U.S. patent application number 12/075208 was filed with the patent office on 2009-02-26 for isolated nucleic acids and polypeptides associated with glucose homeostasis disorders and method of detecting the same.
Invention is credited to Y. T. Chen, Alison J. McVie-Wylie.
Application Number | 20090053713 12/075208 |
Document ID | / |
Family ID | 27737086 |
Filed Date | 2009-02-26 |
United States Patent
Application |
20090053713 |
Kind Code |
A1 |
Chen; Y. T. ; et
al. |
February 26, 2009 |
Isolated nucleic acids and polypeptides associated with glucose
homeostasis disorders and method of detecting the same
Abstract
An isolated and substantially pure nucleic acid sequence located
between D20S119 and D20S178 on human chromosome 20q13, the nucleic
acid sequence including: a nucleic acid sequence coding for a
glucose transporting protein and having the sequence shown in SEQ
ID NO: 1; or a nucleic acid sequence having at least 70% sequence
identity with the nucleic acid sequence shown in SEQ ID NO: 1. The
disclosed nucleic acid sequences map to a locus associated with
human Type II diabetes mellitus and, therefore, therapeutic and
diagnostic screening methods, which accommodate naturally and
artificially occurring polymorphisms, are also disclosed.
Inventors: |
Chen; Y. T.; (Chapel Hill,
NC) ; McVie-Wylie; Alison J.; (Brookline,
MA) |
Correspondence
Address: |
JENKINS, WILSON, TAYLOR & HUNT, P. A.
Suite 1200 UNIVERSITY TOWER, 3100 TOWER BLVD.,
DURHAM
NC
27707
US
|
Family ID: |
27737086 |
Appl. No.: |
12/075208 |
Filed: |
March 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10328198 |
Dec 23, 2002 |
7355023 |
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12075208 |
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PCT/US01/20167 |
Jun 25, 2001 |
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10328198 |
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60215477 |
Jun 29, 2000 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C07K 14/62 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101; G01N 33/564 20130101;
G01N 2500/00 20130101; G01N 2800/042 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-8. (canceled)
9. A method of facilitating a diagnosis of susceptibility to a
glucose homeostasis disorder, the method comprising: (a) obtaining
a biological sample from a subject; (b) determining a sequence of a
target nucleic acid sequence from the biological sample, wherein
the target nucleic acid sequence is a glucose transporter gene
located on chromosome 20q13 between D20S119 and D20S178; and (c)
determining a sequence variation between a wild type nucleic acid
sequence and the target nucleic acid sequence, the presence of a
variation between the wild type nucleic acid sequence and the
target nucleic acid sequence indicating susceptibility of the
subject to a glucose homeostasis-related disorder.
10. The method of claim 9, wherein the target nucleic acid sequence
is a nucleic acid sequence which hybridizes to and is at least 70%
complementary to the glucose transporter gene of SEQ ID NO: 1 and
the wild type nucleic acid sequence is SEQ ID NO: 1.
11. (canceled)
12. The method of claim 9, wherein the disorder affecting glucose
homeostasis is human Type II diabetes mellitus.
13. The method of claim 9, wherein the biological sample is a
tissue sample or a blood sample.
14. The method of claim 9, wherein the determining the sequence is
performed using a dideoxy sequencing method.
15. The method of claim 9, wherein the target nucleic acid sequence
is amplified prior to the determining the sequence.
16-20. (canceled)
21. A method of identifying a subject having a polymorphism of the
nucleic acid sequence shown in SEQ ID NO: 1, the method comprising:
(a) sequencing a target nucleic acid of a sample from a subject by
dideoxy sequencing; and b) identifying a polymorphism by comparing
the nucleic acid sequence of the target nucleic acid sequence to
the DNA sequence shown in SEQ ID NO: 1.
22. The method of claim 21, wherein the sequencing is performed
following amplification of the target nucleic acid.
23-66. (canceled)
67. A method of facilitating a diagnosis of susceptibility to a
glucose homeostasis disorder by determining the presence of a
genetic variation in or near a glucose transporter gene,
comprising: a) obtaining a biological sample from a subject; and b)
performing a mutation analysis of genomic DNA and/or RNA from the
biological sample, wherein performing the mutation analysis
determines whether there is a genetic variation in or near the
glucose transporter gene.
68. The method of claim 23, wherein the subject is a human.
69. The method of claim 23, wherein the disorder affecting glucose
homeostasis is human Type II diabetes mellitus.
70. The method of claim 23, wherein the biological sample is a
tissue sample or a blood sample.
71. The method of claim 23, wherein the genetic variation is one or
more of a polymorphism, a single nucleotide polymorphism (SNP), a
chromosomal translocation, a chromosomal inversion, or a repeat
expansion.
72. The method of claim 23, wherein the mutation analysis is
performed by a technique selected from the group consisting of: a
single-strand conformation polymorphism (SSCP) analysis, a
SSCP/heteroduplex analysis, an enzyme mismatch cleavage, an
allele-specific hybridization, a restriction analysis of the
genomic DNA and/or RNA, and a direct sequence analysis of the
genomic DNA and/or RNA.
73. The method of claim 28, wherein the mutation analysis is
performed by the direct sequence analysis and the genomic DNA
and/or RNA is amplified prior to the direct sequence analysis.
74. The method of claim 28, wherein the direct sequence analysis is
performed using one or more oligonucleotide primers having a design
based on an intronic sequence flanking an exon of the glucose
transporter gene shown in Table 2.
75. The method of claim 28, wherein the direct sequence analysis is
performed using one or more oligonucleotide primers having a design
based on an exon of the glucose transporter gene of SEQ ID NO:
1.
76. The method of claim 28, wherein the genetic variation is a
single nucleotide polymorphism (SNP) and the mutation analysis is
the allele-specific hybridization, the direct sequence analysis of
the genomic DNA and/or RNA, or the restriction analysis of the
genomic DNA and/or RNA.
77. The method of claim 28, wherein the genetic variation is a
repeat expansion and the mutation analysis comprises PCR of the
genomic DNA and/or RNA with one or more oligonucleotide primers
flanking the repeat.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is based on and claims priority to U.S.
Provisional Application Ser. No. 60/215,477, filed Jun. 29, 2000,
herein incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to nucleic acid
sequences coding for sugar transporters useful for screening
against glucose homeostasis disorders, and particularly to nucleic
acid sequences coding for the glucose transporter which map to the
region between D20S119 and D20S178 on human chromosome 20q13.
BACKGROUND ART
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular
cases, to provide additional details respecting the practice, are
incorporated herein by reference, and for convenience, are
referenced by author and date in the text.
[0004] Diabetes mellitus is a condition in which the glucose
homeostasis of a subject becomes unbalanced and leads to a
hyperglycemic systemic condition. There are two forms of the
diabetic condition, Type I and Type II. Type I diabetes usually
occurs in individuals under approximately 20 years of age, is
insulin-dependent, is commonly accompanied by ketoacidosis and
represents about 10% of the diabetic population. Type II diabetes
affects approximately 5 percent of the adult American population
and represents about 90% of the diabetic population. Type II
diabetes is commonly associated with obesity, usually occurs in
individuals over approximately 40 years of age and is non-insulin
dependent. A subset of type II diabetes can occur in younger
individuals and is referred to as maturity onset diabetes of the
young (MODY).
[0005] Interestingly, persons suffering from Type II diabetes can
exhibit normal or even elevated levels of insulin, which helps the
body maintain glucose homeostasis. This suggests that in Type II
diabetes there might be a decreased sensitivity of the body to the
effects of insulin, although defective insulin secretion can also
be involved. Another critical factor in systemic maintenance of
glucose homeostasis is the uptake of glucose by glucose transporter
proteins. Furthermore, other factors include excessive hepatic
glucose production and increased lipolysis in adipose tissue. See,
e.g., Edelman, (1998) Adv. Internal Med. 43: 449-500; Vaag, (1999)
Dan. Med. Bull. 46 (3): 197-234.
[0006] Type II diabetes is typically slow to develop, is hereditary
and is associated with obese individuals. Ongoing research has
demonstrated that there is both a genetic component and an
environmental component that leads to the Type II diabetic
condition. The environmental component can lead to an acquired
resistance to the action of insulin. The genetic component
manifests itself as a condition rendering an individual predisposed
to insulin resistance and more susceptible to the chronic onset of
the diabetic condition. The genetic component can also involve
glucose uptake by glucose transport proteins. Studies of the Pima
Indian population, which has an unusually high incidence of Type II
diabetes, and other populations, have indicated that alterations in
glucose metabolism can be detected in subjects monitored before the
onset of the condition. The genetic component presents a complex
pattern of inheritance that is not fully understood.
[0007] Studies of the genetic component of the condition have
indicated linkage of Type II diabetes susceptibility on many human
chromosomes, including 2, 11, 12 and 20. A locus on chromosome 2
appears to be a major factor in development of Diabetes Mellitus in
Mexican Americans (Hanis et al., (1996) Nat. Genet. 13: 161-66), a
locus on chromosome 12 has been found in a Finnish populations
(Mahtani et al., (1996) Nat. Genet. 14: 90-94) and a locus on
chromosome 11q has been identified in Pima Indians (Hanson et al.,
(1998) Diabetes 46: 494-501). Studies by Zouali et al. suggest the
location of a susceptibility locus on chromosome 20q in the PCK1
region. Zouali et al., (1997) Hum. Mol. Genet. 6:1401-08. At least
three other studies present evidence for linkage of Type II
diabetes on chromosome 20 in sibships. Ghosh et al., (1999) Proc.
Natl. Acad. Sci. USA 96: 2198-2203; Bowden et al., (1997) Diabetes
46: 882-86; Ji et al., (1997) Diabetes 46: 876-81.
[0008] As noted, a hallmark of the Type II diabetic condition is a
hyperglycemic imbalance in body glucose homeostasis. This condition
can arise from a defect in the mechanism by which glucose is
processed by the human body and can occur at various control points
in the mechanism. A defect can occur, for example, during glucose
uptake by the brain, glucose storage in the liver or
insulin-dependent uptake in muscles and adipocytes in the human
body.
[0009] Central to the uptake of glucose and the maintenance of
glucose homeostasis are the glucose transporter proteins. These
proteins control glucose absorption by the above-mentioned tissues.
In mammalian cells, glucose transport is catalyzed by a number of
membrane proteins, including the GLUT family of proteins, GLUT1-5,
GLUT8, GLUTX1 and GLUT9. Glucose transport proteins are found in a
wide variety of species, and share a common structural motif:
glucose transport proteins are characterized by the presence of 12
connected transmembrane helical segments. See, e.g., Ibberson et
al., (2000) J. Biol. Chem. 275: 4607-12; Doege et al., (2000) J.
Biol. Chem. 275: 16275-80. An extracellular loop containing a
glycosylation site is also present.
[0010] Although the precise primary defect in the Type II diabetic
condition is presently unsettled, work in the field suggests that
there is a strong causal link between the condition and glucose
mobilization and metabolism. Zierler, (1999) Am. J. Physiol. 276
(3, pt. 1): E409-26; Shepard and Kahn, (1999) New Engl. J. Med. 341
(4): 248-57. Glucose transporter proteins are, therefore, likely
candidates for analysis when attempting to explain the link between
the Type II diabetic condition, as well as other glucose
homeostatis imbalances, and their genetic components. Indeed, a
mutation in GLUT4 has been found in a patient with Type II
diabetes. Kusari, et al., (1991) JCI 88: 1323-30.
[0011] Clearly, it would be of tremendous value to researchers and
clinicians to have a specific polynucleotide sequence coding for a
glucose transport protein which is identified as being associated
with human Type II diabetes. Such a result would permit diagnostic
and therapeutic treatment of the condition, which if left untreated
can lead to circulatory deficiencies and blindness. Traditional
genetic approaches have proven to be inadequate to accomplish this
goal. Linkage disequilibrium analysis, for example, which has
historically proven helpful in studying complex disease genes, has
not yet yielded results useful for Type II diabetes-related
therapeutic treatments.
[0012] What is needed, therefore, is the identification of a
polynucleotide sequence coding for a glucose transporter that is
associated with human Type II diabetes and other glucose
homeostasis disorders, as well as a method for accurately
diagnosing a subject's susceptibility to, and the early detection
of such conditions. Gene and drug therapy to relieve this
condition, after the condition has manifested, is also needed. The
present invention solves this problem by providing a polynucleotide
and a polypeptide useful for diagnosing the susceptibility of a
subject to human Type II diabetes and other glucose homeostasis
disorders, a method for diagnosing the susceptibility of a subject
to human Type II diabetes and other glucose homeostasis disorders
using the polynucleotide and polypeptide and a method of treating
the condition, all of which take into account the polymorphic
nature of the polynucleotide sequence.
DISCLOSURE OF THE INVENTION
[0013] An isolated and substantially pure nucleic acid sequence
located between D20S119 and D20S178 on human chromosome 20q13 is
disclosed. The nucleic acid sequence comprises a nucleic acid
sequence coding for a novel glucose transporting protein and having
the sequence shown in SEQ ID NO: 1, or a nucleic acid sequence
having at least 70% sequence identity with the nucleic acid
sequence shown in SEQ ID NO: 1. Additionally, a nucleic acid
sequence which hybridizes to and is at least 70% complementary to
the glucose transporter-encoding nucleic acid sequence is
disclosed.
[0014] Also disclosed is a method of facilitating a diagnosis of
susceptibility to a glucose homeostasis disorder. The method
comprises obtaining a biological sample from a subject; isolating a
target nucleic acid sequence located between D20S119 and D20S178 on
human chromosome 20q13, the target nucleic acid sequence encoding a
glucose transporter polypeptide; sequencing the target nucleic acid
sequence; and determining a sequence variation between a wild type
nucleic acid sequence and the isolated target nucleic acid
sequence, the presence of variations between the wild type nucleic
acid sequence and the isolated target nucleic acid sequence
indicating susceptibility in the subject to a disorder affecting
glucose homeostasis.
[0015] A method of screening a biological sample for the presence
of the novel glucose transporter polypeptide is also disclosed. The
presence of polypeptide in the sample is detected by evaluating the
formation and presence of antibody-polypeptide conjugates.
[0016] In another aspect, the present invention provides a method
of screening a biological sample for the presence of antibodies
immunoreactive with a novel glucose transporter polypeptide. In
accordance with such a method, a biological sample is exposed to a
glucose transporter polypeptide under biological conditions and for
a period of time sufficient for antibody-polypeptide conjugate
formation and the formed conjugates are detected.
[0017] A method of a facilitating a diagnosis of a disorder
affecting glucose homeostasis is also disclosed. In one embodiment,
the method comprises: (a) obtaining a biological sample from a
subject; and (b) determining an amount of a glucose transporter
polypeptide present in the biological sample, wherein the presence
of a reduced amount of the glucose transporter polypeptide as
compared to a standard facilitates a diagnosis of a disorder
affecting glucose homeostasis. Optionally, the amount of glucose
transporting protein in the biological sample can be determined by
Western blot analysis.
[0018] In another embodiment, the method comprises: (a) obtaining a
glucose transporter polypeptide from a subject; (b) determining an
activity level of a glucose transporter polypeptide from the
subject; and (c) detecting a variation in glucose transport
activity between a wild type glucose transporter polypeptide and
the glucose transporter polypeptide from the subject, wherein the
presence of a glucose transport activity variation between the wild
type glucose transporter polypeptide and the glucose transporter
polypeptide from the subject facilitates a diagnosis of a disorder
affecting glucose homeostasis.
[0019] The glucose transporter polypeptide can be obtained from a
subject by isolating from the subject a biological sample
comprising the glucose transporter polypeptide. In this case the
method preferably further comprises determining the subcellular
localization of the glucose transporter polypeptide in the
biological sample.
[0020] In still a further embodiment, this invention pertains to
therapeutic methods based upon the modulation of glucose transport
via the polynucleotides and polypeptides described herein. Such
therapeutic methods include gene therapy approaches using an
isolated and purified polynucleotide of the present invention.
[0021] The foregoing aspects and embodiments have broad utility
given the biological significance of glucose transport. By way of
example, the foregoing aspects and embodiments are useful in the
preparation of screening assays and assay kits that are used to
identify compounds that affect or modulate glucose transport, or
that are used to detect the presence of the proteins and nucleic
acids of this invention in biological samples.
[0022] Accordingly, it is an object of the present invention to
provide a novel glucose transporter polypeptide, and to provide a
novel polynucleotide encoding the same. The object is achieved in
whole or in part by the present invention. An object of the
invention having been stated hereinabove, other objects will become
evident as the description proceeds when taken in connection with
the accompanying Figures and Laboratory Examples as best described
herein below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1A depicts a 5' region including the promoter region
(SEQ ID NO: 4) of the glucose transporter-encoding polynucleotide
sequence (SEQ ID NO: 1) found between D20S119 and D20S178 on human
chromosome 20q13;
[0024] FIG. 1B depicts the cDNA polynucleotide sequence (SEQ ID NO:
1) of the glucose transporter found between D20S119 and D20S178 on
human chromosome 20q13 and the corresponding deduced amino acid
sequence (SEQ ID NO: 2);
[0025] FIG. 1C depicts the remaining 3' untranslated DNA sequence
of the glucose transporter polynucleotide sequence (SEQ ID NO: 1)
found between D20S119 and D20S178 on human chromosome 20q13;
[0026] FIG. 2 is a schematic depicting the genomic structure and
organization of the novel glucose transporter of the present
invention;
[0027] FIG. 3 is a schematic depicting the topology of the novel
glucose transporter of the present invention;
[0028] FIG. 4 is an autoradiograph of a Northern blot depicting the
tissue distribution of the glucose transporter-encoding
polynucleotide of the present invention; and
[0029] FIG. 5 is a diagram depicting an amino acid comparison of
human glucose transporter 1 (GLUT1) (SEQ ID NO: 3) and the novel
glucose transporter (SEQ ID NO: 2) of the present invention. Amino
acids highlighted in bold are necessary for sugar transport
function. This diagram also indicates the approximate regions of
the transmembrane domains (TM).
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
[0030] Following long-standing patent law convention, the terms "a"
and "an" mean "one or more" when used in this application,
including the claims.
[0031] As used herein, the term "glucose transporter" carries the
connotation generally recognized by those of skill in the art. The
term therefore includes the ability of a protein to transport
fructose, galactose and other sugars, as well as glucose.
[0032] As used herein, the term "mutation" carries its traditional
connotation and means a change, inherited, naturally occurring or
introduced, in a nucleic acid or polypeptide sequence, and is used
in its sense as generally known to those of skill in the art.
[0033] As used herein, the term "labeled" means the attachment of a
moiety, capable of detection by spectroscopic, radiologic or other
methods, to a probe molecule.
[0034] As used herein, the term "target cell" refers to a cell,
into which it is desired to insert a nucleic acid sequence or
polypeptide, or to otherwise effect a modification from conditions
known to be standard in the unmodified cell. A nucleic acid
sequence introduced into a target cell can be of variable length.
Additionally, a nucleic acid sequence can enter a target cell as a
component of a plasmid or other vector or as a naked sequence.
[0035] As used herein, the term "polymorphism" refers to a
difference in the nucleotide sequence of a given region as compared
to a nucleotide sequence in a homologous region of another
individual, in particular, a difference in the nucleotide sequence
of a given region that differs between individuals of the same
species. Polymorphisms include single nucleotide differences,
differences in sequence of more than one nucleotide, insertions,
inversions and deletions. The term refers to the occurrence of two
or more genetically determined alternative sequences or alleles in
a population. A polymorphic marker is the locus at which divergence
occurs. Preferred markers have at least two alleles, each occurring
at frequency of greater than 1%. A polymorphic locus can be as
small as one base pair.
[0036] As used herein, the term "transcription" means a cellular
process involving the interaction of an RNA polymerase with a gene
that directs the expression as RNA of the structural information
present in the coding sequences of the gene. The process includes,
but is not limited to the following steps: (a) the transcription
initiation, (b) transcript elongation, (c) transcript splicing, (d)
transcript capping, (e) transcript termination, (f) transcript
polyadenylation, (g) nuclear export of the transcript, (h)
transcript editing, and (i) stabilizing the transcript.
