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Localization of the transcript of the CDC7 gene of Saccharomyces cerevisiae

The CDC7 gene of Saccharomyces cerevisiae is a cell division cycle gene, whose gene product appears to act late in GI phase (Pringle & Hartwell, 1981). The gene has recently been cloned in the vector YRp7 (pBR322-Trpl+ -ars, autonomous replicator sequence; Botstein et al., 1979) and found to be contained within a 3.3 kb EcoRILHpul fragment of yeast chromosomal DNA. Northern blotting experiments showed that the central region of this fragment encoded a 1.7 kb mRNA (Meddle et al., 1985). Sequencing of the DNA segment giving rise to this transcript revealed an open reading frame which would code for a protein of M, about 58 000 (J. Rosamond, personal communication). The first aim of the present study was to determine the relationship of the mRNA leader sequence to the proposed translational start codon since it was our intention to make a fusion protein between the C-terminus of E. coli [j-galactosidase and the N-terminus of the CDC7 gene product. A second aim was to sequence the 5’ flanking region of the gene. For the determination of the CDC7 mRNA cap site, S1 nuclease mapping and primer extension studies were performed. High specific activity probes and primers were of single-stranded DNA prepared using 15-mer or 17-mer sequencing primers from appropriate restriction fragments cloned in MI3 mp8 as described by Burke (1984), except that urea/polyacrylamide gels were used for analysis (Sanger et al., 1980). The probe for S1 nuclease mapping was derived from the 687 base pair MluI-EcoRV fragment (see Fig. I), which runs from 156 base pairs within the proposed coding region to 531 base pairs upstream of it. It was internally labelled using [ c ~ ~ ~ P ] ~ C T P . The two single-stranded primers used in the primer extension studies were derived from the 41 base-pair AsuII-ClaI and 75 base-pair HpaII-EcoRV fragments, respectively; both primers were 5’ end-labelled with [y-’*P]ATP (3000 Ci/mmol) and polynucleotide kinase. Because of their method of preparation, the SI probe and the two primers retain MI3 sequences at their 5’ ends. They were hybridized to yeast poly (A+) mRNA between 55 and 65°C. S1 mapping showed several start sites, the most upstream being approximately 103 nucleotides from the proposed translational start codon (+ 1) while primer extension indicated a position between 105 and 110. To define these cap sites more precisely, primer extension