Isolated nucleic acids and polypeptides associated with glucose homeostasis disorders and method of detecting the same
a technology of glucose homeostasis and nucleic acid sequences, applied in the field of nucleic acid sequences coding for sugar transporters, can solve the problems of linkage disequilibrium, hyperglycemic systemic condition, traditional genetic approaches have proven inadequate to achieve this goal, etc., and achieve the effect of facilitating diagnosis
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example 1
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.
example 2
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™ 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.
example 3
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 32P by the random priming method.
[0285]The Northern blot was bought commercially from Clontech of Palo Alto, Calif. Probe conditions were 68° C. hybridization for 1 hr using a hybridization buffer sold under the trademark EXPRESSHYB™ by Clontech of Palo Alto, Calif., followed by 2 washes with 2×SSC, 0.05% SDS for 40 minutes, each at room temperature.
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