[0037] As used herein, the term "expression" generally refers to
the cellular processes by which a biologically active polypeptide
is produced from RNA.
[0038] As used herein, the term "transcription factor" means a
cytoplasmic or nuclear protein which binds to such gene, or binds
to an RNA transcript of such gene, or binds to another protein
which binds to such gene or such RNA transcript or another protein
which in turn binds to such gene or such RNA transcript, so as to
thereby modulate expression of the gene. Such modulation can
additionally be achieved by other mechanisms; the essence of
"transcription factor for a gene" is that the level of
transcription of the gene is altered in some way.
[0039] As used herein, the term "susceptible to Type II diabetes"
means a statistically significant increase in the probability of
developing measurable symptoms and signs of Type II diabetes in an
individual having a particular genetic mutation or polymorphism
compared with the probability in an individual lacking the genetic
mutation or polymorphism.
[0040] As used herein, the term "hybridization" means the binding
of a probe molecule, a molecule to which a detectable moiety has
been bound, to a target sample.
[0041] As used herein, the term "detecting" means confirming the
presence of a target entity by observing the occurrence of a
detectable signal, such as a radiologic or spectroscopic signal
that will appear exclusively in the presence of the target
entity.
[0042] As used herein, the term "sequencing" means the determining
the ordered linear sequence of nucleic acids or amino acids of a
DNA or protein target sample, using conventional manual or
automated laboratory techniques.
[0043] As used herein, the term "isolated" means oligonucleotides
substantially free of other nucleic acids, proteins, lipids,
carbohydrates or other materials with which they can be associated,
such association being either in cellular material or in a
synthesis medium. The term can also be applied to polypeptides, in
which case the polypeptide will be substantially free of nucleic
acids, carbohydrates, lipids and other undesired polypeptides.
[0044] As used herein, the term "substantially pure" means that the
polynucleotide or polypeptide is substantially free of the
sequences and molecules with which it is associated in its natural
state, and those molecules used in the isolation procedure. The
term "substantially free" means that the sample is at least 50%,
preferably at least 70%, more preferably 80% and most preferably
90% free of the materials and compounds with which is it associated
in nature.
[0045] As used herein, the term "parenteral" means intravenous,
intra-muscular, intra-arterial injection, intraventricular,
intrathecal or infusion introduction techniques.
[0046] As used herein, the term "primer" means a sequence
comprising two or more deoxyribonucleotides or ribonucleotides,
preferably more than three, and more preferably more than eight and
most preferably at least about 20 nucleotides of an exonic or
intronic region. Such oligonucleotides are preferably between ten
and thirty bases in length.
[0047] As used herein, the term "DNA segment" means a DNA molecule
that has been isolated free of total genomic DNA of a particular
species. Furthermore, a DNA segment encoding a glucose transporter
polypeptide refers to a DNA segment that contains SEQ ID NO: 1, yet
is isolated away from, or purified free from, total genomic DNA of
a source species, such as Homo sapiens. Included within the term
"DNA segment" are DNA segments and smaller fragments of such
segments, and also recombinant vectors, including, for example,
plasmids, cosmids, phages, viruses, and the like.
[0048] As used herein, the phrase "enhancer-promoter" means a
composite unit that contains both enhancer and promoter elements.
An enhancer-promoter is operatively linked to a coding sequence
that encodes at least one gene product.
[0049] As used herein, the phrase "operatively linked" means that
an enhancer-promoter is connected to a coding sequence in such a
way that the transcription of that coding sequence is controlled
and regulated by that enhancer-promoter. Techniques for operatively
linking an enhancer-promoter to a coding sequence are well known in
the art; the precise orientation and location relative to a coding
sequence of interest is dependent, inter alia, upon the specific
nature of the enhancer-promoter.
B. Polynucleotides and Polypeptides
[0050] In particular embodiments, the invention concerns isolated
DNA segments and recombinant vectors incorporating DNA sequences
which encode a glucose transporting polypeptide that includes
within its amino acid sequence an amino acid sequence of the
present invention. In other particular embodiments, the invention
concerns recombinant vectors incorporating DNA segments that encode
a polypeptide comprising the amino acid sequence of a human glucose
transporting polypeptide; and these vectors also carry the promoter
and enhancer regions associated with the nucleic acid sequence
encoding the glucose transporting polypeptide.
[0051] B1. Polynucleotides
[0052] The present invention pertains to a previously unidentified
gene located between markers D20S119 and D20S178 on chromosome
20q13. A Type II diabetes locus has previously been mapped to this
region. The novel polynucleotide sequence, a preferred embodiment
of which is shown in SEQ ID NO: 1, contains a 1626 bp coding
sequence, and the full-length cDNA is 4.3 kb. The sequence is
highly expressed in the liver and pancreas.
[0053] A preferred nucleic acid sequence of the present invention,
such as that shown in SEQ ID NO: 1, is a glucose transporter
sequence that is isolated from wild type cells. The sequence
represents the glucose transporter nucleic acid sequence occurring
in nature and existing without mutation. Therefore, wild type
cells, as referred to herein, are those cells occurring in nature
that contain non-mutated glucose transporter nucleic acid
sequences. The wild type sequence is the native nucleic acid
sequence and is the sequence against which assessments of
polymorphism and mutation are made.
[0054] The terms "glucose transporter gene", "glucose transporter
gene segment", "glucose transporter gene sequence", "glucose
transporter polynucleotide", "glucose transporter nucleic acid
molecule", and "glucose transporter nucleic acid sequence" refer to
any nucleic acid sequence (e.g. a DNA sequence) that is
substantially identical to a polynucleotide sequence encoding
"glucose transporter gene product", "glucose transporter protein",
"glucose transporter polypeptide", and "glucose transporter
peptide" as defined below, and can also comprise any combination of
associated control sequences. The terms also refer to RNA, or
antisense sequences, complementary to such DNA sequences.
[0055] As used herein, the term "DNA segment" refers to a DNA
molecule that has been isolated free of total genomic DNA of a
particular species. Furthermore, a DNA segment encoding a glucose
transporter polypeptide refers to a DNA segment that contains
glucose transporter coding sequences, yet is isolated away from, or
purified free from, total genomic DNA of a source species, such as
Homo sapiens. Included within the term "DNA segment" are DNA
segments and smaller fragments of such segments, and also
recombinant vectors, including, for example, plasmids, cosmids,
phages, viruses, and the like.
[0056] B2. Polypeptides
[0057] A preferred deduced amino acid sequence of the
polynucleotide sequence, SEQ ID NO: 2, is shown in FIG. 1. Analysis
of the amino acid sequence revealed that a polypeptide comprising
541 residues. Analysis of the polypeptide shows that it contains 12
membrane-spanning helical sequences with a glycosylation site
located on loop 9, which joins helices 9 and 10. This topology is
typical of glucose transporting proteins. Mueckler, (1994) Eur J.
Biochem. 219: 713-25; Baldwin, (1993) Biochim. Biophys. Acta 1154:
17-49. The amino acid sequence also reveals several other motifs
common to glucose transporters, including important amino acids
essential to sugar transport function. Barrett et al., (1999) Curr.
Op. Cell Biol. 11:496-502; Walmsley et al., (1998) Trends Biochem.
Sci. 23:476-481. Representative amino acids implicated in sugar
transport include but are not limited to those set forth in bold in
FIG. 5.
[0058] The terms "glucose transporter gene product", "glucose
transporter protein", "glucose transporter polypeptide", and
"glucose transporter peptide" refer to peptides having amino acid
sequences which are substantially identical to native amino acid
sequences from the organism of interest and which are biologically
active in that they comprise all or a part of the amino acid
sequence of a glucose transporter polypeptide, or cross-react with
antibodies raised against a glucose transporter polypeptide, or
retain all or some of the biological activity of the native amino
acid sequence or protein. Such biological activity can include
immunogenicity.
[0059] The terms "glucose transporter gene product", "glucose
transporter protein", "glucose transporter polypeptide", and
"glucose transporter peptide" also include analogs of a glucose
transporter polypeptide. By "analog" is intended that a DNA or
peptide sequence can contain alterations relative to the sequences
disclosed herein, yet retain all or some of the biological activity
of those sequences. Analogs can be derived from genomic nucleotide
sequences as are disclosed herein or from other organisms, or can
be created synthetically. Those skilled in the art will appreciate
that other analogs, as yet undisclosed or undiscovered, can be used
to design and/or construct glucose transporter analogs. There is no
need for a "glucose transporter gene product", "glucose transporter
protein", "glucose transporter polypeptide", and "glucose
transporter peptide" to comprise all or substantially all of the
amino acid sequence of a glucose transporter polypeptide gene
product. Shorter or longer sequences are anticipated to be of use
in the invention; shorter sequences are herein referred to as
"segments". Thus, the terms "glucose transporter gene product",
"glucose transporter protein", "glucose transporter polypeptide",
and "glucose transporter peptide" also include fusion or
recombinant glucose transporter polypeptides and proteins
comprising sequences of the present invention. Methods of preparing
such proteins are disclosed herein and are known in the art.
[0060] B3. Sequence Similarity and Identity
[0061] As used herein, the term "substantially similar" means that
a particular sequence varies from nucleic acid sequence of SEQ ID
NO: 1, or the amino acid sequence of SEQ ID NO: 2 by one or more
deletions, substitutions, or additions, the net effect of which is
to retain at least some of biological activity of the natural gene,
gene product, or sequence. Such sequences include "mutant" or
"polymorphic" sequences, or sequences in which the biological
activity is altered to some degree but retains at least some of the
original biological activity. In determining nucleic acid
sequences, all subject nucleic acid sequences capable of encoding
substantially similar amino acid sequences are considered to be
substantially similar to a reference nucleic acid sequence,
regardless of differences in codon sequences or substitution of
equivalent amino acids to create biologically functional
equivalents.
[0062] Additionally, nucleic acids that are substantially identical
to SEQ ID NO: 1 and SEQ ID NO: 2, the preferred glucose transporter
sequences, e.g. allelic variants, genetically altered versions of
the gene, etc., bind to the provided glucose sequences under
stringent hybridization conditions. By using probes, particularly
labeled probes of DNA sequences, one can isolate homologous or
related genes. The source of homologous genes can be any species,
e.g. primate species; rodents, such as rats and mice, canines,
felines, bovines, equines, yeast, nematodes, etc.
[0063] Between mammalian species, e.g. human and mouse, homologs
have substantial sequence similarity, i.e. at least 75% sequence
identity between nucleotide sequences. Sequence similarity is
calculated based on a reference sequence, which can be a subset of
a larger sequence, such as a conserved motif, coding region,
flanking region, etc. A reference sequence will usually be at least
about 18 nt long, more usually at least about 30 nt long, and can
extend to the complete sequence that is being compared. Algorithms
for sequence analysis are known in the art, such as BLAST,
described in Altschul et al., (1990) J. Mol. Biol. 215: 403-10.
[0064] Percent identity or percent similarity of a DNA or peptide
sequence can be determined, for example, by comparing sequence
information using the GAP computer program, available from the
University of Wisconsin Geneticist Computer Group. The GAP program
utilizes the alignment method of Needleman et al., (1970) J. Mol.
Biol. 48: 443, as revised by Smith et al., (1981) Adv. Appl. Math.
2:482. Briefly, the GAP program defines similarity as the number of
aligned symbols (i.e., nucleotides or amino acids) which are
similar, divided by the total number of symbols in the shorter of
the two sequences. The preferred parameters for the GAP program are
the default parameters, which do not impose a penalty for end gaps.
See, e.g. Schwartz et al., eds., (1979), Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
357-358; Gribskov et al., (1986) Nucl. Acids. Res. 14: 6745.
[0065] Thus, for sequence comparison, typically one sequence acts
as a reference sequence to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer program, subsequence coordinates are
designated if necessary, and sequence algorithm program parameters
are selected. The sequence comparison algorithm then calculates the
percent sequence identity for the designated test sequence(s)
relative to the reference sequence, based on the selected program
parameters.
[0066] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman (1981) Adv Appl Math 2:482, by the homology alignment
algorithm of Needleman & Wunsch (1970) J Mol Biol 48:443, by
the search for similarity method of Pearson & Lipman (1988)
Proc Natl Acad Sci USA 85:2444-2448, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, Madison, Wis.), or by visual inspection. See
generally, Ausubel et al., 1992.
[0067] A preferred algorithm for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al. (1990) J Mol Biol 215: 403-410.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold. These initial neighborhood
word hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when the cumulative
alignment score falls off by the quantity X from its maximum
achieved value, the cumulative score goes to zero or below due to
the accumulation of one or more negative-scoring residue
alignments, or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength W=11, an expectation E=10,
a cutoff of 100, M=5, N=-4, and a comparison of both strands. For
amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix. See Henikoff & Henikoff (1989) Proc Natl Acad
Sci USA 89:10915.
[0068] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. See, e.g., Karlin and Altschul
(1993) Proc Natl Acad Sci USA 90:5873-5887. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a test nucleic acid sequence is
considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid sequence to
the reference nucleic acid sequence is less than about 0.1, more
preferably less than about 0.01, and most preferably less than
about 0.001.
[0069] The term "similarity" is contrasted with the term
"identity". Similarity is defined as above; "identity", however,
means a nucleic acid or amino acid sequence having the same amino
acid at the same relative position in a given family member of a
gene family. Homology and similarity are generally viewed as
broader terms than the term identity. Biochemically similar amino
acids, for example leucine and isoleucine or glutamate/aspartate,
can be present at the same position--these are not identical per
se, but are biochemically "similar." As disclosed herein, these are
referred to as conservative differences or conservative
substitutions. This differs from a conservative mutation at the DNA
level, which changes the nucleotide sequence without making a
change in the encoded amino acid, e.g. TCC to TCA, both of which
encode serine.
[0070] As used herein, DNA analog sequences are "substantially
identical" to specific DNA sequences disclosed herein if: (a) the
DNA analog sequence is derived from coding regions of the nucleic
acid sequence shown in SEQ ID NO: 1; or (b) the DNA analog sequence
is capable of hybridization of DNA sequences of (a) under stringent
conditions and which encode a biologically active gene product of
the nucleic acid sequence shown in SEQ ID NO: 1; or (c) the DNA
sequences are degenerate as a result of alternative genetic code to
the DNA analog sequences defined in (a) and/or (b). Substantially
identical analog proteins and nucleic acids will have between about
70% and 80%, preferably between about 81% to about 90% or even more
preferably between about 91% and 99% sequence identity with the
corresponding sequence of the native protein or nucleic acid.
Sequences having lesser degrees of identity but comparable
biological activity are considered to be equivalents.
[0071] As used herein, "stringent conditions" means conditions of
high stringency, for example 6.times.SSC, 0.2%
polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin, 0.1%
sodium dodecyl sulfate, 100 .mu.g/ml salmon sperm DNA and 15%
formamide at 68.degree. C. For the purposes of specifying
additional conditions of high stringency, preferred conditions are
salt concentration of about 200 mM and temperature of about
45.degree. C. One example of such stringent conditions is
hybridization at 4.times.SSC, at 65.degree. C., followed by a
washing in 0.1.times.SSC at 65.degree. C. for one hour. Another
exemplary stringent hybridization scheme uses 50% formamide,
4.times.SSC at 42.degree. C.
[0072] In contrast, nucleic acids having sequence similarity are
detected by hybridization under lower stringency conditions. Thus,
sequence identity can be determined by hybridization under lower
stringency conditions, for example, at 50.degree. C. or higher and
0.1.times.SSC (9 mM NaCl/0.9 mM sodium citrate) and the sequences
will remain bound when subjected to washing at 55.degree. C. in
1.times.SSC.
[0073] As used herein, the term "complementary sequences" means
nucleic acid sequences which are base-paired according to the
standard Watson-Crick complementarity rules. The present invention
also encompasses the use of nucleotide segments that are
complementary to the sequences of the present invention. A
particular example of a contemplated complementary nucleic acid
segment is an antisense oligonucleotide.
[0074] Hybridization can also be used for assessing complementary
sequences and/or isolating complementary nucleotide sequences. As
discussed above, 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 about
30.degree. C., typically in excess of about 37.degree. C., and
preferably in excess of about 45.degree. C. Stringent salt
conditions will ordinarily be less than about 1,000 mM, typically
less than about 500 mM, and preferably less than about 200 mM.
However, the combination of parameters is much more important than
the measure of any single parameter. See, e.g., Wetmur &
Davidson, (1968) J. Mol. Biol. 31: 349-70. Determining appropriate
hybridization conditions to identify and/or isolate sequences
containing high levels of homology is well known in the art. See,
e.g., Sambrook et al., (1992), Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y.
[0075] As used herein, the term "functionally equivalent codon" is
used to refer to codons that encode the same amino acid, such as
the ACG and AGU codons for serine. Glucose transporter encoding
nucleic acid sequences having SEQ ID NO: 1 which have functionally
equivalent codons are covered by the invention. Thus, when
referring to the sequence examples presented in SEQ ID NO: 1,
applicants contemplate substitution of functionally equivalent
codons of Table 1 into the sequence examples of SEQ ID NO: 1. Thus,
applicants are in possession of amino acid and nucleic acids
sequences which include such substitutions but which are not set
forth herein in their entirety for convenience.
TABLE-US-00001 TABLE 1 Functionally Equivalent Codons Amino Acids
Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU
Aspartic Acid Asp D GAC GAU Glumatic acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine
His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine
Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S
ACG AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val
V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0076] It will also be understood by those of skill in the art that
amino acid and nucleic acid sequences can include additional
residues, such as additional N- or C-terminal amino acids or 5' or
3' nucleic acid sequences, and yet still be essentially as set
forth in one of the sequences disclosed herein, so long as the
sequence retains biological protein activity where polypeptide
expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences which can, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or can include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0077] B4. Biological Equivalents
[0078] The present invention envisions and includes biological
equivalents to the glucose transporter polypeptide of the present
invention. The term "biological equivalent" refers to proteins
having amino acid sequences which are substantially identical to
the native amino acid sequences in the glucose transporter of the
present invention and which are biologically active in that they
are capable of mediating glucose uptake, or cross-reacting with
anti-glucose transporter antibodies raised against a glucose
transporter polypeptide of the present invention.
[0079] For example, certain amino acids can be substituted for
other amino acids in a protein structure without appreciable loss
of interactive capacity with, for example, structures in the
nucleus of a cell. Since it is the interactive capacity and nature
of a protein that defines that protein's biological functional
activity, certain amino acid sequence substitutions can be made in
a protein sequence (or the nucleic acid sequence encoding it) to
obtain a protein with the same, enhanced, or antagonistic
properties. Such properties can be achieved by interaction with the
normal targets of the native protein, but this need not be the
case, and the biological activity of the invention is not limited
to a particular mechanism of action. It is thus contemplated in
accordance with the present invention that various changes can be
made in the sequence of a glucose transporter polypeptide of the
present invention or underlying nucleic acid sequence without
appreciable loss of biological utility or activity.
[0080] Biologically equivalent peptides, as used herein, are
peptides in which certain, but not most or all, of the amino acids
can be substituted. Representative amino acids that are preferably
not substituted include those implicated in glucose transport. Such
amino acids are set forth in bold in FIG. 5. Thus, when referring
to the sequence examples presented in SEQ ID NO: 1, applicants
envision substitution of codons that encode biologically equivalent
amino acids as described herein into the sequence example of SEQ ID
NO: 1. Thus, applicants are in possession of amino acid and nucleic
acids sequences which include such substitutions but which are not
set forth herein in their entirety for convenience.
[0081] Alternatively, functionally equivalent proteins or peptides
can be created via the application of recombinant DNA technology,
in which changes in the protein structure can be engineered, based
on considerations of the properties of the amino acids being
exchanged, e.g. substitution of Ile for Leu. Changes designed by
man can be introduced through the application of site-directed
mutagenesis techniques, e.g., to introduce improvements to the
antigenicity of the protein or to test a glucose transporter
polypeptide of the present invention in order to glucose transport
activity, or other activity at the molecular level.
