 20
<212> DNA
<213> Mouse
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 304
ccatcctttt tgcctgtctt 20
<210> 305
<211> 20
<212> DNA
<213> Mouse
<400> 305
tctggaggaa catgtgatgg 20
<210> 306
<211> 20
<212> DNA
<213> Mouse
<400> 306
caccatcctt tttgcctgtc 20
<210> 307
<211> 19
<212> DNA
<213> Mouse
<400> 307
gaacatgtga tggggcaac 19
<210> 308
<211> 19
<212> DNA
<213> Mouse
<400> 308
caaagcagca ggaggagtg 19
<210> 309
<211> 20
<212> DNA
<213> Mouse
<400> 309
aaatgtactg gccaggcaac 20
71
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 310
<211> 20
<212> DNA
<213> Mouse
<400> 310
agtgctagac ccagcaccag ~ 20
<210> 311
<211> 20
<212> DNA
<213> Mouse
<400> 311
aaatgtactg gccaggcaac 20
<210> 312
<211> 20
<212> DNA
<213> Mouse
<400> 312
gcactgacca gtctgtcacc 20
<210> 313
<211> 20
<212> DNA
<213> Mouse
<400> 313
gtccccagag aaaagcacag 20
<210> 314
<211> 20
<212> DNA
<213> Mouse
<400> 314
cagtctgtca ccacctctgg 20
<210> 315
<211> 20
72
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 315
cagtggtccc cagagaaaag 20
<210> 316
<211> 20
<212> DNA
<213> Mouse
<400> 316
tactattcgg ggcttgttgg 20
<210> 317
<211> 20
<212> DNA
<213> Mouse
<400> 317
gcagcactat gtgcctggta 20
<210> 318
<211> 20
<212> DNA
<213> Mouse
<400> 318
tactattcgg ggcttgttgg 20
<210> 319
<211> 20
<212> DNA
<213> Mouse
<400> 319
gcctggtatt tgatcgcttt 20
<210> 320
<211> 20
<212> DNA
<213> Mouse
73
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 320
gctcagctag ggatggagaa 20
<210> 321
<211> 20
<212> DNA
<213> Mouse
<400> 321
cagctcaggg acacaatgaa 20
<210> 322
<211> 20
<212> DNA
<213> Mouse
<400> 322
tcctacaggc tagggctcag 20
<210> 323
<211> 20
<212> DNA
<213> Mouse
<400> 323
cagctcaggg acacaatgaa 20
<210> 324
<211> 20
<212> DNA
<213> Mouse
<400> 324
gggactgatg tgtggcttgt 20
<210> 325
<211> 20
<212> DNA
<213> Mouse
<400> 325
aggcgtccca ggaatagaag 20
74
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 326
<211> 21
<212> DNA
<213> Mouse
<400> 326
ggactgatgt gtggcttgtt t 21
<210> 327
<211> 20
<212> DNA
<213> Mouse
<400> 327
aggcgtccca ggaatagaag 20
<210> 328
<211> 20
<212> DNA
<213> Mouse
<400> 328 ,
tgtttctgtt ctggtggctg 20
<210> 329
<211> 20
<212> DNA
<213> Mouse
<400> 329
atctgcaggc aggatcagac 20
<210> 330
<211> 20
<212> DNA
<213> Mouse
<400> 330
ctcagtggtg ggtgacagtg 20
<210> 331
<211> 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 331
atctgcaggc aggatcagac 20
<210> 332
<211> 20
<212> DNA
<213> Mouse
<400> 332
acacacagta ccaaccccgt 20
<210> 333
<211> 20
<212> DNA
<213> Mouse
<400> 333
cctgtggtga tcaagaagca 20
<210> 334
<211> 20
<212> DNA
<213> Mouse
<400> 334
tgcttcttga tcaccacagg 20
<210> 335
<211> 20
<212> DNA
<213> Mouse
<400> 335
gcaacagagt cacaactgcc 20
<210> 336
<211> 20
<212> DNA
<213> Mouse
76
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 336
acacacagta ccaaccccgt 20
<210> 337
<211> 20
<212> DNA
<213> Mouse
<400> 337
gcaacagagt cacaactgcc 20
<210> 338
<211> 20
<212> DNA
<213> Mouse
<400> 338
gggtttatgt ggcaagcact 20
<210> 339
<211> 20
<212> DNA
<213> Mouse
<400> 339
actccatttg ccttttgtgg 20
<210> 340
<211> 20
<212> DNA
<213> Mouse
<400> 340
cgctacttcg cttttatccg 20
<210> 341
<211> 20
<212> DNA
<213> Mouse
<400> 341
atgatgacgt acgacgacga 20
77
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 342
<211> 21
<212> DNA
<213> Mouse
<400> 342
gaaaacaatc ggggagaagt.c 21
<210> 343
<211> 20
<212> DNA
<213> Mouse
<400> 343
tgaaattatc acacgccagg 20
<210> 344
<211> 20
<212> DNA
<213> Mouse
<400> 344
agtgagaggc ccagtctcaa 20
<210> 345
<211> 20
<212> DNA
<213> Mouse
<400> 345
gatctgatgc cctcttctgc 20
<210> 346
<211> 20
<212> DNA
<213> Mouse
<400> 346
gctagccttg aagccaacac 20
<210> 347
<21~1> 20
78
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 347
tgaacagcat gcttacccag 20
<210> 348
<211> 20
<212> DNA
