A subtractive cloning approach to the identification of mRNAs specifically expressed in pancreatic beta-cells.
A polymerase chain reaction-based subtractive hybridization procedure was applied to cDNAs prepared from mouse insulinoma (beta TC3) and glucagonoma (alpha TC2) cell lines to construct a library of cDNAs that are highly expressed in pancreatic beta-cells. An analysis of 555 randomly chosen clones in the library showed that 80 were derived from abundant mRNAs and were accounted for by 29 distinct sequences. Of these, 17 were identical or homologous to known mammalian cDNAs or expressed sequence tags. Genes known to be highly expressed in beta-cells were represented at a high frequency, namely insulin (15 of 80 clones), islet amyloid polypeptide (8 of 80 clones), proinsulin convertase 1 (6 of 80 clones), and neuropeptide Y (2 of 80 clones). Many of the novel cDNA sequences that were highly represented in the library showed a relative specificity to beta-cells compared with other tissues, including glucagonoma, liver, kidney, brain, 3T3 fibroblasts, and AtT20 corticotrophs, and warrant further investigation. When combined with functional or immunological screening procedures, the approach will be useful for the isolation of beta-cell-specific molecules for immunological and genetic investigations of beta-cell function and pathology.
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
P I
PI
Neophytou
E M
EM
Muir
J C
JC
Hutton
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D015723:Gene Library; D007515:Islets of Langerhans / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D017931:DNA Primers; D012333:RNA, Messenger