[0082] Amino acid substitutions, such as those which might be
employed in modifying a glucose transporter polypeptide of the
present invention, are generally based on the relative similarity
of the amino acid side-chain substituents, for example, their
hydrophobicity, hydrophilicity, charge, size, and the like. An
analysis of the size, shape and type of the amino acid side-chain
substituents reveals that arginine, lysine and histidine are all
positively charged residues; that alanine, glycine and serine are
all of similar size; and that phenylalanine, tryptophan and
tyrosine all have a generally similar shape. Therefore, based upon
these considerations, arginine, lysine and histidine; alanine,
glycine and serine; and phenylalanine, tryptophan and tyrosine; are
defined herein as biologically functional equivalents. Other
biologically functionally equivalent changes will be appreciated by
those of skill in the art.
[0083] In making biologically functional equivalent amino acid
substitutions, the hydropathic index of amino acids can be
considered. Each amino acid has been assigned a hydropathic index
on the basis of their hydrophobicity and charge characteristics,
these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine
(+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan
(-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);
glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine
(-3.5); lysine (-3.9); and arginine (-4.5).
[0084] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte & Doolittle, (1982),
incorporated herein by reference). It is known that certain amino
acids can be substituted for other amino acids having a similar
hydropathic index or score and still retain a similar biological
activity. In making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indices are within
.+-.2 of the original value is preferred, those which are within
.+-.1 of the original value are particularly preferred, and those
within .+-.0.5 of the original value are even more particularly
preferred.
[0085] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.
with a biological property of the protein. It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein.
[0086] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0087] In making changes based upon similar hydrophilicity values,
the substitution of amino acids whose hydrophilicity values are
within .+-.2 of the original value is preferred, those which are
within .+-.1 of the original value are particularly preferred, and
those within .+-.0.5 of the original value are even more
particularly preferred.
[0088] While discussion has focused on functionally equivalent
polypeptides arising from amino acid changes, it will be
appreciated that these changes can be effected by alteration of the
encoding DNA, taking into consideration also that the genetic code
is degenerate and that two or more codons can code for the same
amino acid.
[0089] Thus, it will also be understood that this invention is not
limited to the particular nucleic acid and amino acid sequences of
SEQ ID NOs: 1-2. Recombinant vectors and isolated DNA segments can
therefore variously include the glucose transporter
polypeptide-encoding region itself, include coding regions bearing
selected alterations or modifications in the basic coding region,
or include larger polypeptides which nevertheless comprise glucose
transporter polypeptide-encoding regions or can encode biologically
functional equivalent proteins or peptides which have variant amino
acid sequences. Biological activity of a glucose transporter
polypeptide can be determined, for example, by glucose uptake
assays as disclosed herein.
[0090] The nucleic acid segments of the present invention,
regardless of the length of the coding sequence itself, can be
combined with other DNA sequences, such as promoters, enhancers,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length can vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length can
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant DNA
protocol. For example, nucleic acid fragments can be prepared which
include a short stretch complementary to a nucleic acid sequence
set forth in SEQ ID NO: 1, such as about 10 nucleotides, and which
are up to 10,000 or 5,000 base pairs in length, with segments of
3,000 being preferred in certain cases. DNA segments with total
lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100, and
about 50 base pairs in length are also useful.
[0091] The DNA segments of the present invention encompass
biologically functional equivalent glucose transporter
polypeptides. Such sequences can rise as a consequence of codon
redundancy and functional equivalency that are known to occur
naturally within nucleic acid sequences and the proteins thus
encoded. Alternatively, functionally equivalent proteins or
peptides can be created via the application of recombinant DNA
technology, in which changes in the protein structure can be
engineered, based on considerations of the properties of the amino
acids being exchanged. Changes can be introduced through the
application of site-directed mutagenesis techniques, e.g., to
introduce improvements to the antigenicity of the protein or to
test glucose transporter mutants in order to examine activity in
the modulation of calcium transport, or other activity at the
molecular level. Site-directed mutagenesis techniques are known to
those of skill in the art and are disclosed herein.
[0092] The invention further encompasses fusion proteins and
peptides wherein the glucose transporter coding region is aligned
within the same expression unit with other proteins or peptides
having desired functions, such as for purification or
immunodetection purposes.
[0093] Recombinant vectors form important further aspects of the
present invention. Particularly useful vectors are those in which
the coding portion of the DNA segment is positioned under the
control of a promoter. The promoter can be that naturally
associated with the glucose transporter gene, as can be obtained by
isolating the 5' non-coding sequences located upstream of the
coding segment or exon, for example, using recombinant cloning
and/or PCR technology and/or other methods known in the art, in
conjunction with the compositions disclosed herein.
[0094] In other embodiments, certain advantages will be gained by
positioning the coding DNA segment under the control of a
recombinant, or heterologous, promoter. As used herein, a
recombinant or heterologous promoter is a promoter that is not
normally associated with a glucose transporter gene in its natural
environment. Such promoters can include promoters isolated from
bacterial, viral, eukaryotic, or mammalian cells. Naturally, it
will be important to employ a promoter that effectively directs the
expression of the DNA segment in the cell type chosen for
expression. The use of promoter and cell type combinations for
protein expression is generally known to those of skill in the art
of molecular biology (See, e.g., Sambrook et al., 1992,
specifically incorporated herein by reference). The promoters
employed can be constitutive or inducible and can be used under the
appropriate conditions to direct high level expression of the
introduced DNA segment, such as is advantageous in the large-scale
production of recombinant proteins or peptides. Appropriate
promoter systems contemplated for use in high-level expression
include, but are not limited to, the vaccinia virus promoter and
the baculovirus promoter.
[0095] In an alternative embodiment, the present invention provides
an expression vector comprising a polynucleotide that encodes a
biologically active glucose transporter polypeptide in accordance
with the present invention. Also preferably, an expression vector
of the present invention comprises a polynucleotide that encodes a
human glucose transporter polypeptide. More preferably, an
expression vector of the present invention comprises a
polynucleotide that encodes a polypeptide comprising an amino acid
residue sequence of SEQ ID NO: 2. More preferably, an expression
vector of the present invention comprises a polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 1. Even more
preferably, an expression vector of the invention comprises a
polynucleotide operatively linked to an enhancer-promoter. More
preferably still, an expression vector of the invention comprises a
polynucleotide operatively linked to a prokaryotic promoter.
Alternatively, an expression vector of the present invention
comprises a polynucleotide operatively linked to an
enhancer-promoter that is a eukaryotic promoter and the expression
vector further comprises a polyadenylation signal that is
positioned 3' of the carboxy-terminal amino acid and within a
transcriptional unit of the encoded polypeptide.
[0096] In yet another embodiment, the present invention provides a
recombinant host cell transfected with a polynucleotide that
encodes a biologically active glucose transporter polypeptide in
accordance with the present invention. SEQ ID NOs: 1-2 set forth
nucleotide and amino acid sequences from a representative
vertebrate, human. Also contemplated by the present invention are
homologous or biologically functionally equivalent polynucleotides
and glucose transporter polypeptide polypeptides found in other
vertebrates, including particularly mouse and rat homologs.
Preferably, a recombinant host cell of the present invention is
transfected with the polynucleotide that encodes a human glucose
transporter polypeptide. More preferably, a recombinant host cell
of the present invention is transfected with the polynucleotide
sequence set forth in SEQ ID NO: 1. Even more preferably, a
recombinant host cell is a mammalian cell.
[0097] In another aspect, a recombinant host cell of the present
invention is a prokaryotic host cell, including parasitic and
bacterial cells. Preferably, a recombinant host cell of the
invention is a bacterial cell, preferably a strain of Escherichia
coli. More preferably, a recombinant host cell comprises a
polynucleotide under the transcriptional control of regulatory
signals functional in the recombinant host cell, wherein the
regulatory signals appropriately control expression of the glucose
transporter polypeptide in a manner to enable all necessary
transcriptional and post-transcriptional modification.
[0098] In yet another embodiment, the present invention provides a
method of preparing a glucose transporter polypeptide comprising
transfecting a cell with polynucleotide that encodes a biologically
active glucose transporter polypeptide in accordance with the
present invention, to produce a transformed host cell, and
maintaining the transformed host cell under biological conditions
sufficient for expression of the polypeptide. The polypeptide can
be isolated if desired, using any suitable technique. The host cell
can be a prokaryotic or eukaryotic cell. Preferably, the
prokaryotic cell is a bacterial cell of Escherichia coli. More
preferably, a polynucleotide transfected into the transformed cell
comprises the nucleotide base sequence of SEQ ID NO: 1. SEQ ID NOs:
1-2 set forth nucleotide and amino acid sequences for a
representative vertebrate, human. Also provided by the present
invention are homologs or biologically equivalent glucose
transporter polynucleotides and polypeptides found in other
vertebrates, particularly warm-blooded vertebrates, more
particularly mammals, and even more particularly mouse and rat
homologs.
[0099] As mentioned above, in connection with expression
embodiments to prepare recombinant glucose transporter polypeptide
proteins and peptides, it is contemplated that longer DNA segments
will most often be used, with DNA segments encoding the entire
glucose transporter polypeptide protein, functional domains or
cleavage products thereof, being most preferred. However, it will
be appreciated that the use of shorter DNA segments to direct the
expression of glucose transporter polypeptides or core regions,
such as can be used to generate anti-glucose transporter
polypeptide antibodies, also falls within the scope of the
invention.
[0100] DNA segments which encode peptide antigens from about 15 to
about 50 amino acids in length, or more preferably, from about 15
to about 30 amino acids in length are contemplated to be
particularly useful. DNA segments encoding peptides will generally
have a minimum coding length in the order of about 45 to about 150,
or to about 90 nucleotides. DNA segments encoding full length
proteins can have a minimum coding length on the order of about
4,000 or 5,000 nucleotides for a protein in accordance with SEQ ID
NO: 1. DNA segments of the present invention can contain 300, 400,
500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or up
to 5,000 nucleotides. Peptides of the present invention can contain
10, 20, 50, 100, 200, 300, 400, 500, 750, 1,000, or up to 1,500
amino acids.
[0101] B5. Sequence Modification Techniques
[0102] Modifications to the glucose transporter proteins and
peptides described herein can be carried out using techniques known
in the art, including site directed mutagenesis. Site-specific
mutagenesis is a technique useful in the preparation of individual
peptides, or biologically functional equivalent proteins or
peptides, through specific mutagenesis of the underlying DNA. The
technique further provides a ready ability to prepare and test
sequence variants; for example, incorporating one or more of the
foregoing considerations, by introducing one or more nucleotide
sequence changes into the DNA. Site-specific mutagenesis allows the
production of mutants through the use of specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to provide a
primer sequence of sufficient size and sequence complexity to form
a stable duplex on both sides of the deletion junction being
traversed. Typically, a primer of about 17 to 30 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0103] In general, the technique of site-specific mutagenesis is
well known in the art as exemplified by publications (e.g., Adelman
et al., (1983) DNA 2:183; Sambrook et al., 1989) and can be
achieved in a variety of ways generally known to those of skill in
the art.
[0104] B6. Other Structural Equivalents
[0105] The knowledge of the structure of the glucose transporter
polypeptide of the present invention provides a tool for
investigating the mechanism of action of these proteins in a
subject. For example, binding of these proteins to various
substrate molecules can be predicted by various computer models.
Upon discovering that such binding in fact takes place, knowledge
of the protein structure then allows design and synthesis of small
molecules which mimic the functional binding of the glucose
transporter polypeptide to the substrate. This is the method of
"rational" drug design, also described below.
[0106] Use of the isolated and purified glucose transporter
polypeptide of the present invention in rational drug design is
thus provided in accordance with the present invention. Additional
rational drug design techniques are described in U.S. Pat. Nos.
5,834,228 and 5,872,011, herein incorporated in their entirety.
[0107] Thus, in addition to the peptidyl compounds described
herein, the inventors also contemplate that other sterically
similar compounds can be formulated to mimic the key portions of
the peptide structure. Such compounds can be used in the same
manner as the peptides of the invention and hence are also
functional equivalents. The generation of a structural functional
equivalent can be achieved by the techniques of modeling and
chemical design known to those of skill in the art. It will be
understood that all such sterically similar constructs fall within
the scope of the present invention.
C. Introduction of the Novel Glucose Transporter Polypeptide into
an Expression System
[0108] In accordance with the present invention, where the nucleic
acid sequence shown in SEQ ID NO: 1 itself is employed to introduce
the present glucose transporter translation product into a system
for study, a convenient method of introduction will be through the
use of a recombinant vector that incorporates the desired gene,
together with its associated promoter and enhancer sequences. The
preparation of recombinant vectors is well known to those of skill
in the art and described in many references, such as, for example,
Sambrook et al. (1992), incorporated herein in its entirety.
[0109] C1. Vector Construction
[0110] It is understood that the DNA coding sequences to be
expressed, in this case those encoding the present glucose
transporter gene product, are positioned in a vector adjacent to
and under the control of a promoter. It is understood in the art
that to bring a coding sequence under the control of such a
promoter, one generally positions the 5' end of the transcription
initiation site of the transcriptional reading frame of the gene
product to be expressed between about 1 and about 50 nucleotides
"downstream" of (i.e., 3' of) the chosen promoter. One can also
desire to incorporate into the transcriptional unit of the vector
an appropriate polyadenylation site (e.g., 5'-AATAAA-3'), if one
was not contained within the original inserted DNA. Typically,
these poly-A addition sites are placed about 30 to 2000 nucleotides
"downstream" of the coding sequence at a position prior to
transcription termination.
[0111] While use of the control sequences of the present gene will
be preferred, other control sequences can be employed, so long as
they are compatible with the genotype of the cell being treated.
Thus, one can mention other useful promoters by way of example,
including, e.g., an SV40 early promoter, a long terminal repeat
promoter from retrovirus, an actin promoter, a heat shock promoter,
a metallothionein promoter, and the like.
[0112] As is known in the art, a promoter is a region of a DNA
molecule typically within about 100 nucleotide pairs upstream of
(i.e., 5' to) the point at which transcription begins (i.e., a
transcription start site). That region typically contains several
types of DNA sequence elements that are located in similar relative
positions in different genes.
[0113] Another type of discrete transcription regulatory sequence
element is an enhancer. An enhancer imposes specificity of time,
location and expression level on a particular coding region or
gene. A major function of an enhancer is to increase the level of
transcription of a coding sequence in a cell that contains one or
more transcription factors that bind to that enhancer. An enhancer
can function when located at variable distances from transcription
start sites so long as a promoter is present.
[0114] An enhancer-promoter used in a vector construct of the
present invention can be any enhancer-promoter that drives
expression in a cell to be transfected. By employing an
enhancer-promoter with well-known properties, the level and pattern
of gene product expression can be optimized.
[0115] For introduction of, for example, a nucleic acid sequence
coding for a human glucose transporter, a vector construct that
will deliver the gene to the affected cells is desired. Viral
vectors can be used. These vectors will preferably be an
adenoviral, a retroviral, a vaccinia viral vector, adeno-associated
virus or Lentivirus; these vectors are preferred because they have
been successfully used to deliver desired sequences to cells and
tend to have a high infection efficiency. Suitable vector-glucose
transporter gene constructs are adapted for administration as
pharmaceutical compositions, as described herein below. Viral
promoters can also be of use in vectors of the present invention,
and are known in the art.
[0116] Commonly used viral promoters for expression vectors are
derived from polyoma, cytomegalovirus, Adenovirus 2, and Simian
Virus 40 (SV40). The early and late promoters of SV40 virus are
particularly useful because both are obtained easily from the virus
as a fragment that also contains the SV40 viral origin of
replication. Smaller or larger SV40 fragments can also be used,
provided there is included the approximately 250 bp sequence
extending from the HindIII site toward the BglI site located in the
viral origin of replication. Further, it is also possible, and
often desirable, to utilize promoter or control sequences normally
associated with the desired gene sequence, including the promoter
sequence shown in SEQ ID NO: 4, or fragment thereof, provided such
control sequences are compatible with the host cell systems.
[0117] The origin of replication can be provided either by
construction of the vector to include an exogenous origin, such as
can be derived from SV40 or other viral source, or can be provided
by the host cell chromosomal replication mechanism. If the vector
is integrated into the host cell chromosome, the latter is often
sufficient.
[0118] Where a glucose transporter gene itself is employed it will
be most convenient to simply use the sequence shown in SEQ ID NO: 1
directly, and optionally, in conjunction with the promoter sequence
shown in SEQ ID NO: 4. However, it is contemplated that certain
regions of SEQ ID NO: 1 can be employed exclusively without
employing the entire nucleic acid sequence itself. It is proposed
that it will ultimately be preferable to employ the smallest region
needed to modulate biological activity so that one is not
introducing unnecessary DNA into cells which receive a glucose
transporter gene construct.
D. Use of the Novel Polynucleotide Sequence in Predictive
Diagnostic Screening
[0119] In light of the fact that the present nucleic acid sequence
maps to a locus on chromosome 20 known to be associated with Type
II diabetes, the level of expression of the glucose transporter of
the present invention can be used in the investigation of normal,
experimentally perturbed or disease-state metabolism. For example,
the effect of a treatment, e.g. the administration of a drug can be
studied by its effect on expression of the glucose transporter of
the present invention. This approach can be used to optimize or
monitor treatment or to assist in the discovery or evaluation of
new treatments. Such treatments can focus on interactions between
the nucleic acid or amino acid sequences of the present
invention.
[0120] Clearly, the novel polynucleotide sequence has a role in the
diagnostic predictive screening of individuals susceptible to the
Type II diabetic condition. The polynucleotide sequence, or regions
thereof, unique to the present invention can be used as probes to
determine the presence, absence or, since the invention
accommodates the polymorphic nature of the sequence, mutations in
the identified glucose transporter protein. The absence or mutation
of the wild type sequence can alert researchers and clinicians to
the fact that the subject can be abnormally susceptible to Type II
diabetes.
[0121] D1. Preparation of Probes and Conditions for
Hybridization
[0122] As set forth above, in certain aspects, DNA sequence
information provided by the invention allows for the preparation of
probes that specifically hybridize to encoding sequences of the
present glucose transporter DNA sequence. In these aspects, probes
of an appropriate length are prepared based on a consideration of
the encoding sequence for a polypeptide of the present invention.
The ability of such probes to specifically hybridize to other
encoding sequences lends them particular utility in a variety of
embodiments. Most importantly, the probes can be used in a variety
of assays for detecting the presence of complementary sequences in
a given sample. However, other uses are envisioned, including the
use of the sequence information for the preparation of mutant
species primers, or primers for use in preparing other genetic
constructions.
[0123] To provide certain of the advantages in accordance with the
invention, a preferred nucleic acid sequence employed for
hybridization studies or assays includes probe sequences that are
complementary to or mimic at least a 14 to 40 or so long nucleotide
stretch of a nucleic acid sequence of the present invention. A size
of at least 14 nucleotides in length helps to ensure that the
fragment is of sufficient length to form a duplex molecule that is
both stable and selective. Molecules having complementary sequences
over stretches greater than 14 bases in length are generally
preferred, though, to increase stability and selectivity of the
hybrid, and thereby improve the quality and degree of specific
hybrid molecules obtained. One will generally prefer to design
nucleic acid molecules having complementary stretches of 14 to 20
nucleotides, or even longer where desired, such as 30, 40, 50, 60,
100, 200, 300, or 500 nucleotides or up to the full length of the
present DNA sequence. Such fragments can be readily prepared by,
for example, directly synthesizing the fragment by chemical
synthesis, by application of nucleic acid amplification technology,
such as the PCR technology of U.S. Pat. No. 4,683,202, herein
incorporated by reference, or by introducing selected sequences
into recombinant vectors for recombinant production.