<213> Mouse
<400> 348
tccctagagg cctgtctgtc 20
<210> 349
<211> 20
<212> DNA
<213> Mouse
<400> 349
tcgtctcgga gcctcttcta 20
<210> 350
<211> 20
<212> DNA
<213> Mouse
<400> 350
gatagtccct tagccagccc 20
<210> 351
<211> 20
<212> DNA
<213> Mouse
<400> 351
gccatagctc ctcactgctc 20
<210> 352
<211> 20
<212> DNA
<213> Mouse
79
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 352
cagagtgggc tctggtcttc 20
<210> 353
<211> 20
<212> DNA
<213> Mouse
<400> 353
ttgtgttcag atgctcctgc 20
<210> 354
<211> 20
<212> DNA
<213> Mouse
<400> 354
ttatttctgt gctagccgcc 20
<210> 355
<211> 20
<212> DNA
<213> Mouse
<400> 355
atcaagtcaa cgtccccaag 20
<210> 356
<211> 20
<212> DNA
<213> Mouse
<400> 356
acctggcctg tgctaatctc 20
<210> 357
<211> 20
<212> DNA
<213> Mouse
<400> 357
gcaccaaccc taagaaagca 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 358
<211> 22
<212> DNA
<213> Mouse
<400> 358
tcaggctaac ctcaaactca ca 22
<210> 359
<211> 27
<212> DNA
<213> Mouse
<400> 359
aaagaaaaga aaagaaaaag tcagaca 27
<210> 360
<211> 20
<212> DNA
<213> Mouse
<400> 360
cccagaactc catcctcaaa 20
<210> 361
<211> 20
<212> DNA
<213> Mouse
<400> 361
cccaacctgt ggtcagctat 20
<210> 362
<211> 20
<212> DNA
<213> Mouse
<400> 362
ggggcaggtg ggtaataagt 20
<210> 363
<211> 20
81
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 363
caaaagccca actccttgag 20
<210> 364
<211> 20
<212> DNA
<213> Mouse
<400> 364
gctcagtggg taagagcac.c 20
<210> 365
<211> 20
<212> DNA
<213> Mouse
<400> 365
ctaccctgcc gctaatctca 20
<210> 366
<211> 20
<212> DNA
<213> Mouse
<400> 366
cagttagcac cccaccctaa 20
<210> 367
<211> 20
<212> DNA
<213> Mouse
a.
<400> 367
tctgcacctc tgttcacctg 20
<210> 368
<211> 20
<212> DNA
<213> Mouse
82
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 368
acctctaggg tttacgggga 20
<210> 369
<211> 20
<212> DNA
<213> Mouse
<400> 369
cctcaggtag tgcaagctcc 20
<210> 370
<211> 20
<212> DNA
<213> Mouse
<400> 370
tcagttacca agggtttcgg 20
<210> 371
<211> 20
<212> DNA
<213> Mouse
<400> 371
ataggttgtc acaggccagg 20
<210> 372
<211> 20
<212> DNA
<213> Mouse
<400> 372
tcagttacca agggtttcgg 20
<210> 373
<211> 20
<212> DNA
<213> Mouse
<400> 373
ataggttgtc acaggccagg 20
83
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 374
<211> 20
<212> DNA
<213> Mouse
<400> 374
gtggttgctg ggatttgaac 20
<210>375
<211>20
<212>DNA
<213>Mouse
<400> 375
caagcaacca aacaaccaaa 20
<210> 376
<211> 20
<212> DNA
<213> Mouse
<400> 376
tccggaggac cataaatctg 20
<210> 377
<211> 20
<212> DNA
<213> Mouse
<400> 377
cacagtccca gtcattccct 20
<210> 378
<211> 20
<212> DNA
<213> Mouse
<400> 378
gtcccaaaag ctagcacagg 20
<210> 379
<211> 20
84
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 379
tcatgagcca ccatgtgatt 20
<210> 380
<211> 20
<212> DNA
<213> Mouse
<400> 380
gaccttcgga agagcagttg 20
<210> 381
<211> ~20
<212> DNA
<213> Mouse
<400> 381
agtgtgtgtc gccatatcca 20
<210> 382
<211> 20
<212> DNA
<213> Mouse
<400> 382
cctactctct ctccccgctt 20
<210> 383
<211> 20
<212> DNA
<213> Mouse
<400> 383
ggaaaatgtt tggccttgaa 20
<210> 384
<211> 20
<212> DNA
<213> Mouse
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 384
ctggagtgaa aggcaggaag 20
<210> 385
<211> 20
<212> DNA
<213> Mouse
<400> 385
aggcggcacc atatgaataa 20
<210> 386
<211> 21
<212> DNA
<213> Mouse
<400> 386
tgagagtggg aattctgttc a 21
<210> 387
<211> 20
<212> DNA
<213> Mouse
<400> 387
ggatgtaatt ggtggcaagg 20
<210> 388
<211> 20
<212> DNA
<213> Mouse
<400> 388
ctgttggagg aggtggccta 20
<210> 389
<211> 21
<212> DNA
<213> Mouse
<400> 389
tgcttgtatg tttttcctcg t 21
86
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 390
<211> 20
<212> DNA
<213> Mouse
<400> 390
tgagagtgcc ctcctctttg 20
<210> 391
<211> 18
<212> DNA
<213> Mouse
<400> 391
gaacccctga ccccagac 18
<210> 392
<211> 22
<212> DNA
<213> Mouse
<400> 392
