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

Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia

Address all correspondence and requests for reprints to: Dr. David Cram, Burnet Clinical Research Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Australia. E-mail: cram{at}wehi.edu.au

A knowledge of ß cell-specific gene expression provides a basis for identifying proteins potentially involved in ß cell function and pathology. To identify candidate ß cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, ßTC3 and TC1. Following three successive subtractions of TC1 complementary DNA from ßTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported ß cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-ß cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to ßTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in ßTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in ß cell adhesion events during islet ontogeny. In ßTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic ß cell growth and/or differentiation.

In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate ß cell growth and differentiation.

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