[0124] Accordingly, a nucleotide sequence of the present invention
can be used for its ability to selectively form duplex molecules
with complementary stretches of the gene. Depending on the
application envisioned, one employs varying conditions of
hybridization to achieve varying degrees of selectivity of the
probe toward the target sequence. For applications requiring a high
degree of selectivity, one typically employs relatively stringent
conditions to form the hybrids. Such conditions are particularly
selective, and tolerate little, if any, mismatch between the probe
and the template or target strand.
[0125] Of course, for some applications, less stringent
hybridization conditions are typically needed to allow formation of
the heteroduplex; one of ordinary skill in the art will know how to
adjust the hybridization conditions for optimizing particular
procedures. For example, it is generally appreciated that
conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated by one of skill
in the art using known methods to carry out the desired function or
experiment, without undue experimentation.
[0126] D2. Screening Procedure
[0127] The predictive screening process is performed as follows.
The sample of nucleic acid to be probed will be substantially pure
and free of contaminants that could interfere with the screening
process. Nucleic acid purification methods known in the art or
commercially available can be employed to remove contaminants. A
DNA or RNA molecule and particularly a DNA segment or
polynucleotide can be used for hybridization to a DNA or RNA source
or sample suspected of encoding the glucose transporter polypeptide
of the present invention; such molecules are referred to as
"probes," and such hybridization is "probing". Such probes can be
made synthetically. Probes useful in the present invention can be
designed with sequences complementary to the sequence shown in SEQ
ID NO: 1. The probing is accomplished by contacting, or
hybridizing, the oligonucleotide probe to a DNA source suspected of
possessing the glucose transporter DNA sequence of the present
invention. In some cases, the probes constitute only a single
probe, and in others, the probes constitute a collection of probes
based on a certain amino acid sequence or sequences of the
polypeptide and account in their diversity for the redundancy
inherent in the genetic code. Other molecules which are neither DNA
nor RNA but are capable of hybridizing in a similar manner and
which are designed structurally to mimic the DNA or RNA sequence of
the claimed glucose transporter DNA sequence are also provided.
[0128] A suitable sample to examine is a sample that is capable of
expressing a polypeptide of the present invention and can be a
genomic library of a cell line of interest. Alternatively, a source
of DNA or RNA can include total DNA or RNA from the blood of a
patient or a cell line of interest. Once the hybridization process
of the invention has identified a candidate DNA segment, a positive
clone can be confirmed by further hybridization, restriction enzyme
mapping, sequencing and/or expression and testing.
[0129] D3. Analysis of Sample Screening Data
[0130] Screening data obtained using the present invention can be
compared to data obtained from the nucleic acid and polypeptide
sequence of the present invention. For example, DNA sequence data
obtained from a sample can be compared to the sequence shown in SEQ
ID NO: 1. The comparison can be made using manual or automated
methods, the practicality of which will be determined by the size
and quality of obtained data. Variations and abnormalities in
nucleic acid sequence when comparing the sample's sequence and the
sequence shown in SEQ ID NO: 1 can predict the susceptibility of
the subject supplying the sample to Type II diabetes. Variations in
known glucose binding motifs, for example, can be implicated in an
observed decreased biological ability to transport glucose.
Sequence variations can also be predictive or indicative of
improper protein folding, improper insertion of the translated
protein within the membrane or a range of other structural defects
manifested in an observed systemic condition.
[0131] Individuals at risk for a glucose-related disorder can also
be identified by the possession of structural defects in the
claimed glucose transporter gene. A nucleic acid sequence from a
subject can be analyzed, using the claimed invention and
methodology known to those skilled in the art, for gross
chromosomal rearrangements such as deletions, insertions,
translocations, frame shifts or point mutations, as described
above. Gross rearrangements affecting the reading frame of the gene
are also detectable and likely to alter biological glucose
transport activity.
[0132] D4. Uses of the Invention Related to Diagnostic
Screening
[0133] The described DNA molecules can be used in a number of
techniques beyond that described above, including their use as: (1)
diagnostic tools to detect normal and abnormal DNA sequences in DNA
derived from patient's cells; (2) reagents for detecting and
isolating other members of the polypeptide family and related
polypeptides from a DNA library potentially containing such
sequences; (3) primers for hybridizing to related sequences for the
purpose of amplifying those sequences; (4) primers for altering
native glucose transporter DNA sequences; as well as (5) other
techniques which rely on the similarity of the sequences of
interest to those of the sequences herein disclosed.
[0134] D5. Mapping
[0135] FIG. 2 shows a schematic of the genomic structure and
organization of the novel glucose transporter nucleic acid
sequence. The sequence contains five exons and four introns.
Untranslated sequences are found abutting exon 1 and exon 5. FIG. 2
also characterizes the splice sites of the exon/intron junctions.
The exons vary in size from 56 bp to greater than 2597 bp. Spliced
out introns vary in size from 539 to 15300 bp. Only 4 bp of the 5'
coding sequence are present in exon 1 and similarly, 79 bp of the
3' coding sequence are present in exon 5.
[0136] The present invention can be used by researchers in the
field to screen clinical isolates, laboratory-generated clones or
samples for the investigation of Type II diabetes-related
conditions and will aid in genetic mapping studies. The nucleic
acid sequence of the present invention is particularly useful when
employed, either as shown in SEQ ID NO: 1 or as fragments thereof,
as a probe in a screening process. In this role, the nucleic acid
sequence of the present invention can accommodate naturally
occurring or artificially-created polymorphisms within the
sequence. Mutations in the claimed invention can be associated with
an increased susceptibility to Type II diabetes. Exemplary uses of
the sequence in laboratory studies are presented, said uses being
implemented in conjunction with standard laboratory techniques
familiar to one skilled in the art. The uses presented are
non-exhaustive and variations will be apparent to those skilled in
the art.
[0137] The nucleic acid sequence that encodes for the claimed
glucose transporter can be used to generate hybridization probes
that are useful for mapping naturally occurring genomic sequences
and/or disease loci. The sequences can be mapped to a particular
chromosome or to a specific region of the chromosome using
well-known techniques. Such techniques include FISH, FACS, or
artificial chromosome constructions, such as yeast artificial
chromosomes, bacterial artificial chromosomes, bacterial P1
constructions or single chromosome cDNA libraries as reviewed in
Price, C. M. (1993) Blood Rev. 7: 127-134, and Trask, B. J. (1991)
Trends Genet. 7: 149-154.
[0138] FISH (as described in Verma et al. (1988) Human Chromosomes:
A Manual of Basic Techniques, Pergamon Press, New York, N.Y.) can
be correlated with other physical chromosome mapping techniques and
genetic map data. Examples of genetic map data can be found in the
1994 Genome Issue of Science (265:1981f). Correlation between the
location of the gene encoding the claimed glucose transporter on a
physical chromosomal map and a specific disease, or predisposition
to a specific disease, can help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of
the claimed invention can be used to detect differences in gene
sequences between normal, carrier, or affected individuals.
[0139] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers can be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, reveals associated markers also
found in other mammals, such as humans, even if the number or arm
of a particular human chromosome is not known. New sequences can be
assigned to chromosomal arms, or parts thereof, by physical
mapping. This provides valuable information to investigators
searching for disease genes using positional cloning or other gene
discovery techniques. Once the disease or syndrome has been crudely
localized by genetic linkage to a particular genomic region, for
example, a glucose transporter polypeptide of the present invention
to between D20S119 and D20S178, any sequences mapping to that area
can represent associated or regulatory genes for further
investigation. The nucleotide sequences of the present invention
can thus also be used to detect differences in the chromosomal
location due to translocation, inversion, etc. among normal,
carrier, or affected individuals.
[0140] The mapping methods of the present invention also employ
genomic clones of the exons of the claimed glucose transporter.
Coding and genomic sequences for the claimed glucose transporter in
human is set forth in SEQ ID NO: 1 and FIG. 1. Sequences from
exon/intron junctions of the human glucose transporter of the
present invention, are set forth in Table 2.
TABLE-US-00002 TABLE 2 Sequence at Exon/Intron Junction Exon #:
Exon size 5' Splice Donor 3' Splice Acceptor Intron size Amino acid
1 >56 bp ATG G gtaagt . . . ttttttag GC CAC 15,300 bp Gly-2 2
1,284 bp CCA G gtaag . . . ccctag TG ACC 539 bp Val-430 3 123 bp
ATT G gtgagt . . . tttccag GC ACC 2,366 bp Gly-471 4 136 bp AGA CG
gtagg . . . gacag G TTC 4,267 bp Arg-516 5 >2,597 bp
[0141] D6. Use of the Novel Nucleic Acid Sequence in Genetic Assays
and Polymorphism Identification
[0142] The present invention provides genetic assays based on the
genomic sequence of the human glucose transporter gene of the
present invention. The intronic sequence flanking the individual
exons encoding the glucose transporter gene, which are described in
Table 2, is employed in the design of oligonucleotide primers
suitable for the mutation analysis of human genomic DNA. Thus,
intronic primers can be used to screen for genetic variants by a
number of PCR-based techniques, including single-strand
conformation polymorphism (SSCP) analysis (Orita, et al. (1989)
Proc. Natl. Acad. Sci. USA 86 (8): 2766-70), SSCP/heteroduplex
analysis, enzyme mismatch cleavage, and direct sequence analysis of
amplified exons (Kestila, et al. (1998) Mol. Cell. 1 (4): 575-82;
Yuan, et al. (1999) Hum. Mutat. 14 (5): 440-6).
[0143] Automated methods can also be applied to the large-scale
characterization of single nucleotide polymorphisms (Brookes (1999)
Gene 234 (2): 177-186; Wang, et al. (1998) Science 280 (5366):
1077-82) within and near the human glucose transporter gene. Once
genetic variants have been detected in specific patient
populations, e.g. glucose transporter mutations in patients with
Type II diabetes, the present invention provides assays to detect
the mutation by methods such as allele-specific hybridization
(Stoneking, et al. (1991) Am. J. Hum. Genet. 48 (2): 370-82), or
restriction analysis of amplified genomic DNA containing the
specific mutation. Again, these detection methods can be automated
using existing technology (See e.g., Wang, et al. (1998) Science
280 (5366): 1077-82). In the case of genetic disease or human
phenotypes caused by repeat expansion (Lafreniere, et al. (1997)
Nat Genet. 15 (3): 298-302; Timchenko and Caskey (1996) FASEB J 10
(14): 1589-97, the invention provides an assay based on PCR of
genomic DNA with oligonucleotide primers flanking the involved
repeat.
[0144] The provided nucleic acid molecules can be labeled according
to any technique known in the art, such as with radiolabels,
fluorescent labels, enzymatic labels, sequence tags, etc. Such
molecules can be used as allele-specific oligonucleotide probes.
Body samples can be tested to determine whether the nucleic acid
sequence encoding the glucose transporter of the present invention
contains a polymorphism. Suitable body samples for testing include
those comprising DNA, RNA or protein obtained from biopsies,
including liver and pancreatic tissue biopsies; or from blood,
prenatal; or embryonic tissues, for example.
[0145] D6a. Primer Selection and Design
[0146] In one embodiment of the invention two pairs of isolated
oligonucleotide primers are provided. These sets of primers are
optionally derived from one of the glucose transporter exons shown
in FIG. 2. The oligonucleotide primers are useful, for example, in
detecting a polymorphism of the glucose transporter of the present
invention. The primers direct amplification of a target
polynucleotide prior to sequencing. In another embodiment of the
invention isolated allele specific oligonucleotides (ASO) are
provided. The allele specific oligonucleotides are also useful in
detecting a polymorphism of the glucose transporter of the present
invention.
[0147] The primers of the invention embrace oligonucleotides of
sufficient length and appropriate sequence so as to provide
initiation of polymerization on a significant number of nucleic
acids in the polymorphic locus.
[0148] Environmental conditions conducive to synthesis include the
presence of nucleotide triphosphates and an agent for
polymerization, such as DNA polymerase, and a suitable temperature
and pH. The primer is preferably single stranded for maximum
efficiency in amplification, but can be double stranded. If double
stranded, the primer is first treated to separate its strands
before being used to prepare extension products. The primer must be
sufficiently long to prime the synthesis of extension products in
the presence of the inducing agent for polymerization. The exact
length of primer will depend on many factors, including
temperature, buffer, and nucleotide composition. The
oligonucleotide primer typically contains 12-20 or more
nucleotides, although it can contain fewer nucleotides. The primers
should have sufficient complementarity with the 5' and 3' sequences
flanking the transition to hybridize therewith and permit
amplification of the genomic locus.
[0149] Oligonucleotide primers of the invention are employed in the
amplification method, which is an enzymatic chain reaction that
produces exponential quantities of polymorphic locus relative to
the number of reaction steps involved. Typically, one primer is
complementary to the negative (-) strand of the polymorphic locus
and the other is complementary to the positive (+) strand.
Annealing the primers to denatured nucleic acid followed by
extension with an enzyme, such as the large fragment of DNA
polymerase I (Klenow) and nucleotides, results in newly synthesized
(+) and (-) strands containing the target polymorphic locus
sequence. Because these newly synthesized sequences are also
templates, repeated cycles of denaturing, primer annealing, and
extension results in exponential production of the region (i.e.,
the target polymorphic locus sequence) defined by the primers. The
product of the chain reaction is a discreet nucleic acid duplex
with termini corresponding to the ends of the specific primers
employed.
[0150] The oligonucleotide primers of the invention can be prepared
using any suitable method, such as conventional phosphotriester and
phosphodiester methods or automated embodiments thereof. In one
such automated embodiment, diethylphosphoramidites are used as
starting materials and can be synthesized as described by Beaucage
et al., (1981) Tetrahedron Lett. 22:1859-1862. One method for
synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. No. 4,458,066.
[0151] D6b. Amplification Techniques
[0152] Any nucleic acid specimen, in purified or non-purified form,
can be utilized as the starting nucleic acid or acids, providing it
contains, or is suspected of containing, a nucleic acid sequence
containing the polymorphic locus. Thus, the method can amplify, for
example, DNA or RNA, including messenger RNA, wherein DNA or RNA
can be single stranded or double stranded. In the event that RNA is
to be used as a template, enzymes, and/or conditions optimal for
reverse transcribing the template to DNA would be utilized. In
addition, a DNA-RNA hybrid that contains one strand of each can be
utilized. A mixture of nucleic acids can also be employed, or the
nucleic acids produced in a previous amplification reaction herein,
using the same or different primers can be so utilized. The
specific nucleic acid sequence to be amplified, i.e., the
polymorphic locus, can be a fraction of a larger molecule or can be
present initially as a discrete molecule, so that the specific
sequence constitutes the entire nucleic acid. It is not necessary
that the sequence to be amplified be present initially in a pure
form; it can be a minor fraction of a complex mixture, such as
contained in whole human DNA.
[0153] DNA utilized herein can be extracted from a body sample,
such as blood, tissue material (e.g. liver or pancreatic tissue),
and the like by a variety of techniques such as that described by
Sambrook et al. in Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, N.Y., (1992). If the extracted sample is impure, it
can be treated before amplification with an amount of a reagent
effective to open the cells, or animal cell membranes of the
sample, and to expose and/or separate the strand(s) of the nucleic
acid(s). This lysing and nucleic acid denaturing step to expose and
separate the strands will allow amplification to occur much more
readily.
[0154] The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and
dTTP are added to the synthesis mixture, either separately or
together with the primers, in adequate amounts and the resulting
solution is heated to about 90-100.degree. C. from about 1 to 10
minutes, preferably from 1 to 4 minutes. After this heating period,
the solution is allowed to cool, which is preferable for the primer
hybridization. To the cooled mixture is added an appropriate agent
for effecting the primer extension reaction (called herein "agent
for polymerization"), and the reaction is allowed to occur under
conditions known in the art. The agent for polymerization can also
be added together with the other reagents if it is heat stable.
This synthesis (or amplification) reaction can occur at room
temperature up to a temperature above which the agent for
polymerization no longer functions. Thus, for example, if DNA
polymerase is used as the agent, the temperature is generally no
greater than about 40.degree. C. Most conveniently the reaction
occurs at room temperature.
[0155] The agent for polymerization can be any compound or system
that will function to accomplish the synthesis of primer extension
products, including enzymes. Suitable enzymes for this purpose
include, for example, E. coli DNA polymerase I, Klenow fragment of
E. coli DNA polymerase, polymerase muteins, reverse transcriptase,
other enzymes, including heat-stable enzymes (i.e., those enzymes
which perform primer extension after being subjected to
temperatures sufficiently elevated to cause denaturation), such as
Taq polymerase. Suitable enzyme will facilitate combination of the
nucleotides in the proper manner to form the primer extension
products that are complementary to each polymorphic locus nucleic
acid strand. Generally, the synthesis will be initiated at the 3'
end of each primer and proceed in the 5' direction along the
template strand, until synthesis terminates, producing molecules of
different lengths.
[0156] The newly synthesized strand and its complementary nucleic
acid strand will form a double-stranded molecule under hybridizing
conditions described herein and this hybrid is used in subsequent
steps of the method. In the next step, the newly synthesized
double-stranded molecule is subjected to denaturing conditions
using any of the procedures described above to provide
single-stranded molecules.
[0157] The steps of denaturing, annealing, and extension product
synthesis can be repeated as often as needed to amplify the target
polymorphic locus nucleic acid sequence to the extent necessary for
detection. The amount of the specific nucleic acid sequence
produced will accumulate in an exponential fashion. See McPherson
et al., eds., (1992) PCR. A Practical Approach, ILR Press.
[0158] The amplification products can be detected by Southern blot
analysis with or without using radioactive probes. In one such
method, for example, a small sample of DNA containing a very low
level of the nucleic acid sequence of the polymorphic locus is
amplified, and analyzed via a Southern blotting technique or
similarly, using dot blot analysis. The use of non-radioactive
probes or labels is facilitated by the high level of the amplified
signal. Alternatively, probes used to detect the amplified products
can be directly or indirectly detectably labeled, for example, with
a radioisotope, a fluorescent compound, a bioluminescent compound,
a chemiluminescent compound, a metal chelator or an enzyme. Those
of ordinary skill in the art will know of other suitable labels for
binding to the probe, or will be able to ascertain such, using
routine experimentation.
[0159] Sequences amplified by the methods of the invention can be
further evaluated, detected, cloned, sequenced, and the like,
either in solution or after binding to a solid support, by any
method usually applied to the detection of a specific DNA sequence
such as dideoxy sequencing, PCR, oligomer restriction (Saiki et
al., (1985), Bio-Technol. 3: 1008-12) allele-specific
oligonucleotide (ASO) probe analysis (Conner et al., (1983), Proc.
Natl. Acad. Sci. U.S.A. 80: 278), oligonucleotide ligation assays
(OLAs) (Landgren et al., (1988) Science 241: 1007), and the like.
Molecular techniques for DNA analysis have been reviewed (Landgren
et. al., (1988), Science 242: 229-37.
[0160] D6c. Additional Amplification Methods
[0161] Preferably, the method of amplifying is by PCR, as described
herein and in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188;
each of which is hereby incorporated by reference; and as is
commonly used by those of ordinary skill in the art. Alternative
methods of amplification have been described and can also be
employed as long as the locus amplified by PCR using primers of the
invention is similarly amplified by the alternative technique. Such
alternative amplification systems include but are not limited to
self-sustained sequence replication, which begins with a short
sequence of RNA of interest and a T7 promoter. Reverse
transcriptase copies the RNA into cDNA and degrades the RNA,
followed by reverse transcriptase polymerizing a second strand of
DNA.
[0162] Another nucleic acid amplification technique is nucleic acid
sequence-based amplification (NASBA.TM.) which uses reverse
transcription and T7 RNA polymerase and incorporates two primers to
target its cycling scheme. NASBA.TM. amplification can begin with
either DNA or RNA and finish with either, and amplifies to about
10.sup.8 copies within 60 to 90 minutes.