tgaagtgcag atttttacat gg 22
<210> 393
<211> 20
<212> DNA
<213> Mouse
<400> 393
gttttggggt ggaaaaggat 20
<210> 394
<211> 20
<212> DNA
<213> Mouse
<400> 394
ccgtcgacat ttaggtgaca 20
<210> 395
<211> 20
87
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 395
gatactgggg tggtgggtaa 20
<210> 396
<211> 20
<212> DNA
<213> Mouse
<400> 396
ccgtcgacat ttaggtgaca 20
<210> 397
<211> 20
<212> DNA
<213> Mouse
<400> 397
cgtcccagct gtgtaactga 20
<210> 398
<211> 21
<212> DNA
<213> Mouse
<400> 398
ggaagcaaat gctccactaa a 21
<210> 399
<211> 20
<212> DNA
<213> Mouse
<400> 399
tatccctagc cccttgtgtg 20
<210> 400
<211> 20
<212> DNA
<213> Mouse
88
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 400
ccgtcgacat ttaggtgaca 20
<210> 401
<211> 20
<212> DNA
<213> Mouse
<400> 401
gggtcctgtt ggtagtgacc 20
<210> 402
<211> 20
<212> DNA
<213> Mouse
<400> 402
tataagcagc ccctcattgg 20
<210> 403
<211> 20
<212> DNA
<213> Mouse
<400> 403
caggccagac actgcttaca 20
<210> 404
<211> 20
<212> DNA
<213> Mouse
<400> 404
ccttgggatc tggtgtgact 20
<210> 405
<211> 20
<212> DNA
<213> Mouse
<400> 405
tgggtttaga gtacggctgg 20
89
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 406
<211> 20
<212> DNA
<213> Mouse
<400> 406
acccatttcc taatcccctg 20
<210> 407
<211> 20
<212> DNA
<213> Mouse
<400> 407
atctctccag cccctctcag 20
<210> 408
<211> 20
<212> DNA
<213> Mouse
<400> 408
gggctgggaa ttgaacctat 20
<210> 409
<211> 20
<212> DNA
<213> Mouse
<400> 409
tgaatccctt acagccttgc 20
<210> 410
<211> 20
<212> DNA
<213> Mouse
<400> 410
gccccataaa atccactcct 20
<210> 411
<211> 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 411
gctccggaag gctagaagat 20
<210> 412
<211> 20
<212> DNA
<213> Mouse
<400> 412
ggtttgggag tgttaggcaa 20
<210> 413
<211> 20
<212> DNA
<213> Mouse
<400> 413
actcagttgg cctctcctca 20
<210> 414
<211> 19
<212> DNA
<213> Mouse
<400> 414
acagaaatcc ctcatgcga 19
<210> 415
<211> 21
<212 > DNA
<213> Mouse
<400> 415
tcagtgtgga ccagaaagtc c 21
<210>416
<211>22
<212>DNA
<213>Mouse
91
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 416
tctgcaagtc agctcttgat as 22
<210> 417
<211> 23
<212> DNA
<213> Mouse
<400> 417
actcataagg gtcaagctgt ctg ~ 23
<210> 418
<211> 20
<212> DNA
<213> Mouse
<400> 418
tctccccttt taccactccc 20
<210> 419
<211> 20
<212> DNA
<213> Mouse
<400> 419
gcaaggagtc aaaaacagca 20
<210> 420
<211> 20
<212> DNA
<213> Mouse
<400> 420
gctagttggg gaacaaacca 20
<210> 421
<211> 20
<212> DNA
<213> Mouse
<400> 421
actgcaaatg tccaactcca 20
92
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 422
<211> 20
<212> DNA
<213> Mouse
<400> 422
cagttacaca gctgggacga 20
<210> 423
<211> 20
<212> DNA
<213> Mouse
<400> 423
gcaagagcct agcaatccac 20
<210> 424
<211> 20
<212> DNA
<213> Mouse
<400> 424
cagtttagca ccccacccta 20
<210> 425
<211> 20
<212> DNA
<213> Mouse
<400> 425
tctgcacctc tgttcacctg 20
<210> 426
<211> 20
<212> DNA
<213> Mouse
<400> 426
gggttccact tgatgctgat 20
<210> 427
<211> 20
93
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 427
tggtctgttt cctggagctt 20
<210> 428
<211> 21
<212> DNA
<213> Mouse
<400> 428
tgtagggaat gtttctgcac c 21
<210> 429
<211> 20
<212> DNA
<213> Mouse
<400> 429
acatggaaca ggattctggc 20
<210> 430
<211> 20
<212> DNA
<213> Mouse
<400> 430
gcaggcaaac agacagacaa 20
<210> 431
<211> 20
<212> DNA
<213> Mouse
<400> 431
atgggggatc ccttactgac 20
<210> 432
<211> 20
<212> DNA
<213> Mouse
94
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 432
cggtcaggag tagtgtgggt 20
<210> 433
<211> 20
<212> DNA
<213> Mouse
<400> 433
cagcagctga tattgaggca 20
<210> 434
<211> 22
<212> DNA
<213> Mouse
<400> 434
aatgatgaag tgtcagcctc ag 22
<210> 435
<211> 20
<212> DNA
<213> Mouse
<400> 435
caacagaact caaagcctgg 20
<210> 436
<211> 20
<212> DNA
<213> Mouse
<400> 436
agcaggcaca ggtctcttgt 20
<210> 437
<211> 20
<212> DNA
