Nucleic acid molecules encoding GLUTX and uses thereof

Details Nucleic acid molecules encoding GLUTX and uses thereof Nucleic acid molecules encoding GLUTX and uses thereof

2000-07-05

2002-02-12

McGarry, Sean (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S006120, C435S014000, C435S007100

Reexamination Certificate

active

06346374

ABSTRACT:

BACKGROUND OF THE INVENTION
A number of mammalian glucose (hexose) transporters (GLUTs have been identified. High affinity GLUTs are found in nearly every tissue. A low affinity GLUT (GLUT-2) is expressed in tissues which are associated with high glucose flux (e.g., intestine, kidney, and liver). It is thought that the level of expression of high affinity GLUTs influences the rate or glucose uptake. It is also thought that the expression of various GLUTs is regulated by glucose and various hormones (Thorens,
Am. J. Physiol.
270 (Gastrointest. Liver Physiol. 33:G541-G553, 1996). Human GLUT-1 is described by Mucckler et al. (
Science
229:941, 1985). Human GLUT-2 is described by Fukumoto et al. (
Proc. Nat'l Acad. Sci. USA
264:776, 1989). Human GLUT-3 is described by Keller et al. (
J. Biol. Chem.
264:18884, 1989). Human GLUT-4 is described by Fukumoto et al. (
J. Biol. Chem.
264:7776, 1989). Human GLUT-5 is described by Kayano et al. (
Nature
377:151, 1995).
SUMMARY OF THE INVENTION
The invention described herein relates discovery and characterization of a cDNA encoding GLUTX, a human glucose transporter protein. The nucleotide sequence of a cDNA encoding GLUTX is shown in
FIGS. 1A-1E
. The deduced amino acid sequence of GLUTX is shown in
FIGS. 2A-2D
. GLUTX is predicted to include 12 transmembrane domains. The first transmembrane domain extends from about amino acid 52 (intracellular end) to about amino acid 71 (extracellular end). The second transmembrane domain extends from about amino acid 108 (extracellular end) to about amino acid 128 (intracellular end). The third transmembrane domain extends from about amino acid 141 (intracellalar end) to about amino acid 159 (extracellular end). The fourth transmembrane domain extends from about amino acid 166 (extracellular end) to about amino acid 189 (intracellular end). The fifth transmembrane domain extends from about amino acid 204 (intracellular end) o about amino acid 221 (extracellular end). The sixth transmembrane domain extends from about amino acid 233 (extracellular end) to about amino acid 252 (intracellular end). The seventh transmembrane domain extends from about amino acid 317 (intracellular end) to about amino acid 338 (extracellular end). The eighth transmembrane domain extends from about amino acid 355 (extracellular end) to about amino acid 375 (intracellular end). The ninth transmembrane domain extends from about amino acid 383 (intracellular end) to about amino acid 404 (extracellular end). The tenth transmembrane domain extends from about amino acid 413 (extracellular end) to about amino acid 437 (intracellular end). The eleventh transmembrane domain extends from about amino acid 449 (intracellular end) to about amino acid 472 (extracellular end). The twelfth transmembrane domain extends from about amino acid 481 (extracellular end) Ho about amino acid 499 intacelllar end). GLUTX nucleic acids and polypeptides, as well as molecules which increase or decrease expression or activity of GLUTX, are useful in the diagnosis and treatment of disorders associated with aberrant hexose transport.
GLUTX protein has some sequence similarity to a number of known glucose transporters
FIGS. 3A-3D
.
The invention features isolated nucleic acid molecules (i.e., a nucleic acid molecule that is separated from the 5′ and 3′ coding sequences with which to is immediately contiguous in the naturally occurring genome of an organism, also referred to as a recombinant nucleic acid molecule) that encodes a GLUTX Collectide. Within the invention are polypeptides having the sequence of SEQ ID NO:2 or encoded by nucleic acid molecules having the sequence shown in SEQ ID NO:1. However, the invention is not limited to nucleic acid molecules and polypeptides that are identical to those SEQ ID Nos. For example, the invention includes nucleic acid molecules which encode splice variants, allelic variants or mutant forms of GLUTX as well as the proteins encoded by such nucleic acid molecules.
Also within the invention are nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO:1. Such molecules include, for example, nucleic acid molecules encoding allelic variants of GLUTX or mutant forms of GLUTX. As described further below, molecules that are substantially identical to those of SEQ ID Nos. 1 and 2 are also encompassed by the invention.
The term “substantially pure” as used herein in reference to a given compound (e.g., a GLUTX polypeptide) means that the compound is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the compound may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). When chemically synthesized, a compound of the invention is substantially pure when it is substantially free from the chemical compounds used in the process of its synthesis. Polypeptides or other compounds of interest are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferable at east 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that s completely identical o a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
For polypeptides, the length of the reference polypeptide sequence will generally be at least it amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleocides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides (e.g., 150, 200, 250, or 300 nucleotides).
In the case of polypeptide sequences that are less than 100% Identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 with the default parameters as specified therein.
The BLAST programs, provided as a service by the National Center for Biotechnology Information, are very useful for making sequence comparisons. The programs are described in detail by Karlin et al., (
Proc. Natl. Acad. Sci. USA
87:2264-68, 1990 and 90:5873-7, 1993) and Altschul et al., (Nucl. Acids Res. 25:3389-3402, 1997) and are available on the internet.
The invention also features a host cell that harbors an isolated nucleic acid molecule encoding GLUTX (either alone or in conjunction with a heterologous polyptide, such as a detectable marker) or a nucleic acid

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