[0163] Alternatively, nucleic acid can be amplified by ligation
activated transcription (LAT). LAT works from a single-stranded
template with a single primer that is partially single-stranded and
partially double-stranded. Amplification is initiated by ligating a
cDNA to the promoter oligonucleotide and within a few hours,
amplification is about 10.sup.8 to about 10.sup.9 fold. The QB
replicase system can be utilized by attaching an RNA sequence
called MDV-1 to RNA complementary to a DNA sequence of interest.
Upon mixing with a sample, the hybrid RNA finds its complement
among the specimen's mRNAs and binds, activating the replicase to
copy the tag-along sequence of interest.
[0164] Another nucleic acid amplification technique, ligase chain
reaction (LCR), works by using two differently labeled halves of a
sequence of interest which are covalently bonded by ligase in the
presence of the contiguous sequence in a sample, forming a new
target. The repair chain reaction (RCR) nucleic acid amplification
technique uses two complementary and target-specific
oligonucleotide probe pairs, thermostable polymerase and ligase,
and DNA nucleotides to geometrically amplify targeted sequences. A
2-base gap separates the oligo probe pairs, and the RCR fills and
joins the gap, mimicking normal DNA repair.
[0165] Nucleic acid amplification by strand displacement activation
(SDA) utilizes a short primer containing a recognition site for
HincII with short overhang on the 5' end which binds to target DNA.
A DNA polymerase fills in the part of the primer opposite the
overhang with sulfur-containing adenine analogs. HincII is added
but only cuts the unmodified DNA strand. A DNA polymerase that
lacks 5' exonuclease activity enters at the site of the nick and
begins to polymerize, displacing the initial primer strand
downstream and building a new one which serves as more primer.
[0166] SDA produces greater than about a 10.sup.7-fold
amplification in 2 hours at 37.degree. C. Unlike PCR and LCR, SDA
does not require instrumented temperature cycling. Another
amplification system useful in the method of the invention is the
QB Replicase System. Although PCR is the preferred method of
amplification if the invention, these other methods can also be
used to amplify any nucleic acid sequence of the present invention
as described in the method of the invention. Thus, the term
"amplification technique" as used herein and in the claims is meant
to encompass all the foregoing methods.
[0167] D6d. Detection of Polymorphisms
[0168] In another embodiment of the present invention, a method is
provided for identifying a subject having a polymorphism of a
nucleic acid sequence encoding a glucose transporter polynucleotide
of the present invention, comprising sequencing a target nucleic
acid of a sample from a subject by dideoxy sequencing, preferably
following amplification of the target nucleic acid.
[0169] In another embodiment of the present invention a method is
provided for identifying a subject having a polymorphism of the
nucleic acid sequence shown in SEQ ID NO: 1, comprising contacting
a target nucleic acid of a sample from a subject with a reagent
that detects the presence of a polymorphism in the nucleic acid
sequence shown in SEQ ID NO: 1 and detecting the reagent. A number
of hybridization methods are disclosed herein and are well known to
those skilled in the art. Many of them are useful in carrying out
the invention.
[0170] D6e. Hybridization Conditions and Detection
[0171] As discussed above, 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 of ordinary skill in the art.
Stringent temperature conditions will generally include
temperatures in excess of 30.degree. C., typically in excess of
37.degree. C., and preferably in excess of 45.degree. C. Stringent
salt conditions will ordinarily be less than 1,000 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. (See, e.g., (1968) Wetmur & Davidson,
J. Mol. Biol. 31: 349-70).
[0172] Accordingly, a nucleotide sequence of the present invention
can be used for its ability to selectively form duplex molecules
with complementary stretches of the nucleic acid sequence shown in
SEQ ID NO: 1. Depending on the application envisioned, one employs
varying conditions of hybridization to achieve varying degrees of
selectivity of the probe toward the target sequence. For
applications requiring a high degree of selectivity, one typically
employs relatively stringent conditions to form the hybrids. For
example, one selects relatively low salt and/or high temperature
conditions, such as provided by 0.02M-0.15M salt at temperatures of
about 50.degree. C. to about 70.degree. C. including particularly
temperatures of about 55.degree. C., about 60.degree. C. and about
65.degree. C. Such conditions are particularly selective, and
tolerate little, if any, mismatch between the probe and the
template or target strand.
[0173] Of course, for some applications, for example, where one
desires to prepare mutants employing a mutant primer strand
hybridized to an underlying template or where one seeks to isolate
polypeptide coding sequences from related species, functional
equivalents, or the like, less stringent hybridization conditions
are typically needed to allow formation of the heteroduplex. Under
such circumstances, one employs conditions such as 0.15M-0.9M salt,
at temperatures ranging from about 20.degree. C. to about
55.degree. C., including particularly temperatures of about
25.degree. C., about 37.degree. C., about 45.degree. C., and about
50.degree. C. Cross-hybridizing species can thereby be readily
identified as positively hybridizing signals with respect to
control hybridizations. In any case, it is generally appreciated
that conditions can be rendered more stringent by the addition of
increasing amounts of formamide, which serves to destabilize the
hybrid duplex in the same manner as increased temperature. Thus,
hybridization conditions can be readily manipulated, and thus will
generally be a method of choice depending on the desired
results.
[0174] In certain embodiments, it is advantageous to employ a
nucleic acid sequence of the present invention in combination with
an appropriate technique, such as a label, for determining
hybridization. A wide variety of appropriate indicator reagents are
known in the art, including radioactive, enzymatic or other
ligands, such as avidin/biotin, which are capable of giving a
detectable signal. In preferred embodiments, one likely employs an
enzyme tag such a urease, alkaline phosphatase or peroxidase,
instead of radioactive or other environmentally undesirable
reagents. In the case of enzyme tags, calorimetric indicator
substrates are known which can be employed to provide a reagent
visible to the human eye or spectrophotometrically, to identify
specific hybridization with complementary nucleic acid-containing
samples.
[0175] In general, it is envisioned that the hybridization probes
described herein are useful both as reagents in solution
hybridization as well as in embodiments employing a solid phase. In
embodiments involving a solid phase, the sample containing test DNA
(or RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to specific hybridization with selected probes under desired
conditions. The selected conditions depend inter alia on the
particular circumstances based on the particular criteria required
(depending, for example, on the G+C content, type of target nucleic
acid, source of nucleic acid, size of hybridization probe, etc.).
Following washing of the hybridized surface so as to remove
nonspecifically bound probe molecules, specific hybridization is
detected, or even quantified via the label.
[0176] The materials for use in the method of the invention are
ideally suited for the preparation of a screening kit. Such a kit
can comprise a carrier having compartments to receive in close
confinement one or more containers such as vials, tubes, and the
like, each of the containers comprising one of the separate
elements to be used in the method. For example, one of the
containers can comprise an amplifying reagent for amplifying the
DNA shown in SEQ ID NO: 1, such as the necessary enzyme(s) and
oligonucleotide primers for amplifying target DNA from the
subject.
[0177] Oligonucleotide primers comprising target flanking 5' and 3'
polynucleotide sequence have substantially the sequence set forth
in the flanking 5' and 3' portions of SEQ ID NO: 1 and Table 2, and
sequences substantially complementary or homologous thereto. Other
oligonucleotide primers for amplifying a target sequence will be
known or readily ascertainable to those of skill in the art given
the disclosure of the present invention presented herein.
E. Use of the Novel Polypeptide in Predictive Diagnostic
Screening
[0178] The present invention provides a method of screening a
biological sample for the presence of the novel glucose transporter
polypeptide disclosed herein. In accordance with a screening assay
method, a biological sample is exposed to an antibody
immunoreactive with the polypeptide whose presence is being
assayed. Typically, exposure is accomplished by forming an
admixture in a liquid medium that contains both the antibody and
the candidate polypeptide. Either the antibody or the sample with
the polypeptide can be affixed to a solid support (e.g., a column
or a microtiter plate). Additional details of methods for such
assays are known in the art. The presence of polypeptide in the
sample is detected by evaluating the formation and presence of
antibody-polypeptide conjugates. Techniques for detecting such
antibody-antigen conjugates or complexes are well known in the art
and include but are not limited to centrifugation, affinity
chromatography and the like, and binding of a secondary antibody to
the antibody-candidate receptor complex.
[0179] In one embodiment, detection is accomplished by detecting an
indicator affixed to the antibody. Exemplary and well-known
indicators include radioactive labels (e.g., .sup.32P, .sup.125I,
.sup.14C), a second antibody or an enzyme such as horseradish
peroxidase. Techniques for affixing indicators to antibodies are
known in the art.
[0180] In another aspect, the present invention provides a method
of screening a biological sample for the presence of antibodies
immunoreactive with a novel glucose transporter polypeptide.
Preferably, the antibody so identified has activity in the
modulation of glucose transporter polypeptide biological activity
in accordance with the present invention. In accordance with such a
method, a biological sample is exposed to a glucose transporter
polypeptide under biological conditions and for a period of time
sufficient for antibody-polypeptide conjugate formation and the
formed conjugates are detected.
[0181] A method of a facilitating a diagnosis of a disorder
affecting glucose homeostasis is provided in accordance with the
present invention. In one embodiment, the method comprises: (a)
obtaining a biological sample from a subject; and (b) determining
an amount of a glucose transporter polypeptide present in the
biological sample, wherein the presence of a reduced amount of the
glucose transporter polypeptide as compared to a standard
facilitates a diagnosis of a disorder affecting glucose
homeostasis. Optionally, the amount of glucose transporting protein
in the biological sample can be determined by Western blot
analysis.
[0182] In another embodiment, the method comprises: (a) obtaining a
glucose transporter polypeptide from a subject; (b) determining an
activity level of a glucose transporter polypeptide from the
subject; and (c) detecting a variation in glucose transport
activity between a wild type glucose transporter polypeptide and
the glucose transporter polypeptide from the subject, the presence
of a glucose transport activity variation between the wild type
glucose transporter polypeptide and the glucose transporter
polypeptide from the subject facilitating a diagnosis of a disorder
affecting glucose homeostasis.
[0183] The glucose transporter polypeptide can be obtained from a
subject by isolating from the subject a biological sample
comprising the glucose transporter polypeptide. In this case the
method preferably further comprises determining the subcellular
localization of the glucose transporter polypeptide in the
biological sample.
[0184] A biological sample to be screened can be a biological fluid
such as extracellular or intracellular fluid (e.g. blood), or a
cell or tissue extract or homogenate. A biological sample can also
be an isolated cell (e.g., in culture) or a collection of cells
such as in a tissue sample or histology sample. A tissue sample can
be suspended in a liquid medium or fixed onto a solid support such
as a microscope slide.
[0185] A preferred screening system to facilitate a diagnosis of a
disorder affecting glucose homeostasis in a subject involves
expression of a glucose transport polypeptide of the present
invention from the subject in Xenopus oocytes (Gould and Lienhard
(1989), Biochem. 28, 9447-57), followed by a glucose uptake assay.
The Xenopus oocytes are transfected or microinjected with mRNA
encoding the glucose transporter polypeptide of the present
invention that has been isolated from a biological sample from the
subject. This is another approach for obtaining the glucose
transporter polypeptide from the subject.
[0186] The activity of a glucose transporter polypeptide present in
the Xenopus oocytes is then determined by monitoring labeled
glucose uptake. The glucose label can comprise any suitable label,
but is preferably a radiolabel. Variations in glucose transport
activity between a wild type glucose transporter polypeptide and
the glucose transporter polypeptide from the subject are then
detected. The presence of glucose transport activity variations
between the wild type glucose transporter polypeptide and the
isolated glucose transporter polypeptide facilitate the diagnosis
of a disorder affecting glucose homeostasis in the subject.
[0187] Another preferred screening system to facilitate a diagnosis
of a disorder affecting glucose homeostasis in a subject is a
glucose uptake assay system. The rate of glucose uptake is
monitored and variations from known parameters can indicate the
presence of a glucose homeostatis disorder. Glucose uptake assays
are also useful in monitoring cell culture glucose uptake in either
transfected cells or in cells obtained from a patient, such as skin
fibroblasts or lymphocytes, that comprise a glucose transporter
polypeptide of the present invention. In addition, the subcellular
localization of a glucose transporter polypeptide in a sample
obtained from the subject is determined. The subcellular
localization of the protein is determined because it has been shown
that some glucose transporter polypeptides translocate between
intracellular compartments and the plasma membrane in response to
certain stimuli, such as insulin or exercise. Therefore, it is
possible that a subject with a glucose transport disorder might
localize the protein incorrectly within cells in the sample. An
assessment of translocation can be made using standard
immunocytochemical methodology well known to one of skill in the
art, such as by immunofluorescent staining using an anti-glucose
transporter polypeptide antibody.
F. Method of Screening for Chemical and Biological Modulators of
the Biological Activity of the Glucose Transporter of the Present
Invention
[0188] A representative method of screening candidate substances
for their ability to modulate the biological activity of the
glucose transporter of the present invention comprises: (a)
establishing replicate test and control samples that comprise a
biologically active glucose transporter polypeptide; (b)
administering a candidate substance to a test sample; (c) measuring
the biological activity of the polypeptide in the test and the
control samples; and (d) determining whether the candidate
substance modulates biological activity relative to an appropriate
control. By "modulate" is intended an increase, decrease, or other
alteration of any or all biological activities or properties of the
glucose transporter polypeptide. A representative polypeptide is
disclosed in SEQ ID NO: 2.
[0189] A candidate substance identified according to the screening
assay described herein has an ability to modulate the biological
activity of a glucose transporter polypeptide. Such a candidate
compound has utility in the treatment of disorders and conditions
associated with the biological activity of a glucose transporter
polypeptide. Candidate compounds can be hydrophobic, polycyclic, or
both, molecules, and are typically about 500-1,000 daltons in
molecular weight.
[0190] In a cell-free system, the method comprises the steps of
establishing a control system comprising a glucose transporter
polypeptide and a ligand to which the polypeptide is capable of
binding; establishing a test system comprising a glucose
transporter polypeptide, the ligand, and a candidate compound; and
determining whether the candidate compound modulates the activity
of the polypeptide by comparison of the test and control systems. A
representative ligand comprises a monoclonal antibody, and in this
embodiment, the biological activity or property screened includes
binding affinity.
[0191] In another embodiment of the invention, the glucose
transporter polypeptide or a catalytic or immunogenic fragment or
oligopeptide thereof, can be used for screening libraries of
compounds in any of a variety of drug screening techniques. The
fragment employed in such screening can be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. The formation of binding complexes, between the
glucose transporter polypeptide and the agent being tested, can be
measured. In a preferred embodiment, the glucose transporter
polypeptide has an amino acid sequence of SEQ ID NO: 2. In a more
preferred embodiment, the glucose transporter polypeptide is
encoded by a polynucleotide of SEQ ID NO: 1.
[0192] Another technique for drug screening which can be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO 84/03564, herein incorporated by
reference. In this method, as applied to a polypeptide of the
present invention, large numbers of different small test compounds
are synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with the polypeptide,
or fragments thereof, and washed. Bound polypeptide is then
detected by methods well known in the art. The purified polypeptide
can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support.
[0193] In one embodiment, a method of screening for a modulator of
a glucose transport polypeptide encoded by a nucleic acid sequence
located between D20S119 and D20S178 on human chromosome 20q13
comprises: providing cells from a cDNA expression library on a
substrate, the cells comprising cDNA coding for the glucose
transporter polypeptide; causing protein synthesis by the cells;
subjecting the cells to a library of test samples; detecting an
interaction between a test sample and a cell expressing the glucose
transporter polypeptide; identifying a test sample that interacts
with a cell expressing the glucose transporter polypeptide; and
isolating the test sample that interacts with the glucose
transporter polypeptide. In this method, cells can be derived from
a prokaryote or a eukaryote, including Homo sapiens.
[0194] In another embodiment, a method of screening for a modulator
of a glucose transport polypeptide encoded by a nucleic acid
sequence located between D20S119 and D20S178 on human chromosome
20q13 comprises: affixing distinct colonies of cells from a cDNA
expression library on a substrate, the cells comprising cDNA coding
for the glucose transporter polypeptide; causing protein synthesis
by said colonies on the substrate; subjecting the colonies of cells
to a library of test samples; detecting an interaction between a
test sample and a cell expressing the glucose transporter
polypeptide; identifying a test sample that interacts with a cell
expressing a glucose transporter polypeptide; and isolating a test
sample that interacts with a glucose transporter polypeptide. In
this method, the cells can also be derived from a prokaryote or a
eukaryote, including Homo sapiens.
[0195] In yet another embodiment, a method of screening for a
modulator of a glucose transport polypeptide encoded by a nucleic
acid sequence located between D20S119 and D20S178 on human
chromosome 20q13, comprises: providing a library of test samples;
contacting a glucose transporter polypeptide with each test sample;
detecting an interaction between a test sample and a glucose
transporter polypeptide; identifying a test sample that interacts
with a glucose transporter polypeptide; and isolating a test sample
that interacts with a glucose transporter polypeptide.
[0196] In each of the foregoing embodiments, an interaction can be
detected spectrophotometrically, radiologically or immunologically.
An interaction between the glucose transporter polypeptide and a
test sample can also be quantified. Such an interaction can be
quantified by determining glucose transport activity.
[0197] A screening assay of the present invention can also involve
determining the ability of a candidate substance to modulate, i.e.
inhibit or promote glucose transporter biological activity and
preferably, to thereby modulate the biological activity of the
glucose transporter of the present invention in target cells.
Target cells can be either naturally occurring cells known to
contain the polypeptide gene product of the present invention or
transformed cells produced in accordance with a process of
transformation set forth herein above. The test samples can further
comprise a cell or cell line that expresses the polypeptide gene
product of the present invention, for example, Xenopus oocytes
expressing a foreign glucose transporter as discussed above. The
present invention also provides a recombinant cell line suitable
for use in the exemplary method. Such cell lines can be mammalian,
or human, or they can from another organism, including but not
limited to yeast. Representative assays include genetic screening
assays and molecular biology screens such as a yeast two-hybrid
screen that will effectively identify genes related to a
susceptibility to Type II diabetes and those genes important for
proper glucose transport and other glucose transport-mediated
cellular process. One version of the yeast two-hybrid system has
been described (Chien et al., (1991), Proc. Natl. Acad. Sci. USA,
88: 9578-82) and is commercially available from Clontech (Palo
Alto, Calif.).
[0198] As is well known in the art, a screening assay can provide a
cell under conditions suitable for testing the modulation of the
biological activity of a glucose transporter polypeptide. These
conditions include but are not limited to pH, temperature,
tonicity, the presence of relevant metabolic factors (e.g., metal
ions such as for example Ca.sup.++, growth factor, interleukins, or
colony stimulating factors), and relevant modifications to the
polypeptide such as glycosylation or prenylation. A glucose
transporter polypeptide of the present invention can be expressed
and utilized in a prokaryotic or eukaryotic cell. The host cell can
also be fractionated into sub-cellular fractions where a structure
of interest can be found. For example, cells expressing the
polypeptide can be fractionated into the nuclei, the endoplasmic
reticulum, vesicles, or the membrane surfaces of the cell.
[0199] F1. Rational Drug Design
[0200] A method of identifying modulators of the activity of the
glucose transporter polypeptide of the present invention using
rational drug design is provided in accordance with the present
invention. The method comprises the steps of designing a potential
modulator for the glucose transporter polypeptide of the present
invention that will form non-covalent bonds with amino acids in the
substrate binding site based upon the structure of the glucose
transporter polypeptide of the present invention; synthesizing the
modulator; and determining whether the potential modulator
modulates the activity of the glucose transporter polypeptide of
the present invention. Modulators can be synthesized using
techniques known in the art.
[0201] The determination of whether the modulator modulates the
biological activity of the glucose transporter polypeptide of the
present invention is made in accordance with the screening methods
disclosed herein, or by other screening methods known in the art.
Preferably, the glucose transporter polypeptide comprises the amino
acid sequence of SEQ ID NO: 2. More preferably, the glucose
transporter polypeptide is encoded by a nucleic acid having the
sequence of SEQ ID NO: 1.
[0202] F2. Method of Screening for Modulators of Levels and/or
Activity of Glucose Transporter
[0203] In accordance with the present invention there are also
provided methods for screening candidate compounds for the ability
to modulate in vivo glucose transporter levels and/or activity.