<213> Mouse
<400> 437
aagaacagga cagtggtggg 20
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 438
<211> 20
<212> DNA
<213> Mouse
<400> 438
cagcgattgg ctcttctctt 20
<210> 439
<211> 20
<212> DNA
<213> Mouse
<400> 439
ggggcttcct ttctgaggta 20
<210> 440
<211> 20
<212> DNA
<213> Mouse
<400> 440
agctcaggtc cagcttggta 20
<210> 441
<211> 20
<212> DNA
<213> Mouse
<400> 441
attttcccct cctgcttctc 20
<210> 442
<211> 20
<212> DNA
<213> Mouse
<400> 442
ccaagcctct gctggttatc 20
<210> 443
<211> 20
96
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 443
tgagggtgga gaatggaaag 20
<210> 444
<211> 20
<212> DNA
<213> Mouse
<400> 444
gccccataaa atccactcct 20
<210> 445
<211> 20
<212> DNA
<213> Mouse
<400> 445
ttgcctaaca ctcccaaacc 20
<210> 446
<211> 20
<212> DNA
<213> Mouse
<400> 446
cagttacaca gctgggacga 20
<210> 447
<211> 20
<212> DNA
<213> Mouse
<400> 447
gcaagagcct agcaatccac 20
<210> 448
<211> 20
<212> DNA
<213> Mouse
97
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 448
cagcaccttc ctctggtctc 20
<210> 449
<211> 20
<212> DNA
<213> Mouse
<400> 449
tgtctccaga ggttctgcct 20
<210> 450
<211> 24
<212> DNA
<213> Mouse
<400> 450
tggtggtgta atactattcc tttg 24
<210> 451
<211> 26
<212> DNA
<213> Mouse
<400> 451
tctttaattt ttggcttttt gataca 26
<210> 452
<211> 20
<212> DNA
<213> Mouse
<400> 452
cagctgtgtg catgttgacc 20
<210> 453
<211> 20
<212> DNA
<213> Mouse
<400> 453
catcatgaag actcagggca 20
98
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 454
<211> 20
<212> DNA
<213> Mouse
<400> 454
gtccacacct ggcttttgtt 20
<210> 455
<211> 20
<212> DNA
<213> Mouse
<400> 455
cagcactcag tgaggttcca 20
<210> 456
<211> 20
<212> DNA
<213> Mouse
<400> 456
atgtaatgga agggctgctg 20
<210> 457
<211> 20
<212> DNA
<213> Mouse
<400> 457
cagcactcag tgaggttcca 20
<210> 458
<211> 21
<212> DNA
<213> Mouse
<400> 458
aaacaggcat gaaactcagg a 21
<210> 459
<211>~ 20
99
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 459
gggtatcatt gtcacctcca 20
<210> 460
<211> 20
<212> DNA
<213> Mouse
<400> 460
cacaggccaa gttgttgttg 20
<210> 461
<211> 20
<212> DNA
<213> Mouse
<400> 461
caggggacct tctgaatgat 20
<210> 462
<211> 20
<212> DNA
<213> Mouse
<400> 462
agctcaggtc cagcttggta 20
<210> 463
<211> 20
<212> DNA
<213> Mouse
<400> 463
accacaaaat tttcccctcc 20
<210> 464
<211> 20
<212> DNA
<213> Mouse
100
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 464
cgggacctaa aactggacaa 20
<210> 465
<211> 20
<212> DNA
<213> Mouse
<400> 465
tggggacagt taccaggaag 20
<210> 466
<211> 20
<212> DNA
<213> Mouse
<400> 466
ccggaggacc ataaatctga 20
<210> 467
<211> 20
<212> DNA
<213> Mouse
<400> 467
cctcaaaaac aagcctgagc 20
<210> 468
<211> 22
<212> DNA
<213> Mouse
<400> 468
ccttcagaaa tgtgtttgga ca 22
<210> 469
<211> 20
<212> DNA
<213> Mouse
<400> 469
tcctgagttc aaatcccagc 20
101
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 470
<211> 20
<212> DNA
<213> Mouse
<400> 470
ctttccattc tccaccctca 20
<210> 471
<211> 20
<212> DNA
<213> Mouse
<400> 471
aggtcctagg gagaggtcca 20
<210> 472
<211> 20
<212> DNA
<213> Mouse
<400> 472
aggcctaccc aaggacatct 20
<210> 473
<211> 20
<212> DNA
<213> Mouse
<400> 473
gcagtgagct gcagagtttg ~ 20
<210> 474
<211> 20
<212> DNA
<213> Mouse
<400> 474
agacacccta ggtcctgctg 20
<210> 475
<211> 22
102
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 475
tgatctttcc aaacgcataa ga 22
<210> 476
<211> 20
<212> DNA
<213> Mouse
<400> 476
gcaagcaacc tgaacatgaa 20
<210> 477
<211> 20
<212> DNA
<213> Mouse
<400> 477
gcttacgatg gtcgtgaggt 20
<210> 478
<211> 20
<212> DNA
<213> Mouse
<400> 478
acatgcctgc ctatctttgc 20
<210> 479
<211> 20
<212> DNA
<213> Mouse
<400> 479
ggaacctgtt ttccatggtg . 20
<210> 480
<211> 20
<212> DNA
<213> Mouse
103
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 480
accttgttcc tggtgtgagc 20
<210> 481
<211> 20
