Sequence variation and chromosomal mapping of the murine Cdkn2a tumor suppressor gene.

Abstract

Twenty-nine mouse strains were examined for sequence variation of the Cdkn2a (p16 xNK4~) tumor suppressor gene by single-strand conformation polymorphism (SSCP) and direct sequence analyses. Polymorphisms were observed at nucleotides 53 and 61 of exon lot and at nucleotide 33 of exon 113; the polymorphism of nucleotide 53 of exon let resulted in an amino acid substitution. The mouse strains were distributed into three groups of variants. The polymorphisms in Elet segregated into two groups which cosegregated with those of El13 for all but one strain, the latter of which comprised the third group. With recombinant inbred strains derived from intervariant crosses, the Cdkn2a gene was mapped to the same approximate location as microsatellite markers D4MIT77 and D4MIT245 on Chromosome (Chr) 4. The Cdkn2a gene encodes a tumor suppressor that functions as an inhibitor of the cyclin D-dependent kinases, CDK4 and CDK6 (Kamb et al. 1994; Nobori et al. 1994; Quelle et al. 1995a; Serrano et al. 1993). These kinases regulate the transition of cells through the G1 phase of the cell cycle by controlling the functional status of the retinoblastoma protein (pRB), also a tumor suppressor (Serrano et al. 1993). As components of a common pathway for regulation of G1 passage, the inactivation of either pRb or Cdkn2a appears to be selected in various cancers (Otterson et al. 1994). The Cdkn2a gene was identified at the site of common deletion in several types of cancer on human Chr 9p21 and the syntenic region of mouse Chr 4 (Kamb et al. 1994; Nobori et al. 1994, Quelle et al. 1995a). Both the human and mouse homologs of the Cdkn2a yield two unique transcripts that utilize alternative exons 1 (Elet and El13) and incorporate exon 2 in alternative reading frames via the same splice acceptor site (Mao et al. 1995; Quelle et al. 1995b; Stone et al. 1995). The et form encodes the tumor suppressor Cdkn2a, while the 13-form encodes a 19-kDa protein (p19ARF), which is a suspected tumor suppressor (Quelle et al. 1995b). Each of the three exons of the Cdkn2a gene were polymerase chain reaction (PCR)-amplified from normal lung DNA of 29 mouse strains (27 inbred, 1 congenic, and 1 wild). When examined by SSCP and direct sequence analyses, sequence polymorphisms within Elet and El13 were revealed (Fig. 1). The polymorphisms of Elet and El13 cosegregated as three allelic forms. One form consisted of an adenine and a cytodine at nucleotides 53 (codon 18) and 61 (codon 21) of Elet, respectively, and a thymidine at nucleotide 33 (codon 11) of El13 and was present in 20 inbred strains, the congenic strain B10.D2(58N)Sn and the wild strain Mus spretus. The second form was observed in one strain (MA/M4J) and possessed the same El13 sequence as form 1, but possessed a cytidine and adenine at nucleotides 53 and 61 of Elet, respectively. Six inbred strains possessed form 3, which also had a cytidine and an adenine at nucleotides 53 and 61 of Elet, respectively, and a cyElct 10 60 allele 1 ' OCTGCAOACAGACTCKT~CAGCrC~GCrCCOCCCA~CGTGTGCATGACGTGCGG allele 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . A . . allele 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . C . .