Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing

Abstract

SummaryTo 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. [Diabetologia (1995) 38: 381-386] Key words Random cDNA sequencing, MIN6, pancreatic islet beta cell.Understanding the molecular basis of pancreatic islet beta cells is essential in order to develop a logical treatment for diabetes. In addition to glucokinase [1][2][3] and mitochondrial genes [4][5], which are known to be aetiological for non-insulin-dependent diabetes or maturity-onset diabetes of the young, there are others which are related to islet beta-cell Received: 12 July 1994 and in revised form: 20 September 1994Corresponding author: Professor M. Itakura, Otsuka Department of Clinical and Molecular Nutrition, School of Medicine, University of Tokushima, Kuromoto, Tokushima-city, Tokushima, 770, Japan Abbreviations: DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; AGPC, acid guanidinium thiocyanatephenol-chloroform; bp, base pair(s); IAPR islet amyloid polypeptide; HUMMHBA 123, human MHC protein homologous to chicken B complex protein; PACAR pituitary adenylate cyclase activating polypeptide precursor. function and diabetes. It is speculated that the highly-differentiated functions of islet beta cells are based on a group of cell-specific genes. Recently, large,scale cDNA analysis has become a valuable way to examine and register expressed genes in various tissues by determining expressed sequence tags [6][7][8][9]. Abundant transcripts, which are frequently encountered in random sequencing, are assumed to play relatively more important roles than rare transcripts with exceptions including transacting factors and rate-limiting enzymes among others. With this in mind we randomly sequenced cDNA to clone unknown genes which are abundantly expressed and are presumably important for islet beta-cell functions. In this study we report the acquisition of a group of islet beta-cell-related genes by combining a random cDNA sequencing of a total of 1,773 clones from a m...

Expression of Neuronal Traits in Pancreatic Beta Cells

“…Takeda et al (40) have constructed a cDNA library from human pancreatic islets and randomly sequenced 1000 clones. Tanaka et al (41) have sequenced nearly 2000 clones derived from cDNA libraries prepared from a mouse beta cell line. Finally, Neophitou et al (42) have used a subtractive cloning approach between insulin-and glucagon-producing cell lines to identify mRNAs specifically expressed in pancreatic beta cells.…”

Regulatable production of insulin from primary-cultured hepatocytes: insulin production is up-regulated by glucagon and cAMP and down-regulated by insulin

“…Thus, insulin production from non-neuroendocrine cells may correlate more closely to intermediateacting or long-acting insulin. The most desirable regulator of insulin production, even at the transcription level, is glucose, which regulates the expression of many genes in pancreatic ␤ cells and hepatocytes, [16][17][18] both of which possess Glut2 and glucokinase for responding to a physiological range of glucose concentrations. 2,5 Initially, we attempted to use glucose-responsive promoters for the rat S-14 mRNA gene and rat L-type pyruvate kinase, 19,20 but we were unsuccessful in using these promoters to regulate insulin production from rat hepatocytes because the glucose activation of these promoters was weak.…”

Identification of Pancreatic β Cell-Related Genes by Representational Difference Analysis1

“…In one study, Tanaka et al (2) sequenced randomly selected clones from a human islet complementary DNA (cDNA) library and catalogued several hundred genes. However, only a few have attempted to systematically and comprehensively analyze gene expression in ␤ cells.…”