<212> DNA
<213> Mouse
<400> 481
tagctgggac gtggtatggt 20
<210> 482
<211> 20
<212> DNA
<213> Mouse
<400> 482
ccatgggaga ccagaaggta 20
<210> 483
<211> 20
<212> DNA
<213> Mouse
<400> 483
tgagtgtcct ctgcctgatg 20
<210> 484
<211> 20
<212> DNA
<213> Mouse
<400> 484
gcgctgacat cctcctatgt 20
<210> 485
<211> 20
<212> DNA
<213> Mouse
<400> 485
cccactatgg tcccagagaa 20
104
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 486
<211> 20
<212> DNA
<213> Mouse
<400> 486
ttgcacgtct ttgtttcgag 20
<210> 487
<211> 24
<212> DNA
<213> Mouse
<400> 487
aaaggggaat agacctgagt agaa 24
<210> 488
<211> 20
<212> DNA
<213> Mouse
<400> 488
ccaagagtca gccttggagt 20
<210> 489
<211> 20
<212> DNA
<213> Mouse
<400> 489
ggacaggtag ctcacccaac 20
<210> 490
<211> 19
<212> DNA
<213> Mouse
<400> 490
tgccagcttt ggctatcat 19
<210> 491
<211>~20
105
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 491
ttcattgtgt ccctgagctg 20
<210> 492
<211> 24
<212> DNA
<213> Mouse
<400> 492
agctttggct atcatgggtc tcag 24
<210> 493
<211> 22
<212> DNA
<213> Mouse
<400> 493
accaccgcca ctgttctcat ct 22
<210> 494
<211> 20
<212> DNA
<213> Mouse
<400> 494
tgtgggggaa gaacatagaa 20
<210> 495
<211> 22
<212> DNA
<213> Mouse
<400> 495
tgatgtgtgg cttgtttctc tt 22
<210> 496
<211> 20
<212> DNA
<213> Mouse
106
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 496
ataggtgggg agggagctaa 20
<210> 497
<211> 22
<212> DNA
<213> Mouse
<400> 497
tgatgtgtgg cttgtttctc tt 22
<210> 498
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 498
tgtgcctgtc acagcaactt 20
<210> 499
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 499
catgctagca ccgtagctga 20
<210> 500
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 500
ggagaccttc ccctccttct 20
<210> 501
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 501
gctgtagttg aagagggcgt 20
107
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 502
<211> 18
<212> DNA
<213> Homo Sapiens
<400> 502
gtgcttggct tcctccag 18
<210> 503
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 503
caggtcgtac tccatgtcca 20
<210> 504
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 504
tggagtacga cctgaagctg 20
<210>505
<211>20
<212>DNA
<213>Homo Sapiens
<400> 505
actcatcctg gccacaaaag . 20
<210> 506
<211> 19
<212> DNA
<213> Homo Sapiens
<400> 506
gaacaggagg acgctgagg 19
<210> 507
<211> 20
108
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Homo Sapiens
<400> 507
cttttgtggc caggatgagt 20
<210> 508
<211> 20
<212> DNA
<213> Homo Sapiens
<400> 508
tcacctcacc tggttgtcag 20
<210> 509
<211> 20
<212> DNA
<213> Homo sapiens
<400> 509
gtacgacctg aagctgtggg 20
<210> 510
<211> 27
<212> DNA
<213> Homo Sapiens
<400> 510
ggctgagatc acagggttgg gtcactc 27
<210> 511
<211> 27
<212> DNA
<213> Homo Sapiens
<400> 511
ccgtgcctgt tggaagttgc ctctgcc 27
<210> 512
<211> 20
<212> DNA
<213> Mouse
109
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 512
aattcccagc aaccactcac 20
<210> 513
<211> 20
<212> DNA
<213> Mouse
<400> 513
cagacactcc agaagagggc 20
<210> 514
<211> 20
<212> DNA
<213> Mouse
<400> 514
tgactgctct tccgaaggtt 20
<210> 515
<211> 20
<212> DNA
<213> Mouse
<400> 515
tttgtggaat agccaaagcc 20
<210> 516
<211> 20
<212> DNA
<213> Mouse
<400> 516
tctctcctct cttctccccc 20
<210> 517
<211> 20
<212> DNA
<213> Mouse
<400> 517
agcagggtgc atcaccttat 20
110
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 518
<211> 20
<212> DNA
<213> Mouse
<400> 518
taggagtgcc ccataggttg 20
<210> 519
<211> 20
<212> DNA
<213> Mouse
<400> 519
tcattgtacc cagccagtca 20
<210> 520
<211> 20
<212> DNA
<213> Mouse
<400> 520
aggactgagc ctggatgaga 20
<210> 521
<211> 20
<212> DNA
<213> Mouse
<400> 521
ctgggcgttt tgttttgttt 20
<210> 522
<211> 20
<212> DNA
<213> Mouse
<400> 522
cttcctcctg cagctaccac 20
<210> 523
<211> 20
111
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 523