Representative modulators of the level of the glucose transporter
polypeptide of the present invention can comprise modulators of
transcription or expression. Pharmaceuticals that increase or
decrease the transcription or expression levels of the glucose
transporter polypeptide of the present invention have important
clinical application for the modulation of the biological activity
of a glucose transporter polypeptide of the present invention. This
modulation can affect glucose homeostasis. Preferably, the glucose
transporter polypeptide comprises the amino acid sequence of SEQ ID
NO: 2. More preferably, the glucose transporter polypeptide is
encoded by a nucleic acid having the sequence of SEQ ID NO: 1.
[0204] The present invention thus includes a method for discovery
of compounds that modulate the expression levels of the glucose
transporter polypeptide of the present invention, and describes the
use of such compounds. The general approach is to screen compound
libraries for substances that increase or decrease the expression
of the polypeptide.
[0205] In accordance with the present invention there is also
provided a method of identifying a candidate compound or molecule
that is capable of modulating the transcription level of the gene
encoding the glucose transporter polypeptide of the present
invention and thus is capable of acting as a therapeutic agent in
the modulation of the effects of the polypeptide. This modulation
can affect glucose homeostasis. Such modulation can be direct,
i.e., through binding of a candidate molecule directly to the
nucleotide sequence, whether DNA or RNA transcript, or such
modulation can be achieved via one or more intermediaries, such as
proteins other than the glucose transporter polypeptide of the
present invention which are affected by the candidate compound and
ultimately modulate transcription by any mechanism, including
direct binding, phosphorylation or dephosphorylation, etc.
[0206] This method comprises contacting a cell or nucleic acid
sample with a candidate compound or molecule to be tested. These
samples contain nucleic acids which can contain elements that
modulate transcription and/or translation of the nucleic acid
sequence encoding a glucose transporter polypeptide (e.g. a nucleic
acid sequence of SEQ ID NO: 1), such as an operatively linked
promoter or putative upstream regulatory region (SEQ ID NO: 4), and
a DNA sequence encoding a polypeptide that can be detected in some
way. Thus, the polypeptide can be described as a "reporter" or
"marker." Preferably, the candidate compound directly and
specifically transcriptionally modulates expression of a nucleic
acid sequence encoding the glucose transporter polypeptide of the
present invention. Such compounds are anticipated to have
therapeutic or pharmaceutical uses in treating glucose
homeostasis-related diseases and/or disorders.
[0207] The DNA sequence is coupled to and under the control of the
promoter, under conditions such that the candidate compound or
molecule, if capable of acting as a transcriptional modulator of
the nucleic acid sequence, causes a glucose transporter polypeptide
of the present invention to be expressed and so produces a
detectable signal, which can be assayed quantitatively and compared
to an appropriate control. Candidate compounds or molecules of
interest can include those which have the ability to increase or
decrease, i.e., modulate, transcription from the promoter
operatively linked to the nucleic acid sequence. The reporter gene
can encode a reporter known in the art, such as luciferase.
[0208] In certain embodiments of the invention, the polypeptide so
produced is capable of complexing with an antibody or is capable of
complexing with biotin. In this case the resulting complexes can be
detected by methods known in the art. The detectable signal of this
assay can also be provided by messenger RNA produced by
transcription of said reporter gene. Exactly how the signal is
produced and detected can vary and is not the subject of the
present invention; rather, the present invention provides a nucleic
acid sequence for use in such an assay. The molecule to be tested
in these methods can be a purified molecule, a homogenous sample,
or a mixture of molecules or compounds. Further, in the method of
the invention, the DNA in the cell can comprise more than one
modulatable transcriptional regulatory sequence.
[0209] In accordance with the present invention there is also
provided a rapid and high throughput screening method that relies
on the methods described above. This screening method comprises
separately contacting each of a plurality of substantially
identical samples. In such a screening method the plurality of
samples preferably comprises more than about 10.sup.4 samples, or
more preferably comprises more than about 5.times.10.sup.4
samples.
G. Therapeutic Applications
[0210] In accordance with the present invention, a variety of
therapeutic applications are provided. The purified sequence can,
for example, be used to alleviate or ameliorate the Type II
diabetic condition using existing gene therapy techniques. Other
applications provided by the present invention include drug
therapy, wherein a compound is administered to a subject in order
to affect the activity of the glucose transporter of the claimed
invention. An additional therapy provided by the present invention
includes the administration of the glucose transporter polypeptide
of the present invention, or a biologically active fragment or
analog thereof.
[0211] G1. Dosages of A Drug Modulating Glucose Transport
Activity
[0212] As used herein, an "effective" dose refers to one that is
administered in doses tailored to each individual patient
manifesting symptoms of glucose transporter malfunction sufficient
to cause an improvement therein. After review of the disclosure
herein of the present invention, one of ordinary skill in the art
can tailor the dosages to an individual patient, taking into
account the particular formulation and method of administration to
be used with the composition as well as patient height, weight,
severity of symptoms, and stage of the disorder to be treated.
[0213] An effective dose and a therapeutically effective dose are
generally synonymous. However, compounds can be administered to
patients having reduced symptoms or even administered to patients
as a preventative measure. Hence, the composition can be effective
in therapeutic treatment even in the absence of symptoms of the
disorder.
[0214] A unit dose can be administered, for example, 1 to 4 times
per day. The dose depends on the route of administration and the
formulation of a composition containing the compound or compounds.
Further, it will be appreciated by one of ordinary skill in the art
after receiving the disclosure of the present invention that it
might be necessary to make routine adjustments or variations to the
dosage depending on the combination of agents employed, on the age
and weight of the patient, and on the severity of the condition to
be treated.
[0215] Such adjustments or variations, as well as evaluation of
when and how to make such adjustments or variations, are well known
to those of ordinary skill in the art of medicine. Evaluation
parameters and techniques can vary with the patient and the
severity of the disease. Particularly useful evaluative techniques
include but are not limited to the glucose uptake assays disclosed
herein.
[0216] G2. Methods of Transcriptionally Modulating In Vivo Glucose
Transporter Levels in the Treatment of Related Diseases and
Disorders
[0217] A method for transcriptionally modulating, in a
multicellular organism, the expression of a gene encoding a glucose
transporter in order to modulate glucose transporter biological
activity in a warm-blooded vertebrate subject is also provided in
accordance with the present invention. This method comprises
administering to the warm-blooded vertebrate subject a compound at
a concentration effective to transcriptionally modulate expression
of glucose transporter or transporters.
[0218] In accordance with the present invention, the envisioned
compound can optionally comprise an antibody or polypeptide
prepared as described above and which transcriptionally modulates
expression of glucose transporters. Optionally, the antibody or
polypeptide directly binds to DNA or RNA, or directly binds to a
protein involved in transcription.
[0219] Particularly envisioned chemical entities (e.g. small
molecule mimetics) for use in accordance with the present invention
do not naturally occur in any cell, whether of a multicellular or a
unicellular organism. Even more particularly, the chemical entity
is not a naturally occurring molecule, e.g. it is a chemically
synthesized entity. Optionally, the compound can bind a modulatable
transcription sequence of the gene. For example, the compound can
bind a promoter region upstream of the claimed nucleic acid
sequence encoding.
[0220] In the methods above, modulation of transcription results in
either upregulation or downregulation of expression of the gene
encoding the protein of interest, depending on the identity of the
molecule that contacts the cell.
[0221] G2a. Gene Therapy
[0222] A nucleic acid sequence of the present invention, coding for
glucose transporter, can be used for gene therapy in accordance
with the present invention. The general strategy of gene therapy is
the insertion and incorporation of an introduced non-native
sequence of DNA into an organism's native DNA in order to
facilitate a biological change. For example, the nucleic acid
sequence shown in SEQ ID NO: 1 can be use to transform a cell and
produce a cell in which a copy of the cell's defective genomic copy
of a glucose transporting polypeptide-encoding nucleic acid
sequence has been replaced by the transformed nucleic acid
sequence. This approach can be used with cells capable of being
grown in culture in order to study the function of the nucleic acid
sequence. Representative gene therapy methods, including liposomal
transfection of nucleic acids into host cells, are described in
U.S. Pat. Nos. 5,279,833; 5,286,634; 5,399,346; 5,646,008;
5,651,964; 5,641,484; and 5,643,567, the contents of each of which
are herein incorporated by reference.
[0223] Briefly, gene therapy directed toward modulation of glucose
transporter activity, to thereby affect or modulate the biological
activity of glucose transporter in a target cell is described. In
one embodiment, a therapeutic method of the present invention
provides a method for modulation of glucose transporter levels
comprising: (a) delivering to the cell an effective amount of a DNA
molecule comprising a polynucleotide that encodes a polypeptide
that modulates the biological activity of one or more than one
glucose transporter; and (b) maintaining the cell under conditions
sufficient for expression of said polypeptide.
[0224] In a preferred embodiment, the delivered polypeptide
comprises the sequence shown in SEQ ID NO: 2. Delivery can be
accomplished by injecting the DNA molecule into the cell. Where the
cell is in a subject, administering comprises the steps of: (a)
providing a vehicle that contains the DNA molecule; and (b)
administering the vehicle to the subject.
[0225] A vehicle is preferably a cell transformed or transfected
with the DNA molecule or a transfected cell derived from such a
transformed or transfected cell. A representative transformed or
transfected cell is a lymphocyte. Techniques for transforming or
transfecting a cell with a DNA molecule of the present invention
are set forth above.
[0226] Alternatively, the vehicle is a virus or an antibody that
specifically infects a target cell or an antibody that immunoreacts
with an antigen of a target cell. Retroviruses used to deliver the
constructs to the host target tissues generally are viruses in
which the 3'-LTR (linear transfer region) has been inactivated.
That is, these are enhancerless 3'-LTR's, often referred to as SIN
(self-inactivating viruses) because after productive infection into
the host cell, the 3'-LTR is transferred to the 5'-end and both
viral LTR's are inactive with respect to transcriptional activity.
A use of these viruses well known to those skilled in the art is to
clone genes for which the regulatory elements of the cloned gene
are inserted in the space between the two LTR's. An advantage of a
viral infection system is that it allows for a very high level of
infection into the appropriate recipient cell.
[0227] Antibodies have been used to target and deliver DNA
molecules. An N-terminal modified poly-L-lysine (NPLL)-antibody
conjugate readily forms a complex with plasmid DNA. A complex of
monoclonal antibodies against a cell surface thrombomodulin
conjugated with NPLL was used to target a foreign plasmid DNA to an
antigen-expressing mouse lung endothelial cell line and mouse lung.
Those targeted endothelial cells expressed the product encoded by
that foreign DNA.
[0228] It is also envisioned that this embodiment of the present
invention can be practiced using alternative viral or phage
vectors, including retroviral vectors, adenoviral vectors and
vaccinia viruses whose genome has been manipulated in alternative
ways so as to render the virus non-pathogenic. Methods for creating
such a viral mutation are set forth in detail in U.S. Pat. No.
4,769,331, incorporated herein by reference.
[0229] By way of specific example, the human glucose
transporter-encoding polynucleotide of the present invention, or a
glucose transporter-encoding polynucleotide homolog from another
warm-blooded vertebrate is introduced into isolated liver cells or
other relevant cells. The re-injection of the transgene-carrying
cells into the liver or other relevant tissues provides a treatment
for susceptibility to impaired glucose transport function or other
relevant diseases in human and animals.
[0230] G2a1. Gene Therapy Vector Construct Dosing
[0231] A nucleotide sequence of the present invention can be
introduced into cells by introducing a virus containing a vector
construct bearing the sequence of interest directly into a subject.
This process requires the construction of a suitable vector. A
suitable vector will contain the sequence of interest, as well as
other functional sequences known to those skilled in the art to be
required for viable transformation and transfection. A preferred
procedure for alleviating a glucose transporter-related condition
using a vector construct designed according to the described
strategy is as follows.
[0232] The maximally tolerated dose (MTD) of vector construct when
administered directly into the affected tissue is determined.
Primary endpoints are: 1) the rate of transduction in abnormal
and/or normal cells, 2) the presence and stability of this vector
in the systemic circulation and in affected cells, and 3) the
nature of the systemic (fever, myalgias) and local (infections,
pain) toxicities induced by the vector. A secondary endpoint is the
clinical efficacy of the vector construct.
[0233] For example, a 4 ml serum-free volume of viral (e.g.
adenoviral, retroviral, etc.) vector construct (containing up to
5.times.10.sup.7 viral particles in AIM V media) is administered
daily per session. During each session, 1 ml of medium containing
the appropriate titer of vector construct is injected into 4
regions of the affected tissue for a total of 4 ml per session in a
clinical examination room. This is repeated daily for 4 days (4
sessions). This 16 ml total inoculum volume over 4 days is
proportionally well below the one safely tolerated by nude mice
(0.5 ml/20 g body weight).
[0234] Patient evaluation includes history and physical examination
prior to initiation of therapy and daily during the 4-day period of
vector construct injection. Toxicity grading is done using the ECOG
Common Toxicity Criteria. CBC, SMA-20, urinalysis, and conventional
studies are performed daily during this period. Evaluation will
include a regular determination of glucose transporter activity and
an assessment of the progression, if any, of, for example, a Type
II diabetic condition.
[0235] G2a2. Dose Escalation and MTD
[0236] Patients are treated with 3.times.10.sup.6 viral
particles.times.4. Once they have all recovered from all grade 2 or
less toxicities (except alopecia), and as long as grade 3-4
toxicity is not encountered, a subsequent dose level is initiated
in patients. As one grade 3 or 4 toxicity occurs at a given dose
level, a minimum of 6 patients are enrolled at that level. As only
1 of 6 patients has grade 3 or 4 toxicity, dose escalation
continues. The MTD of vector construct is defined as the dose where
2 of 6 patients experience grade 3 or 4 toxicity. If 2 of 3, or if
3 of 6 patients experience grade 3 or 4 toxicity, the MTD is
defined as the immediately lower dose level.
[0237] The following escalation schema is followed: 1) level 1,
3.times.10.sup.6 viral particles; 2) level 2, 1.times.10.sup.7; 3)
level 3, 3.times.10.sup.7; 4) level 4, 5.times.10.sup.7. Patients
with measurable disease are evaluated for a clinical response to
vector construct. Histology and local symptoms are followed.
[0238] G2b. Antisense Oligonucleotide Therapy
[0239] In accordance with the present invention, expression of a
glucose transporter can be modulated in a vertebrate subject
through the administration of an antisense oligonucleotide derived
from a nucleic acid molecule encoding the glucose transporter of
the claimed invention. Therapeutic methods utilizing antisense
oligonucleotides have been described in the art, for example, in
U.S. Pat. Nos. 5,627,158 and 5,734,033, the contents of each of
which are herein incorporated by reference.
[0240] Antisense oligodeoxynucleotides are short (usually about 30
bases) single-stranded synthetic DNAs having a nucleic acid
sequence complementary to the target mRNA and the ability to form a
hybrid duplex by hydrogen bonded base pairing. The formation of a
hybrid duplex can prevent expression of the target mRNA code into
its protein product and thus preclude subsequent deleterious
effects of the polypeptide product. Because the mRNA sequence
expressed by the gene is termed the sense sequence, the
complementary sequence is termed the antisense sequence. Inhibition
of mRNA is more efficient than inhibition of an enzyme's active
site, since one mRNA molecule can give rise to multiple protein
copies.
[0241] Synthetic oligodeoxynucleotides complementary to (antisense)
mRNA of the c-myc oncogene have been used to specifically inhibit
production of c-myc protein, thus arresting the growth of human
leukemic cells in vitro, (See, e.g., Holt et al., (1988), Mol.
Cell. Biol. 8: 963-973; and Wickstrom et al., (1988) Proc. Natl.
Acad. Sci. USA, 85: 1028-32). Oligodeoxynucleotides have also been
employed as specific inhibitors of retroviruses, including the
human immunodeficiency virus (HIV-I). (See, e.g., Zamencik and
Stephenson, (1978) Proc. Natl. Acad. Sci. USA, 75: 280-84 and
Zamencik et al., (1986) Proc. Natl. Acad. Sci. USA, 83:
4143-46).
[0242] Antisense nucleotide sequences can be introduced into a
subject in a variety of ways, preferably by incubation of cells in
the presence of the antisense nucleotide, more preferably through
the use of liposomes and most preferably by in vitro or in vivo
transfection of host cells by viruses.
[0243] G3. Formulation of Therapeutic Compositions
[0244] The glucose transporter biological activity modulating
substances, gene therapy vectors, chemical agents and substances
that inhibit or promote expression of the glucose transporter of
the present invention are adapted for administration as a
pharmaceutical composition as described herein. Additional
formulation and dose preparation techniques have been described in
the art, see for example, those described in U.S. Pat. No.
5,326,902 issued to Seipp et al. on Jul. 5, 1994, U.S. Pat. No.
5,234,933 issued to Marnett et al. on Aug. 10, 1993, and PCT
Publication WO 93/25521 of Johnson et al. published Dec. 23, 1993,
the entire contents of each of which are herein incorporated by
reference.
[0245] For the purposes described above, the identified substances
can normally be administered systemically or partially, usually by
oral or parenteral administration. The doses to be administered are
determined depending upon age, body weight, symptom, the desired
therapeutic effect, the route of administration, and the duration
of the treatment, etc.; one of skill in the art of therapeutic
treatment will recognize appropriate procedures and techniques for
determining the appropriate dosage regimen for effective therapy.
Various compositions and forms of administration are contemplated
and are generally known in the art. Other compositions for
administration include liquids for external use, and endermic
lineaments (ointment, etc.), suppositories and pessaries which
comprise one or more of the active substance(s) and can be prepared
by known methods.
[0246] Thus, the present invention provides pharmaceutical
compositions comprising a polypeptide, polynucleotide, or molecule
or compound of the present invention and a physiologically
acceptable carrier. More preferably, a pharmaceutical composition
comprises a compound discovered via the screening methods described
herein.
[0247] A composition of the present invention is typically
administered parenterally in dosage unit formulations containing
standard, well-known nontoxic physiologically acceptable carriers,
adjuvants, and vehicles as desired.
[0248] Injectable preparations, for example sterile injectable
aqueous or oleaginous suspensions, are formulated according to the
known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol.
[0249] Among the acceptable vehicles and solvents that can be
employed are water, Ringer's solution, and isotonic sodium chloride
solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid find use
in the preparation of injectables.
[0250] Preferred carriers include neutral saline solutions buffered
with phosphate, lactate, Tris, and the like. Of course, one
purifies the vector sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering adenovirus
particles or endotoxins and other pyrogens such that it does not
cause any untoward reactions in the individual receiving the vector
construct. A preferred technique of purifying the vector involves
the use of buoyant density gradients, such as cesium chloride
gradient centrifugation.
[0251] A transfected cell can also serve as a carrier of the
therapeutic agent. By way of example, a liver cell can be removed
from an organism, transfected with a polynucleotide of the present
invention using methods set forth above and then the transfected
cell returned to the organism (e.g. injected intra-vascularly).
[0252] The polypeptides, nucleic acid sequences, chemical agents
and substances described above can further be administered to a
mammal, particularly a human, using the methods described above. A
sustained release formulation using a biodegradable polymer,
micelles, gels, liposomes or transgenic technique is also provided
in accordance with the present invention.
H. Generation of Genetically Modified Non-Human Species
[0253] The present invention contemplates the creation of
transgenic non-human species expressing a glucose transporter of
the present invention, including as a preferred embodiment, the
nucleic acid sequence shown in SEQ ID NO: 1. Representative host
species include rat and mouse. Mouse is especially preferred
because a number of genetically modified mice already exist, e.g.
the NOD mouse, the ob/ob mouse, the SCID mouse, etc. These modified
mice are uniquely suited to the present invention because their
genetic and corresponding expressed phenotypic traits add
additional depth and dimension to the study of glucose homeostasis
disorders.
