Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing.
To understand the molecular basis of glucose concentration-responsive insulin synthesis and secretion from pancreatic islet beta cells, a group of pancreatic islet beta-cell-related cDNAs was cloned. A pair of cDNA libraries was constructed from a mouse pancreatic islet beta-cell line of MIN6, which was cultured in either high glucose or low glucose media. By applying a random cDNA sequencing approach, 503 and 395 independent species were obtained from a total of 1,011 and 762 clones in the high glucose and low glucose library, respectively. The unknown genes comprised the majority of about 70% independent clones in both libraries. In Northern blot analysis, 311 (69.4%) of 448 independent clones showed positive signals within 72 h of autoradiographic exposure. Surprisingly, 150 (48.2%) out of 311 positive clones showed positive signals to MIN6 cells, but not to NIH/3T3 fibroblasts. The expression level of three unknown clones were glucose-concentration dependent. Combination of a random cDNA sequencing approach and Northern blot analysis is useful to obtain a large number of novel genes and islet beta-cell-related genes.
Tanaka
M
M
Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Japan.
Katashima
R
R
Murakami
D
D
Adzuma
K
K
Takahashi
Y
Y
Tomonari
A
A
Iwahana
H
H
Yoshimoto
K
K
Itakura
M
M
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D018076:DNA, Complementary / Q000737:chemistry*; D015870:Gene Expression; D015723:Gene Library; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000096:biosynthesis* / Q000235:genetics; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011897:Random Allocation
D000952:Antigens, Polyomavirus Transforming; D017931:DNA Primers; D018076:DNA, Complementary; D007328:Insulin; D008565:Membrane Proteins; D009687:Nuclear Proteins; D005947:Glucose