accctgctac aacgcagact 20
<210> 524
<211> 20
<212> DNA
<213> Mouse
<400> 524
tccaaccttg acacccattt 20
<210> 525
<211> 20
<212> DNA
<213> Mouse
<400> 525
agccagggct acacagagaa 20
<210> 526
<211> 20
<212> DNA
<213> Mouse
<400> 526
ctgcttttcc tcagcaactg 20
<210> 527
<211> 20
<212> DNA
<213> Mouse
<400> 527
attcgccgtt agaagctagg 20
<210> 528
<211> 20
<212> DNA
<213> Mouse
112
P
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 528
aactgtacgt ggctgctggt 20
<210> 529
<211> 20
<212> DNA
<213> Mouse
<400> 529
attcgccgtt agaagctagg 20
<210> 530
<211> 20
<212> DNA
<213> Mouse
<400> 530
gccaggtgac ccttatgaaa 20
<210> 531
<211> 20
<212> DNA
<213> Mouse
<400> 531
gagagatggc agacagaggc 20
<210> 532
<211> 20
<212> DNA
<213> Mouse
<400> 532
agctctctgt ccctggtgaa 20
<210> 533
<211> 20
<212> DNA
<213> Mouse
<400> 533
tgccaaccac tagcctctct 20
113
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 534
<211> 20
<212> DNA
<213> Mouse
<400> 534
ctgaaccctc cactctcctg 20
<210> 535
<211> 20
<212> DNA
<213> Mouse
<400> 535
agccagggct acacagagaa 20
<210> 536
<211> 20
<212> DNA
<213> Mouse
<400> 536
agccagggct acacagagaa 20
<210> 537
<211> 20
<212> DNA
<213> Mouse
<400> 537
accctgctac aacgcagact 20
<210> 538
<211> 20
<212> DNA
<213> Mouse
<400> 538
gcaagtttca ggagctaggg 20
<210> 539
<211> 20
114
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 539
ccccagaacc agagaccata , 20
<210> 540
<211> 20
<212> DNA
<213> Mouse
<400> 540
ccccagaacc agagaccata 20
<210> 541
<211> 20
<212> DNA
<213> Mouse
<400> 541
ctaggggact ctgccaagtg 20
<210> 542
<211> 20
<212> DNA
<213> Mouse
<400> 542
caagacaccc agtcccaact 20
<210> 543
<211> 20
<212> DNA
<213> Mouse
<400> 543
tacttcccct ttcccgaact 20
<210> 544
<211> 20
<212> DNA
<213> Mouse
115
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 544
tccttggtgc ttaccctcac 20
<210> 545
<211> 20
<212> DNA
<213> Mouse
<400> 545
tgttcctgag ttcacaacgc 20
<210> 546
<211> 20
<212> DNA
<213> Mouse
<400> 546
attcccagca actacatggc 20
<210> 547
<211> 20
<212> DNA
<213> Mouse
<400> 547
acatgtccac tgtggcaaaa 20
<210> 548
<211> 20
<212> DNA
<213> Mouse
<400> 548
tgtcatgagt ttgaggccag 20
<210> 549
<211> 20
<212> DNA
<213> Mouse
<400> 549
atcagacagc ccacaacctc 20
116
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 550
<211> 20
<212> DNA
<213> Mouse
<400> 550
tatgtgccac cacacctgtc 20
<210> 551
<211> 20
<212> DNA
<213> Mouse
<400> 551
gctcaaggaa ggacacacct 20
<210> 552
<211> 22
<212> DNA
<213> Mouse
<400> 552
tgctcttaac attttgagcc at 22
<210> 553
<211> 20
<212> DNA
<213> Mouse
<400> 553
gctcagcccc tgaatcaata 20
<210> 554
<211> 20
<212> DNA
<213> Mouse
<400> 554
gggatctgcc tgtcttacca 20
<210> 555
<211> 20
117
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 555
ggaaggtagg gcctggtaat 20
<210> 556
<211> 20
<212> DNA
<213> Mouse
<400> 556
gctccaagat ctgtgcgatt 20
<210> 557
<211> 20
<212> DNA
<213> Mouse
<400> 557
ttagcgttag ggtgagggtg 20
<210> 558
<211> 20
<212> DNA
<213> Mouse
<400> 558
ggagactacg gacttgtggc 20
<210> 559
<211> 20
<212> DNA
<213> Mouse
<400> 559
cagttcttcc cgaaaaccac 20
<210> 560
<211> 20
<212> DNA
<213> Mouse
118
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 560
tttctgggaa ctgagatggc 20
<210> 561
<211> 20
<212> DNA
<213> Mouse
<400> 561
gttggggctg ctcatagaaa 20
<210> 562
<211> 20
<212> DNA
<213> Mouse
<400> 562
gctgtggctc tcttggagtt 20
<210> 563
<211> 20
<212> DNA
<213> Mouse
<400> 563
ctctgatttc ccacatgcct 20
<210> 564
<211> 20
<212> DNA
<213> Mouse
<400> 564
aagagggagc actgaggaca 20
<210> 565
<211> 20
<212> DNA
<213> Mouse
<400> 565
cagcagcaaa tgacctttca 20
119
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 566
<211> 20
<212> DNA
<213> Mouse
<400> 566
gaggcaggca gatttctgag 20
<210> 567
<211> 20
<212> DNA
<213> Mouse
<400> 567