[0254] Techniques for the preparation of transgenic animals are
known in the art. Exemplary techniques are described in U.S. Pat.
No. 5,489,742 (transgenic rats); U.S. Pat. Nos. 4,736,866,
5,550,316, 5,614,396, 5,625,125 and 5,648,061 (transgenic mice);
U.S. Pat. No. 5,573,933 (transgenic pigs); U.S. Pat. No. 5,162,215
(transgenic avian species) and U.S. Pat. No. 5,741,957 (transgenic
bovine species), the entire contents of each of which are herein
incorporated by reference.
[0255] Additionally, a nucleic acid sequence encoding the glucose
transporter polypeptide of the present invention can be used to
transform a cell. For example, a mutant form of the glucose
transporter can be used to produce a cell in which a copy of the
cell's genomic glucose transporter has been replaced by the
transformed gene. This process can produce a cell which contains,
for example, a modified or deleted for a copy of the gene. This
approach can be used with cells capable of being grown in culture
and in animals and is very useful when investigating the in vivo
function and mechanism of the gene.
[0256] Thus, a genetically modified animal of the present invention
can comprise a targeted modification of the glucose transporter
gene. Animal strains with complete or partial functional
inactivation of the present glucose transporter gene are generated
using standard techniques of site-specific recombination in
embryonic stem cells. Capecchi, M. R. (1989) Science 244 (4910):
1288-92; Thomas, K. R., and Capecchi, M. R. (1990) Nature 346
(6287):847-50; Delpire, E., et al. (1999) Nat Genet. 22 (2):192-5.
Procedures analogous to those employed in the generation of a
"knock-out" animal can be applied in the generation of a
"knock-out" cell line.
[0257] Alternatives include the use of anti-sense or ribozyme
glucose transporter constructs, driven by a universal or
tissue-specific promoter, to reduce levels of the present glucose
transporter, thus achieving a "knock-down" of the isoform (Luyckx,
V. A., et al. (1999) Proc. Natl. Acad. Sci. USA 96 (21): 12174-79).
The invention also provides the generation of animal strains with
conditional or inducible inactivation of individual or the glucose
transporter gene (Sauer, B. (1998) Methods 14 (4): 381-92; Ding,
Y., et al. (1997) J. Biol. Chem. 272 (44): 28142-48).
[0258] The present invention also provides animal strains with
specific "knocked-in" modifications in the glucose transporter
gene. This includes animals with genetically (Forlino, A., et al.
(1999) J. Biol. Chem. 274 (53): 37923-31) and functionally (Kissel,
H., et al. (2000) Embo. J: 19 (6): 1312-1326) relevant point
mutations in the gene, in addition to manipulations such as the
insertion of disease-specific repeat expansions (White, J. K., et
al. (1997) Nat Genet. 17 (4):404-10).
I. Generation of Antibodies
[0259] In still another embodiment, the present invention provides
an antibody immunoreactive with a polypeptide of the present
invention. Preferably, an antibody of the invention is a monoclonal
antibody. Techniques for preparing and characterizing antibodies
are well known in the art (See, e.g., Antibodies A Laboratory
Manual, E. Howell and D. Lane, Cold Spring Harbor Laboratory,
1988).
[0260] Briefly, a polyclonal antibody is prepared by immunizing an
animal with an immunogen comprising a polypeptide or polynucleotide
of the present invention, and collecting antisera from that
immunized animal. A wide range of animal species can be used for
the production of antisera. Because of the relatively large blood
volume of rabbits, a rabbit is a preferred choice for production of
polyclonal antibodies.
[0261] As is well known in the art, a given polypeptide or
polynucleotide can vary in its immunogenicity. It is often
necessary therefore to couple the immunogen (e.g., a polypeptide or
polynucleotide) of the present invention) with a carrier. Exemplary
and preferred carriers are keyhole limpet hemocyanin (KLH) and
bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse
serum albumin or rabbit serum albumin can also be used as
carriers.
[0262] Reagents for conjugating a polypeptide or a polynucleotide
to a carrier protein are well known in the art and include
glutaraldehyde, N-maleimidobencoyl-N-hydroxysuccinimide ester,
carbodiimide and bis-biazotized benzidine.
[0263] As is also well known in the art, immunogenicity to a
particular immunogen can be enhanced by the use of non-specific
stimulators of the immune response known as adjuvants. Exemplary
and preferred adjuvants include complete Freund's adjuvant,
incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
[0264] The amount of immunogen used of the production of polyclonal
antibodies varies, inter alia, upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the immunogen, e.g. subcutaneous,
intramuscular, intradermal, intravenous and intraperitoneal. The
production of polyclonal antibodies is monitored by sampling blood
of the immunized animal at various points following immunization.
When a desired level of immunogenicity is obtained, the immunized
animal can be bled and the serum isolated and stored.
[0265] In another aspect, the present invention provides a method
of producing an antibody immunoreactive with the glucose
transporter polypeptide, the method comprising: (a) transfecting
recombinant host cells with a polynucleotide that encodes that
polypeptide; (b) culturing the host cells under conditions
sufficient for expression of the polypeptide; (c) recovering the
polypeptide; and (d) preparing antibodies to the polypeptide.
Preferably, the glucose transporter polypeptide is capable of
modulating calcium levels within or outside of cells in accordance
with the present invention.
[0266] A monoclonal antibody of the present invention can be
readily prepared through use of well-known techniques such as the
hybridoma techniques exemplified in U.S. Pat. No. 4,196,265 and the
phage-displayed techniques disclosed in U.S. Pat. No. 5,260,203,
the contents of which are herein incorporated by reference.
[0267] A typical technique involves first immunizing a suitable
animal with a selected antigen (e.g., a polypeptide or
polynucleotide of the present invention) in a manner sufficient to
provide an immune response. Rodents such as mice and rats are
preferred animals. Spleen cells from the immunized animal are then
fused with cells of an immortal myeloma cell. Where the immunized
animal is a mouse, a preferred myeloma cell is a murine NS-1
myeloma cell.
[0268] The fused spleen/myeloma cells are cultured in a selective
medium to select fused spleen/myeloma cells from the parental
cells. Fused cells are separated from the mixture of non-fused
parental cells, for example, by the addition of agents that block
the de novo synthesis of nucleotides in the tissue culture media.
This culturing provides a population of hybridomas from which
specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants for reactivity with an antigen-polypeptides.
The selected clones can then be propagated indefinitely to provide
the monoclonal antibody.
[0269] By way of specific example, to produce an antibody of the
present invention, mice are injected intraperitoneally with between
about 1-200 .mu.g of an antigen comprising a polypeptide of the
present invention. B lymphocyte cells are stimulated to grow by
injecting the antigen in association with an adjuvant such as
complete Freund's adjuvant (a non-specific stimulator of the immune
response containing killed Mycobacterium tuberculosis). At some
time (e.g., at least two weeks) after the first injection, mice are
boosted by injection with a second dose of the antigen mixed with
incomplete Freund's adjuvant.
[0270] A few weeks after the second injection, mice are tail bled
and the sera titered by immunoprecipitation against radiolabeled
antigen. Preferably, the method of boosting and titering is
repeated until a suitable titer is achieved. The spleen of the
mouse with the highest titer is removed and the spleen lymphocytes
are obtained by homogenizing the spleen with a syringe.
[0271] Mutant lymphocyte cells known as myeloma cells are obtained
from laboratory animals in which such cells have been induced to
grow by a variety of well-known methods. Myeloma cells lack the
salvage pathway of nucleotide biosynthesis. Because myeloma cells
are tumor cells, they can be propagated indefinitely in tissue
culture, and are thus "immortal". Numerous cultured cell lines of
myeloma cells from mice and rats, such as murine NS-1 myeloma
cells, have been established.
[0272] Myeloma cells are combined under conditions appropriate to
foster fusion with the normal antibody-producing cells from the
spleen of the mouse or rat injected with the antigen/polypeptide of
the present invention. Fusion conditions include, for example, the
presence of polyethylene glycol. The resulting fused cells are
hybridoma cells. Like myeloma cells, hybridoma cells grow
indefinitely in culture.
[0273] Hybridoma cells are separated from unfused myeloma cells by
culturing in a selection medium such as HAT media (hypoxanthine,
aminopterin, and thymidine). Unfused myeloma cells lack the enzymes
necessary to synthesize nucleotides from the salvage pathway
because they are killed in the presence of aminopterin,
methotrexate, or azaserine. Unfused lymphocytes also do not
continue to grow in tissue culture. Thus, only cells that have
successfully fused (hybridoma cells) can grow in the selection
media.
[0274] Each of the surviving hybridoma cells produces a single
antibody. These cells are then screened for the production of the
specific antibody immunoreactive with an antigen/polypeptide of the
present invention. Single cell hybridomas are isolated by limiting
dilutions of the hybridomas. The hybridomas are serially diluted
many times and, after the dilutions are allowed to grow, the
supernatant is tested for the presence of the monoclonal antibody.
The clones producing that antibody are then cultured in large
amounts to produce an antibody of the present invention in
convenient quantity.
[0275] By use of a monoclonal antibody of the present invention,
specific polypeptides and polynucleotide of the invention can be
recognized as antigens, and thus identified. Once identified, those
polypeptides and polynucleotide can be isolated and purified by
techniques such as antibody-affinity chromatography. In
antibody-affinity chromatography, a monoclonal antibody is bound to
a solid substrate and exposed to a solution containing the desired
antigen. The antigen is removed from the solution through an
immunospecific reaction with the bound antibody. The polypeptide or
polynucleotide is then easily removed from the substrate and
purified.
J. Clinical Diagnosis of Type II Diabetes and Other Glucose
Homeostasis-Related Imbalances
[0276] The pattern of expression of one or a combination of glucose
transporters can be used to diagnose persons at risk of diabetes or
other glucose homeostasis disorders. This can be accomplished by
monitoring the expression of the mRNA or the glucose transporter
polypeptide of the present invention. As disclosed herein, the
glucose transporter of the present invention maps to a locus on
human chromosome 20 known to be associated with glucose homeostasis
imbalances. Therefore, expression of this protein can be used to
facilitate a diagnosis of the susceptibility and risk of an
individual to Type II diabetes and other glucose
homeostasis-related disorders.
[0277] The present invention, therefore, allows the identification
of the presence or absence of a polymorphism in a human Type II
diabetes gene and can therefore be used in the diagnosis of Type II
diabetes or in the genetic counseling of individuals that have a
family history of Type II diabetes, although the general population
can also be screened. Such screening has the benefit of not only
alerting an individual to his or her susceptibility to the Type II
diabetic condition, but also allowing a practitioner to offer
accurate advice regarding transmission of the condition to
offspring. This early warning mechanism can provide invaluable
assistance to susceptible offspring because it can allow him or her
to prepare for the onset of the condition. It is possible that,
with the knowledge of an individual's susceptibility to the
condition, the individual can augment certain environmental factors
to prevent or significantly delay the onset of the condition.
[0278] With further study centering around the present invention,
the biological effects of a specific mutation are determined. Early
detection and treatment of such a mutation can prevent later
development of the condition. In addition, the financial costs
difficulties associated with current treatments of glucose
homeostasis-related disorders can be alleviated.
LABORATORY EXAMPLES
[0279] The following Laboratory Examples have been included to
illustrate preferred modes of the invention. Certain aspects of the
following Laboratory Examples are described in terms of techniques
and procedures found or contemplated by the present inventors to
work well in the practice of the invention. These Laboratory
Examples are exemplified through the use of standard laboratory
practices of the inventors. In light of the present disclosure and
the general level of skill in the art, those of skill will
appreciate that the following Laboratory Examples are intended to
be exemplary only and that numerous changes, modifications and
alterations can be employed without departing from the spirit and
scope of the invention.
Laboratory Example 1
Search of the EST Databases
[0280] The NCBI and TIGR expressed sequence tag (EST) databases
were searched for novel isoforms of sugar transport proteins, using
the polypeptide sequence of known sugar transporters, GLUTs 1-5. A
BLAST-type search was performed, based on protein sequence motifs
known to be present in the GLUT family of proteins. The search
criteria included either the entire polypeptide sequence or
keywords such as "sugar transporter". Candidate glucose transporter
sequences were identified (e.g. a 306 base pair (bp) EST named
EST183920 with locus and accession number AA313045) in the search
and used in the generation of the full-length cDNA.
Laboratory Example 2
Generation of the Full-Length cDNA and Chromosomal Localization
[0281] The 5' and 3' cDNA sequences were obtained using 5' and 3'
rapid amplification of cDNA ends (RACE). The full-length cDNA was
then amplified by long range PCR from normal human liver cDNA using
the high fidelity Expand.TM. enzyme (Boehringer Mannheim, Mannheim,
Germany).
[0282] The chromosomal localization and gene structure were
determined by the identification of a BAC clone containing the
genomic DNA sequence of the novel GLUT. The BAC clone was
identified from the databases and had Locus number HS28H20 and
accession number AL031055. It was 127,418 bp in length and had been
localized to chromosome 20q13.1.
Laboratory Example 3
Tissue Distribution of the Novel Polynucleotide Sequence
[0283] FIG. 4 is an autoradiograph of a Northern blot showing the
tissue distribution of the novel polynucleotide sequence. Heart,
brain, placenta, lung, liver, skeletal muscle, kidney and pancreas
tissue were probed for the presence of the novel sequence. Of the
tissues probed, the sequence is most pronounced in liver and
pancreas tissue.
[0284] Conditions for the Northern blot were as follows. The probe
was a PCR product corresponding to residues 1394-1636 of SEQ ID NO:
1. The probe was labeled with .sup.32P by the random priming
method.
[0285] The Northern blot was bought commercially from Clontech of
Palo Alto, Calif. Probe conditions were 68.degree. C. hybridization
for 1 hr using a hybridization buffer sold under the trademark
EXPRESSHYB.TM. by Clontech of Palo Alto, Calif., followed by 2
washes with 2.times.SSC, 0.05% SDS for 40 minutes, each at room
temperature.
Laboratory Example 4
Clinical Detection and Subsequent Isolation of the Nucleic Acid
Sequence
[0286] A young male with histopathological and biochemical findings
indicative of lysosomal glycogen storage disease with normal acid
a-glucosidase (GAA) was studied. It was speculated that defective
transport of glucose out of the lysosomes might be responsible. To
investigate this, the coding region of GLUT4, a known
muscle/adipose tissue glucose transporter, was isolated from a
tissue sample taken from the patient. The nucleic acid sequence was
sequenced and found to have no changes from wild type. This led to
the consideration of the possible existence of an as yet
undescribed glucose transporter.
[0287] A search of the EST databases using the amino acid sequences
of the known GLUT isoforms, and subsequent cloning of the
full-length cDNA, resulted in the identification of a novel glucose
transporter protein. The identified isoform contains 541 amino
acids, has a coding region of 1626 bp and it is highly expressed in
the liver and pancreas. The polynucleotide and amino acid sequence
are presented as SEQ ID NO: 1 and SEQ ID NO: 2 respectively.
[0288] The polypeptide gene product of the nucleic acid sequence
shown in SEQ ID NO: 1 contains 12 transmembrane helices and
contains several conserved motifs that are consistent with the
protein's function as a sugar transporter. Mutation analysis
indicated that this gene is not responsible for the lysosomal
storage disease with normal GAA. This novel glucose transporter
gene is located between D20S119 and D20S178 on chromosome 20q13, a
region of a chromosome where a diabetes mellitus type II locus has
been previously mapped. The function, tissue distribution and
chromosome localization, indicated that this novel glucose
transporter is a gene implicated in the pathogenesis of
diabetes.
[0289] It will be understood that various details of the invention
may be changed without departing from the scope of the invention.
Furthermore, the foregoing description is for the purpose of
illustration only, and not for the purpose of limitation--the
invention being defined by the claims.
