Summary
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.
Article PDF
Similar content being viewed by others
Abbreviations
- DMEM:
-
Dulbecco's modified Eagle's medium
- FCS:
-
fetal calf serum
- AGPC:
-
acid guanidinium thiocyanate-phenol-chloroform
- bp:
-
base pair(s)
- IAPP:
-
islet amyloid polypeptide
- HUMMHBA 123:
-
human MHC protein homologous to chicken B complex protein
- PACAP:
-
pituitary adenylate cyclase activating polypeptide precursor
References
Froguel Ph, Vaxillaire M, Sun F et al. (1992) Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus. Nature 356: 162–164
Hattersley AT, Turner RC, Permutt MA et al. (1992) Linkage of type 2 diabetes to the glucokinase gene. Lancet 339: 1307–1310
Vionnet N, Stoffel M, Takeda J et al. (1992) Nonsense mutation in the glucokinase gene causes early-onset non-insulin-dependent diabetes mellitus. Nature 356: 721–722
Ballinger SW, Shoffner JM, Hedaya EV et al. (1992) Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion. Nature Genet 1: 11–15
van den Quweland JMW, Lemkes HHPJ, Ruitenbeek W et al. (1992) Mutation in mitochondrial tRNA gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nature Genet 1: 368–371
Okubo K, Hori N, Matoba R et al. (1992) Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature Genet 2: 173–179
Okubo K, Hori N, Matoba R et al. (1991) A novel system for large-scale sequencing of cDNA by PCR amplification. DNA Sequence 2: 137–144
Adams MD, Kerlavage AR, Fields C et al. (1993) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet 4: 256–267
Adams MD, Kelley JM, Gocayne MD et al. (1991) Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252: 1651–1656
Miyazaki J, Araki K, Yamato E, Ikegami H et al. (1990) Establishment of a pancreatic Β cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127: 126–132
Xie W, Rothblum LI (1991) Rapid, small scale RNA isolation from tissue culture cells. BioTechniques 11: 125–127
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444–2448
Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 227: 1435–1441
Wentworth BM, Schaefer IM, Villa-Komaroff L et al. (1986) Characterization of the two nonallelic genes encoding mouse preproinsulin. J Mol Evol 23: 305–312
Bibb MJ, van Etten RA, Wright CT et al. (1981) Sequence and gene organization of mouse mitochondrial DNA. Cell 26: 167–180
Nishi M, Chan SJ, Nagamatsu S et al. (1989) Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone. Proc Natl Acad Sci USA 86: 5738–5742
Takeda J, Yano H, Eng S et al. (1993) A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones. Human Molecular Genetics 2: 1793–1798
Hara E, Kato T, Nakada S et al. (1991) Subtractive cDNA cloning using Oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonic carcinoma cells. Nucl Acids Res 25: 7097–7104
Lisitsyn N, Lisitsyn N, Wigler M (1993) Cloning the differences between two complex genomes. Science 259: 946–951
Liang P, Pardee AB (1992) Differential display of eukaryotic messenger by means of the polymerase chain reaction. Science 257: 967–971
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tanaka, M., Katashima, R., Murakami, D. et al. Molecular cloning of a group of mouse pancreatic islet beta-cell-related genes by random cDNA sequencing. Diabetologia 38, 381–386 (1995). https://doi.org/10.1007/BF00410274
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00410274