gtttcacatg ttgtggtggc 20
<210> 568
<211> 20
<212> DNA
<213> Mouse
<400> 568
gggacctttg ggatagcatt 20
<210> 569
<211> 20
<212> DNA
<213> Mouse
<400> 569
i
tcagacatct ctggcctcct 20
<210>570 "
<211>20
<212>DNA
<213>Mouse
<400> 570
ttcactaagt tgcccaggct 20
<210> 571
<211> 22
120
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 571
tgcctttttc tcacattgtc tc 22
<210> 572
<211> 20
<212> DNA
<213> Mouse
<400> 572
ttagaagcag aggcagaggc 20
<210> 573
<211> 20
<212> DNA
<213> Mouse
<400> 573
gacctttgga agagcagtcg 20
<210> 574
<211> 20
<212> DNA
<213> Mouse
<400> 574
tggcagctca caatgtcttt 20
<210> 575
<211> 20
<212> DNA
<213> Mouse
<400> 575
ggtgtggtgt aggggaagaa 20
<210> 576
<211> 22
<212> DNA
<213> Mouse
121
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 576
tttcaactgc aaacacaaac ag 22
<210> 577
<211> 19
<212> DNA
<213> Mouse
<400> 577
agggccaagg aaggagaat 19
<210> 578
<211> 24
<212> DNA
<213> Mouse
<400> 578
gcaaatatat agggtaccga gctg 24
<210> 579
<211> 20
<212> DNA
<213> Mouse
<400> 579
cagattctcc agctgtcagg 20
<210> 580
<211> 19
<212> DNA
<213> Mouse
<400> 580
ctgtgtttcc gcaccaagt 19
<210> 581
<211> 20
<212> DNA
<213> Mouse
<400> 581
ctgcccgtcc ttatcttctg 20
122
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<220> 582
<211> 20
<212> DNA
<213> Mouse
<400> 582
acgcacgctc actcatacac 20
<210> 583
<211> 20
<212> DNA
<213> Mouse
<400> 583
cagcagaggt gatgggttct 20
<210> 584
<211> 22
<212> DNA
<213> Mouse
<400> 584
ttgtcacaca gtggttaaat gc 22
<210> 585
<211> 20
<212> DNA
<213> Mouse
<400> 585
tagaaccgtg gctgaggact 20
<210> 586
<211> 24
<212> DNA
<213> Mouse
<400> 586
ccgtaagata tgaaagaact tgga 24
<210> 587
<211> 20
123
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 587
taatcctggc ttagcgcttg 20
<210> 588
<211> 20
<212> DNA
<213> Mouse
<400> 588
tagaaagcac aggggacagg 20
<210> 589
<211> 20
<212> DNA
<213> Mouse
<400> 589
ccttcctcgt ctgagctgtt 20
<210> 590
<211> 20
<212> DNA
<213> Mouse
<400> 590
ttgggacgtg acctgagaat 20
<210> 591
<211> 20
<212> DNA
<213> Mouse
<400> 591
tatgtgtctg gccgttgttc 20
<210> 592
<211> 19
<212> DNA
<213> Mouse
124
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 592
gatgtgggtg caggtgaag 19
<210> 593
<211> 20
<212> DNA
<213> Mouse
<400> 593
ccccttctgg agtgtctgaa 20
<210> 594
<211> 21
<212> DNA
<213> Mouse
<400> 594
tctaggcagg gctacctttt t 21
<210> 595
<211> 19
<212> DNA
<213> Mouse
<400> 595
gctgagcagc ctctagcaa 19
<210> 596
<211> 20
<212> DNA
<213> Mouse
<400> 596
accatggctt ttcccagtaa 20
<210> 597
<211> 20
<212> DNA
<213> Mouse
<400> 597
ctgtgccttt ggtgatcaga 20
125
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 598
<211> 20
<212> DNA
<213> Mouse
<400> 598
tgtggcactc tacggcataa 20
<210> 599
<211> 23
<212> DNA
<213> Mouse
<400> 599
tgcatcacta ttaagcctca acc 23
<210> 600
<211> 23
<212> DNA
<213> Mouse
<400> 600
aagaatttgc aaagactgtg aga 23
<210> 601
<211> 20
<212> DNA
<213> Mouse
<400> 601
ctggaccttt ggaagagcag 20
<210> 602
<211> 20
<212> DNA
<213> Mouse
<400> 602
ggtggctcaa accatccata 20
<210> 603
<211> 20
126
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 603
gagggcaatg agcaaaatgt 20
<210> 604
<211> 20
<212> DNA
<213> Mouse
<400> 604
ggtcctgtct ctggttcagg 20
<210> 605
<211> 20
<212> DNA
<213> Mouse
<400> 605
taacacccac atcaggcaac 20
<210> 606
<211> 22
<212> DNA
<213> Mouse
<400> 606
tttcatttcc tggtgttcct tt 22
<210> 607
<211> 20
<212> DNA
<213> Mouse
<400> 607
aaacacaggc ggaacgatag 20
<210> 608
<211> 20
<212> DNA
<213> Mouse
127
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 608
ctatcgttcc gcctgtgttt 20
<210> 609
<211> 21
<212> DNA
<213> Mouse
<400> 609
aaggaagagg atggagaaag a 21
<210> 610
<211> 20
<212> DNA
<213> Mouse
<400> 610
cgggtcttaa tggagcagag 20
<210> 611
<211> 20
<212> DNA
<213> Mouse
<400> 611
tcctccccag ttacctagca 20