Sequence CWU 1
1
414075DNAHomo sapiensCDS(53)..(1678) 1atgcgcgccc ggcccctcag
cgcccccagc acgccgccga gtcccgctcg cc atg ggc 58Met Gly1cac tcc cca
cct gtc ctg cct ttg tgt gcc tct gtg tct ttg ctg ggt 106His Ser Pro
Pro Val Leu Pro Leu Cys Ala Ser Val Ser Leu Leu Gly5 10 15ggc ctg
acc ttt ggt tat gaa ctg gca gtc ata tca ggt gcc ctg ctg 154Gly Leu
Thr Phe Gly Tyr Glu Leu Ala Val Ile Ser Gly Ala Leu Leu20 25 30cca
ctg cag ctt gac ttt ggg cta agc tgc ttg gag cag gag ttc ctg 202Pro
Leu Gln Leu Asp Phe Gly Leu Ser Cys Leu Glu Gln Glu Phe Leu35 40 45
50gtg ggc agc ctg ctc ctg ggg gct ctc ctc gcc tcc ctg gtt ggt ggc
250Val Gly Ser Leu Leu Leu Gly Ala Leu Leu Ala Ser Leu Val Gly
Gly55 60 65ttc ctc att gac tgc tat ggc agg aag caa gcc atc ctc ggg
agc aac 298Phe Leu Ile Asp Cys Tyr Gly Arg Lys Gln Ala Ile Leu Gly
Ser Asn70 75 80ttg gtg ctg ctg gca ggc agc ctg acc ctg ggc ctg gct
ggt tcc ctg 346Leu Val Leu Leu Ala Gly Ser Leu Thr Leu Gly Leu Ala
Gly Ser Leu85 90 95gcc tgg ctg gtc ctg ggc cgc gct gtg gtt ggc ttc
gcc att tcc ctc 394Ala Trp Leu Val Leu Gly Arg Ala Val Val Gly Phe
Ala Ile Ser Leu100 105 110tcc tcc atg gct tgc tgt atc tac gtg tca
gag ctg gtg ggg cca cgg 442Ser Ser Met Ala Cys Cys Ile Tyr Val Ser
Glu Leu Val Gly Pro Arg115 120 125 130cag cgg gga gtg ctg gtg tcc
ctc tat gag gca ggc atc acc gtg ggc 490Gln Arg Gly Val Leu Val Ser
Leu Tyr Glu Ala Gly Ile Thr Val Gly135 140 145atc ctg ctc tcc tat
gcc ctc aac tat gca ctg gct ggt acc ccc tgg 538Ile Leu Leu Ser Tyr
Ala Leu Asn Tyr Ala Leu Ala Gly Thr Pro Trp150 155 160gga tgg agg
cac atg ttc ggc tgg gcc act gca cct gct gtc ctg caa 586Gly Trp Arg
His Met Phe Gly Trp Ala Thr Ala Pro Ala Val Leu Gln165 170 175tcc
ctc agc ctc ctc ttc ctc cct gct ggt aca gat gag act gca aca 634Ser
Leu Ser Leu Leu Phe Leu Pro Ala Gly Thr Asp Glu Thr Ala Thr180 185
190cac aag gac ctc atc cca ctc cag gga ggt gag gcc ccc aag ctg ggc
682His Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu Ala Pro Lys Leu
Gly195 200 205 210ccg ggg agg cca cgg tac tcc ttt ctg gac ctc ttc
agg gca cgc gat 730Pro Gly Arg Pro Arg Tyr Ser Phe Leu Asp Leu Phe
Arg Ala Arg Asp215 220 225aac atg cga ggc cgg acc aca gtg ggc ctg
ggg ctg gtg ctc ttc cag 778Asn Met Arg Gly Arg Thr Thr Val Gly Leu
Gly Leu Val Leu Phe Gln230 235 240caa cta aca ggg cag ccc aac gtg
ctg tgc tat gcc tcc acc atc ttc 826Gln Leu Thr Gly Gln Pro Asn Val
Leu Cys Tyr Ala Ser Thr Ile Phe245 250 255agc tcc gtt ggt ttc cat
ggg gga tcc tca gcc gtg ctg gcc tct gtg 874Ser Ser Val Gly Phe His
Gly Gly Ser Ser Ala Val Leu Ala Ser Val260 265 270ggg ctt ggc gca
gtg aag gtg gca gct acc ctg acc gcc atg ggg ctg 922Gly Leu Gly Ala
Val Lys Val Ala Ala Thr Leu Thr Ala Met Gly Leu275 280 285 290gtg
gac cgt gca ggc cgc agg gct ctg ttg cta gct ggc tgt gcc ctc 970Val
Asp Arg Ala Gly Arg Arg Ala Leu Leu Leu Ala Gly Cys Ala Leu295 300
305atg gcc ctg tcc gtc agt ggc ata ggc ctc gtc agc ttt gcc gtg ccc
1018Met Ala Leu Ser Val Ser Gly Ile Gly Leu Val Ser Phe Ala Val
Pro310 315 320atg gac tca ggc cca agc tgt ctg gct gtg ccc aat gcc
acc ggg cag 1066Met Asp Ser Gly Pro Ser Cys Leu Ala Val Pro Asn Ala
Thr Gly Gln325 330 335aca ggc ctc cct gga gac tct ggc ctg ctg cag
gac tcc tct cta cct 1114Thr Gly Leu Pro Gly Asp Ser Gly Leu Leu Gln
Asp Ser Ser Leu Pro340 345 350ccc att cca agg acc aat gag gac caa
agg gag cca atc ttg tcc act 1162Pro Ile Pro Arg Thr Asn Glu Asp Gln
Arg Glu Pro Ile Leu Ser Thr355 360 365 370gct aag aaa acc aag ccc
cat ccc aga tct gga gac ccc tca gcc cct 1210Ala Lys Lys Thr Lys Pro
His Pro Arg Ser Gly Asp Pro Ser Ala Pro375 380 385cct cgg ctg gcc
ctg agc tct gcc ctc cct ggg ccc cct ctg ccc gct 1258Pro Arg Leu Ala
Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu Pro Ala390 395 400cgg ggg
cat gca ctg ctg cgc tgg acc gca ctg ctg tgc ctg atg gtc 1306Arg Gly
His Ala Leu Leu Arg Trp Thr Ala Leu Leu Cys Leu Met Val405 410
415ttt gtc agt gcc ttc tcc ttt ggg ttt ggg cca gtg acc tgg ctt gtc
1354Phe Val Ser Ala Phe Ser Phe Gly Phe Gly Pro Val Thr Trp Leu
Val420 425 430ctc agc gag atc tac cct gtg gag ata cga gga aga gcc
ttc gcc ttc 1402Leu Ser Glu Ile Tyr Pro Val Glu Ile Arg Gly Arg Ala
Phe Ala Phe435 440 445 450tgc aac agc ttc aac tgg gcg gcc aac ctc
ttc atc agc ctc tcc ttc 1450Cys Asn Ser Phe Asn Trp Ala Ala Asn Leu
Phe Ile Ser Leu Ser Phe455 460 465ctc gat ctc att ggc acc atc ggc
ttg tcc tgg acc ttc ctg ctc tac 1498Leu Asp Leu Ile Gly Thr Ile Gly
Leu Ser Trp Thr Phe Leu Leu Tyr470 475 480gga ctg acc gct gtc ctc
ggc ctg ggc ttc atc tat tta ttt gtt cct 1546Gly Leu Thr Ala Val Leu
Gly Leu Gly Phe Ile Tyr Leu Phe Val Pro485 490 495gaa aca aaa ggc
cag tcg ttg gca gag ata gac cag cag ttc cag aag 1594Glu Thr Lys Gly
Gln Ser Leu Ala Glu Ile Asp Gln Gln Phe Gln Lys500 505 510aga cgg
ttc acc ctg agc ttt ggc cac agg cag aac tcc act ggc atc 1642Arg Arg
Phe Thr Leu Ser Phe Gly His Arg Gln Asn Ser Thr Gly Ile515 520 525
530ccg tac agc cgc atc gag atc tct gcg gcc tcc tga ggtcttttgg
1688Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala Ser535 540gagtggcccc
tgcccccaaa ggtggtctgc ttttgctggg gtaaaaagga tgaaagtctg
1748agaatgccca actcttcatt ttgagtctca ggccctgaag gttcctgagg
atctagcttc 1808atgcctcagt ttccccattg acttgcacat ctctgcagta
tttataagaa gaatattcta 1868tgaagtcttt gttgcaccat ggacttttct
caaagaatct caagggtacc aatcctggca 1928ggaagtctct cccgatatca
cccctaaatc caaatgagga tatcatcttt tctaatctct 1988tttttcaact
ggctgggaca ttttcggaag ggggaagtct ctttttttac tcttatcatt
2048tttttttttt gaggtggagt ctcattctgt tgcccaggct ggcctgatct
tggctcactg 2108caacctccac ctcctgagtt caagcgattc ttgtgcctca
gcctcctaag cagctgggac 2168tacaggcgca tgcaaccata cccagctaat
ttatttttag cagagatggg gtttcactgt 2228gttggccagg ctggtcgtga
actcctgagc tcaagtgatc cacccacctc agcctcccag 2288agtgctagga
ttacaggcct tttgactctt ttatctgagt tttattgacc cctctaattc
2348tcttacccag aatatttatc cttcaccagc aactctgact ctttgacggg
aggcctcagt 2408tctagtcctt ggtctgctgg tgtcattgct gtaggaatga
ccacgggcct cagtttcccc 2468atttgtataa tgggaagcct gtaccaggtc
attcttaaga tttctcctga ctccagtgag 2528ctggaattct aaatgctggt
ctaggagctg tctccaggat ggtgcaggat ggctttgcgg 2588aaaggagatg
ggtttggagg ccaacaaacc tgcttgtcaa tattgccttt gcctcttggc
2648agcccttgaa cttgagtaaa taacaactcc ctgaacctca gtttcctcat
ctgcagaatg 2708gggataatta tgtcccaggg gtatatttag accctgtttc
ctttcaggag ggtccccagc 2768tggtccaggg cctgggaaat ttctacttat
cctcattacc caggtccctc ctttggaccc 2828tgtaaagggt cagggtgaat
cagatggggg actgagcaag tagctatgac tgcagatcat 2888gtaaggaagg
gactgacaag aagctcccag atgctgggga gaatgaagag ctaaaataga
2948tcctaggtgc tggatgcttt gtcatccatg cgtgcacata tgggtgctgg
cagagccccc 3008aaggactctg gcctctcgag ttctcctatc ttctccattc
tagatgcttc ccttgtatcc 3068agtgatgtgc tggagctggc tttgccaagc
ttgtgagagc tggttgctac attttcagga 3128tttttacaag ttggtaaaca
cagccattat aaaaaattaa atgatttaaa tttataatta 3188agtaaattac
attaaaacaa aaaaattata ctcaaaattc attacttaat tttactacct
3248gttactatta tctgtgcttt tgaggctatt tctacatagt aactcttatg
gagacctagg 3308ggagacaccg cgcatctctt cctgattccc cactcaatga
catcatgtta gtctttggtt 3368gcttaactgg ctgtggggag tgtttttgta
tcacaaagat tagagaggac tacacatcag 3428ggcttgattt attgtttgtt
gattttctag acttcagaac atgctggata aaatgtcagt 3488aatgcaaatt
aaactttaaa gtatgtcttg tttgtagcca atacatggtg tatagcacca
3548aaaaatggag ggattattct tccagtagtt gaacactgtc atccgtttca
gctgacagct 3608gctcaaatca tttaagaagg agttctgaca ttcattttca
ttgttttact tttgtcttcc 3668tcactagtgt aaacaaaaat ttcaaccagc
attcatgccg aacctatacc cattcttcag 3728tgcctagctg tacagttatc
agggattttt atttgtagtc taattttgtc aaatcatggc 3788caaatcgcag
tgatagttga ctttggatac aaggtttggc aaaaaaaaaa atattaacaa
3848aatattctgt aagaatcaat tgtctatatg gaatttagga taaagaatat
ttacaataaa 3908gaatatttac aataaagagt ttattattat ttgtaagttg
tgtgcaacaa acataccctt 3968tatctctgta aaatttatac acacaaaaat
taacaaaaga ttctgtaaga attaattggc 4028tatatggaat ttaggataga
atatttacaa taaagagtat ttacaat 40752541PRTHomo sapiens 2Met Gly His
Ser Pro Pro Val Leu Pro Leu Cys Ala Ser Val Ser Leu 1 5 10 15Leu
Gly Gly Leu Thr Phe Gly Tyr Glu Leu Ala Val Ile Ser Gly Ala20 25
30Leu Leu Pro Leu Gln Leu Asp Phe Gly Leu Ser Cys Leu Glu Gln Glu35
40 45Phe Leu Val Gly Ser Leu Leu Leu Gly Ala Leu Leu Ala Ser Leu
Val50 55 60Gly Gly Phe Leu Ile Asp Cys Tyr Gly Arg Lys Gln Ala Ile
Leu Gly65 70 75 80Ser Asn Leu Val Leu Leu Ala Gly Ser Leu Thr Leu
Gly Leu Ala Gly85 90 95Ser Leu Ala Trp Leu Val Leu Gly Arg Ala Val
Val Gly Phe Ala Ile100 105 110Ser Leu Ser Ser Met Ala Cys Cys Ile
Tyr Val Ser Glu Leu Val Gly115 120 125Pro Arg Gln Arg Gly Val Leu
Val Ser Leu Tyr Glu Ala Gly Ile Thr130 135 140Val Gly Ile Leu Leu
Ser Tyr Ala Leu Asn Tyr Ala Leu Ala Gly Thr145 150 155 160Pro Trp
Gly Trp Arg His Met Phe Gly Trp Ala Thr Ala Pro Ala Val165 170
175Leu Gln Ser Leu Ser Leu Leu Phe Leu Pro Ala Gly Thr Asp Glu
Thr180 185 190Ala Thr His Lys Asp Leu Ile Pro Leu Gln Gly Gly Glu
Ala Pro Lys195 200 205Leu Gly Pro Gly Arg Pro Arg Tyr Ser Phe Leu
Asp Leu Phe Arg Ala210 215 220Arg Asp Asn Met Arg Gly Arg Thr Thr
Val Gly Leu Gly Leu Val Leu225 230 235 240Phe Gln Gln Leu Thr Gly
Gln Pro Asn Val Leu Cys Tyr Ala Ser Thr245 250 255Ile Phe Ser Ser
Val Gly Phe His Gly Gly Ser Ser Ala Val Leu Ala260 265 270Ser Val
Gly Leu Gly Ala Val Lys Val Ala Ala Thr Leu Thr Ala Met275 280
285Gly Leu Val Asp Arg Ala Gly Arg Arg Ala Leu Leu Leu Ala Gly
Cys290 295 300Ala Leu Met Ala Leu Ser Val Ser Gly Ile Gly Leu Val
Ser Phe Ala305 310 315 320Val Pro Met Asp Ser Gly Pro Ser Cys Leu
Ala Val Pro Asn Ala Thr325 330 335Gly Gln Thr Gly Leu Pro Gly Asp
Ser Gly Leu Leu Gln Asp Ser Ser340 345 350Leu Pro Pro Ile Pro Arg
Thr Asn Glu Asp Gln Arg Glu Pro Ile Leu355 360 365Ser Thr Ala Lys
Lys Thr Lys Pro His Pro Arg Ser Gly Asp Pro Ser370 375 380Ala Pro
Pro Arg Leu Ala Leu Ser Ser Ala Leu Pro Gly Pro Pro Leu385 390 395
400Pro Ala Arg Gly His Ala Leu Leu Arg Trp Thr Ala Leu Leu Cys
Leu405 410 415Met Val Phe Val Ser Ala Phe Ser Phe Gly Phe Gly Pro
Val Thr Trp420 425 430Leu Val Leu Ser Glu Ile Tyr Pro Val Glu Ile
Arg Gly Arg Ala Phe435 440 445Ala Phe Cys Asn Ser Phe Asn Trp Ala
Ala Asn Leu Phe Ile Ser Leu450 455 460Ser Phe Leu Asp Leu Ile Gly
Thr Ile Gly Leu Ser Trp Thr Phe Leu465 470 475 480Leu Tyr Gly Leu
Thr Ala Val Leu Gly Leu Gly Phe Ile Tyr Leu Phe485 490 495Val Pro
Glu Thr Lys Gly Gln Ser Leu Ala Glu Ile Asp Gln Gln Phe500 505
510Gln Lys Arg Arg Phe Thr Leu Ser Phe Gly His Arg Gln Asn Ser
Thr515 520 525Gly Ile Pro Tyr Ser Arg Ile Glu Ile Ser Ala Ala
Ser530 535 5403492PRTHomo sapiens 3Met Glu Pro Ser Ser Lys Lys Leu
Thr Gly Arg Leu Met Leu Ala Val 1 5 10 15Gly Gly Ala Val Leu Gly
Ser Leu Gln Phe Gly Tyr Asn Thr Gly Val20 25 30Ile Asn Ala Pro Gln
Lys Val Ile Glu Glu Phe Tyr Asn Gln Thr Trp35 40 45Val His Arg Tyr
Gly Glu Ser Ile Leu Pro Thr Thr Leu Thr Thr Leu50 55 60Trp Ser Leu
Ser Val Ala Ile Phe Ser Val Gly Gly Met Ile Gly Ser65 70 75 80Phe
Ser Val Gly Leu Phe Val Asn Arg Phe Gly Arg Arg Asn Ser Met85 90
95Leu Met Met Asn Leu Leu Ala Phe Val Ser Ala Val Leu Met Gly
Phe100 105 110Ser Lys Leu Gly Lys Ser Phe Glu Met Leu Ile Leu Gly
Arg Phe Ile115 120 125Ile Gly Val Tyr Cys Gly Leu Thr Thr Gly Phe
Val Pro Met Tyr Val130 135 140Gly Glu Val Ser Pro Thr Ala Phe Arg
Gly Ala Leu Gly Thr Leu His145 150 155 160Gln Leu Gly Ile Val Val
Gly Ile Leu Ile Ala Gln Val Phe Gly Leu165 170 175Asp Ser Ile Met
Gly Asn Lys Asp Leu Trp Pro Leu Leu Leu Ser Ile180 185 190Ile Phe
Ile Pro Ala Leu Leu Gln Cys Ile Val Leu Pro Phe Cys Pro195 200
205Glu Ser Pro Arg Phe Leu Leu Ile Asn Arg Asn Glu Glu Asn Arg
Ala210 215 220Lys Ser Val Leu Lys Lys Leu Arg Gly Thr Ala Asp Val
Thr His Asp225 230 235 240Leu Gln Glu Met Lys Glu Glu Ser Arg Gln
Met Met Arg Glu Lys Lys245 250 255Val Thr Ile Leu Glu Leu Phe Arg
Ser Pro Ala Tyr Arg Gln Pro Ile260 265 270Leu Ile Ala Val Val Leu
Gln Leu Ser Gln Gln Leu Ser Gly Ile Asn275 280 285Ala Val Phe Tyr
Tyr Ser Thr Ser Ile Phe Glu Lys Ala Gly Val Gln290 295 300Gln Pro
Val Tyr Ala Thr Ile Gly Ser Gly Ile Val Asn Thr Ala Phe305 310 315
320Thr Val Val Ser Leu Phe Val Val Glu Arg Ala Gly Arg Arg Thr
Leu325 330 335His Leu Ile Gly Leu Ala Gly Met Ala Gly Cys Ala Ile
Leu Met Thr340 345 350Ile Ala Leu Ala Leu Leu Phe Gln Leu Pro Trp
Met Ser Tyr Leu Ser355 360 365Ile Val Ala Ile Phe Gly Phe Val Ala
Phe Phe Glu Val Gly Pro Gly370 375 380Pro Ile Pro Trp Phe Ile Val
Ala Glu Leu Phe Ser Gln Gly Pro Arg385 390 395 400Pro Ala Ala Ile
Ala Val Ala Gly Phe Ser Asn Trp Thr Ser Asn Phe405 410 415Ile Val
Gly Met Cys Phe Gln Tyr Val Glu Gln Leu Cys Gly Pro Tyr420 425
430Val Phe Ile Ile Phe Thr Val Leu Leu Val Leu Phe Phe Ile Phe
Thr435 440 445Tyr Phe Lys Val Pro Glu Thr Lys Gly Arg Thr Phe Asp
Glu Ile Ala450 455 460Ser Gly Phe Arg Gln Gly Gly Ala Ser Gln Ser
Asp Lys Thr Pro Glu465 470 475 480Glu Leu Phe His Pro Leu Gly Ala
Asp Ser Gln Val485 49041955DNAHomo sapiens 4ctcaaaacat ggcagctcaa
ttgcttcatc agagcaaaca agtgagagat ctcgaaactg 60aacaacacag aaatcattta
taacctaatc cgaaaatgac atccaccata tttgccgtct 120tctattcatt
agacgttgct aagtgatact tctagtgaca tctagaagag aagaggatgc
180cactaggcca cgaataccag gagggtctca gtgtaatcag actttttata
tgaaggctca 240gggatccagg caccagtatt ccagtgaccc agcctcagaa
gttacacagt gtcactctgc 300aggctactga tcaaaccagt cacaagccca
cttttatttc aagggaagga acaacgaatt 360ctggagccat gttttcaaac
tgccccagct attattattt ttgaaactgt gcaaggatcc 420cctggttcag
aggtcttatg gatgctgtca tctttgctga gatacctgct tgtgccttca
480gcatggaaga atgcctgtgt atccacctgt acggtagggg tcgctgtgac
tttgactggt 540gagggtacag ccactggtgc acatgcaaag gtgcctatct
gtgaacacgt attgagaggc 600tggataaggc tgcgcccatg tgagtgctgg
gcttgtacgt gcatttttgc ctgagtgagc 660attagtggca gtgtccccag
cctacccctt tcctgaatcc caggctcata gccaactgcc 720cacctatttc
cacgtggatg cctgctgagc acctcaaatg tcacacagcc aagacagaac
780tctggatctc ctttcccagc cacaagctgc ccctcttcca gtctgtaagt
tcttacggag 840catatatatg tgatctgcct acttttctcc aacctcacca
cagtgacatg agcccaaacc 900aacttctcac cttgcaacag cctcccaggt
gggaaggctg agtattctgg cccttaacca 960gttagaactc cccagttatc
tgtcctgctg atggggttga aatctacatt cctgaccctg 1020gcccaccaaa
gcccctccct tagctcccat ctccctcctc tctccctgtc ttctcctctg
1080ctccagacac tctggcttca tttctgcgtt ttttgtaccc cataagctcc
ttcccacccc 1140ggggcctttg cctttgctgt tccccctgcg gggaatgccg
gatctctgct cagatatcct 1200cttctcagat cagcaagcta aagcagccac
ctgtgtctgc ctaacccacc accgtagttt 1260aactttctgc ctagtcttta
tcactagctg atatttctca ggatccttta gttacttctt 1320tttcgtcttc
ccctcctaga atgtaaactc ttcccctcct agaaggtaaa caaaagacct
1380gttctgtttt gttcttcggc ccatcccaag cctagcgtag tgcctggtat
gtggtggtgt 1440ccaaacccaa gcgtggagtg aatgagggat gaatccatga
gagagtgagc ggctccagtg 1500ggtatgcgcg agtgtctcac tcggtgtaga
tgtgtgtgtt gtgtgtgttg tgtgtgtgcg 1560cacgctgggg aggccagaca
agtgtggacc agtgattggg
gcacctcttc cctgcaaaga 1620ggccagggga agacagtgcg tgtggggtct
tctaccaggg aggatggctt gctggtgtgt 1680cccccccagg ggaggactac
caacgaaggg gacccgggag atggcgggtg ggggcccccg 1740ggaggacagt
gggcgaggga gggggtcctt gccaggcctg gggcggccgg gggcggtcct
1800gggctcccct ccgtcccgcc tccaggcctc ggggcctggc tggccgacgt
ggcgttggcg 1860gcgctgcgcg cgggagggca gggcaggagg gacagaggcg
ggggcgggcc ggaaagtttg 1920tccggcggca gcggcgttgg ggactccggc ggggg
1955
* * * * *
References