High level of single-nucleotide polymorphism in the rat cyclin B1 gene.

Abstract

Inbred rat strains have distinct susceptibilities to carcinogeninduced mammary cancer. Wistar-Furth (WF) rats are highly susceptible, while Copenhagen (COP) and Wistar-Kyoto (WKY) rats are resistant to this carcinoma and the Fischer-344 (F344) rats have an intermediate susceptibility (Isaacs 1986; Haag et al. 1992). Genetic breeding studies showed that this phenotype is a polygenic trait (Shepel et al. 1998). One of the susceptibility loci, Mcs1, identified at the centromeric end of rat Chr 2 (2q1), has been involved in the modulation of tumor number (Hsu et al. 1994; Shepel et al. 1998). The cyclin B1 gene (Ccnb1) is located in or close to the Mcs1-containing region (Shepel et al. 1998; Szpirer et al. 1999). Since cyclins are key components of the cell cycle progression machinery, a disregulation can potentially lead to uncontrolled cell growth and hence to cancer. Indeed, overexpression of several cyclins has been demonstrated to be associated with cancer (Keyomarsi and Pardee 1993). The rat Ccnb1 gene can thus be considered as an Mcs1 candidate. In order to test this hypothesis, we sequenced the Ccnb1 cDNA in the four strains mentioned (WF, COP, WKy, and F344). Our results do not argue in favor of the candidacy of Ccnb1, and, unexpectedly, disclosed a high level of sequence variation in the expressed region of the gene. Two rat Ccnb1 cDNA isoforms have been described so far. Trembley and colleagues (1994) isolated a 1375-bp cDNA from testes, while Markiewicz and associates (1994) isolated a 2348-bp cDNA from fibroblasts. These cDNAs differ mainly in the two untranslated regions (UTRs). The 58 UTRs have different lengths and also show divergent sequences at their 58 end, while their 38 end (last 65 positions, preceding the translation start) are almost identical, suggesting that the two isoforms are produced by alternative splicing involving distinct exons 1. The two 38 UTRs differ in the use of the polyadenylation site (first site used in the testes form, and second site used in the fibroblast form). Poly(A)-enriched RNA was isolated from mammary glands of WF, COP, WKy, F344 rats (purchased from Harlan), and RTPCR was done with four primer pairs spread along the Ccnb1 cDNA sequence, generating four fragments, which were sequenced in each of the four strains (Fig. 1). Two sequences were obtained, as shown in Fig. 2. Both combine the 58 UTR sequence