<210> 612
<211> 19
<2l2> DNA
<213> Mouse
<400> 612
cagcaggcaa gatgacctc 19
<210> 613
<211> 20
<212> DNA
<213> Mouse
<400> 613
gtccctcacc agccatgtta 20
128
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 614
<211> 20
<212> DNA
<213> Mouse
<400> 614
agcctgggct aagttgtgtg 20
<210> 615
<211> 20
<212> DNA
<213> Mouse
<400> 615
tatgggccaa tgttgttcct 20
<210> 616
<211> 20
<212> DNA
<213> Mouse
<400> 616
atggtggctc acaaccatct 20
<210> 617
<211> 20
<212> DNA
<213> Mouse
<400> 617
ttgtcctctg attgcagcat 20
<210> 618
<211> 20
<212> DNA
<213> Mouse
<400> 618
cttgggtcat caggctttgt 20
<210> 619
<211> 20
129
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 619
aagctgccct gctctctctap 20
<210> 620
<211> 20
<212> DNA
<213> Mouse
<400> 620
atgctcagcc tgctttgttt 20
<210> 621
<211> 20
<212> DNA
<213> Mouse
<400> 621
gctgatagcc ctgggttcta 20
<210> 622
<211> 21
<212> DNA
<213> Mouse
<400> 622
tgtacgcaca aattgacttg c 21
<210> 623
<211> 21
<212> DNA
<213> Mouse
<400> 623
gaatccacat tgcaaagcct a 21
<210> 624
<211> 20
<212> DNA
<213> Mouse
130
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 624
cacaggcaaa tgaagggaag 20
<210> 625
<211> 20
<212> DNA
<213> Mouse
<400> 625
ccagacttct ccagctctcc 20
<210> 626
<211> 21
<212> DNA
<213> Mouse
<400> 626
tcctcgagag gctctaggtt t 21
<210> 627
<211> 20
<212> DNA
<213> Mouse
<400> 627
tgcctagtca accacaggag 20
<210> 628
<211> 21
<212> DNA
<213> Mouse
<400> 628
v
cctgtggttg actaggcaga a 21
<210> 629
<211> 20
<212> DNA
<213> Mouse
<400> 629
gcctgatagc ctggaataca 20
131
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 630
<211> 20
<212> DNA
<213> Mouse
<400> 630
aaagggatgt gtggcgtaag 20
<210> 631
<211> 20 l
<212> DNA
<213> Mouse .
<400> 631
caaaacccaa ccttctcagc 20
<210> 632
<211> 20
<212> DNA
<213> Mouse
<400> 632
tgcactgacc gtgatagagg 20
<210> 633
<211> 20
<212> DNA
<213 >_ Mouse
<400> 633
eggtgtagct ctggctgtct 20
<210> 634
<211> 20
<212> DNA
<213> Mouse
<400> 634
catctcacca actcgcactt 20
<210> 635
<211> 21
132
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212 > DNA
<213> Mouse
<400> 635
tttctgggaa caaagaggct a 21
<210> 636
<211> 20
<212 > DNA
<213> Mouse
<400> 636
gaacccaagt gttggggtaa 20
<210> 637
<211> 20
<212 > DNA
<213> Mouse
<400> 637
tggaagccca tctgtctctt 20
<210> 638
<211> 20
<212 > DNA
<213> Mouse
<400> 638
aaatgcaagt gggtgcttct 20
<210> 639
<211> 19
<212> DNA
<213> Mouse
<400> 639
ccagaagagg gcgtcagat 19
<210> 640
<211> 20
<212> DNA
<213> Mouse
133
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<400> 640
ggtgtgcacc accatattca 20
<210> 641
<211> 21
<212> DNA
<213> Mouse
<400> 641
gggaattatc agccaaaaag c 21
<210> 642
<211> 20
<212> DNA
<213> Mouse
<400> 642
gcccaactga aagctcaact 20
<210> 643
<211> 21
<212> DNA
<213> Mouse
<400> 643
ggaaggggga taacaattga a 21
<210> 644
<211> 23
<212> DNA
<213> Mouse
<400> 644
tgctaatttc aagcacagtg aga 23
<210> 645
<211> 20
<212> DNA
<213> Mouse
<400> 645
agcttgacac cttgacagca 20
134
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<210> 646
<211> 20
<212> DNA
<213> Mouse
<400> 646
aacctgcaga gaggagacca 20
<210> 647
<211> 20
<212> DNA
<213> Mouse
<400> 647
ctccaagggg aggactcatt 20
<210> 648
<211> 24
<212> DNA
<213> Mouse
<400> 648
ttcaattgag tttctctcct ctga 24
<210> 649
<211> 20
<212> DNA
<213> Mouse
<400> 649
tgcaggacca agaagtaggc 20
<210> 650
<211> 20
<212> DNA
<213> Mouse
<400> 650
cgagatctga tgccctcttc 20
<210> 651
<211> 20
135
CA 02406999 2002-10-18
WO 01/83749 PCT/USO1/13387
<212> DNA
<213> Mouse
<400> 651
tgctgagagc agaaaaggaa 20
<210> 652
<211> 166
<212> DNA
<213> Mouse
<400> 652
gcagtgagct gcagagtttg cagaatgagg gcactctaaa ctcatcaagt gaggaggccc 60
ttccctcaca ctccagatgg ctgataggtg gcattacatg gtccancgcg cgcacgcgct 120
cagatgcaat ctccacattc ataaccagat gtccttgggt aggcct 166
136
