A Chromosomal Translocation Causing Overproduction

The CYC7-1 mutation in the yeast Saccharomyces cereuisiae causes the production of approximately 30 times the normal amount of iso-2-cytochrome c. Genetic analysis established that the CYC7-I mutation is a reciprocal translocation involving the left arm of chromosome V and the right arm of chromosome XVZ. The chromosome V arm was broken adjacent to the gene CYC7, which determines the primary structure of iso-&-cytochrome c, and this fragment containing the CYC7 gene was joined to the segment of chromosome XVZ. It appears as though the elevation of iso-2-cytochrome c is caused by an abnormal controlling region adjacent to the structural region of the CYC7 gene. HE yeast Saccharomyces cerevisiae contains two distinct forms of cytoTchrome c, iso-1-cytochrome c and iso-2-cytochrome c, which differ in primary structure at 21 residue positions (STEWART and PUTTERMAN, in preparation). Genetic analyses of strains containing mutationally altered forms of cytochrome c have established that the primary structure of iso-1-cytochrome c is determined by the CYCl gene (SHERMAN et al. 1966) , which is located on the right arm of chromosome X (LAWRENCE et at, 1975), and that the primary structure of is0-2cytochrome c is determined by the CYC7 gene (DOWNIE et al. 1977), which is located on the left arm of chromosome V (SHERMAN et al. 1978). Iso-1-cytochrome c and iso-2-cytochrome c normally constitute, respectively, approximately 95 % and 5 % of the total amount of cytochrome c in aerobically grown derepressed cells. Mutants containing elevated amounts of isol-2-cytochrome c are readily obtained on lactate medium after plating cycl strains, which lack iso-I-cytochrome c, and examining the revertant clones ( CLAVILIER, PBRB and SLONIMSKI 1969; CLAVILIER et a1 1976; STEWART et al. 1972; SHERMAN et al. 1974). Mutation of at least five genes can cause elevated amounts of iso-2-cytochrome c. We have reported that the CYC7-2 mutant contains an approximately 20-fold increase of iso-2-cytochrome c due to a mutation in the regulatory region that is believed to be contiguous with the structural region of the CYC7 gene (SHERMAN et al. 1978). Similarly, the mutational alteration that caused an approximate 30-fold increase of iso-%cytochrome c in the CYC7-I mutants was Genetics 88: 689-707 April, 1978. 690 F. SHERMAN AND C. HELMS demonstrated to be near the structural gene CYC7, although the nature of the mutation was not identified (DOWNIE, STEWART and SHERMAN 1977; DOWNIE et al. 1977). In this investigation the mutational change in the CYC7-I mutant is shown to be associated with a chromosome translocation in which one of the breakpoints occurred adjacent to the structural gene CYC7. It is believed that the unusually high level of iso-2-cytochrome c in the CYC7-1 translolcation is due to the abnormal region regulating the structural portion of the CYC7 gene. MATERIALS AND METHODS Strains carrying the CYC7-I translocation, the CYC7-2 regulatory mutant gene, and other related strains have been described by DOWNIE, STEWART and SHERMAN (1977), DOWNIE et al. (1977) and SHERMAN et al. (1978). Conventional yeast genetic procedures of crossing, sporulation, and tetrad analysis were used for meiotic analysis and to construct strains with desired markers (SHERMAN and LAWRENCE 1974). Nutritional markers, the canavanine-resistant marker (canl ) , and the inability to grow on glycerol medium were scored by dispensing small drops of cell suspension onto appropriate types of standard media (SHERMAN and LAWRENCE 1974). Strains carrying mak genes, which prevent the maintenance o r replication of the killer plasmid (WICKER and LEIBOWITZ 1976), were scored by spotting cell suspensions onto buffered nutrient medium containing methylene blue and containing a lawn of sensitive cells [KIL-01, a procedure similar to that first described by SOMERS and BEVAN (1969). Strains carrying the mini gene, which causes sensitivity to methionine and other nutrients (MEURIS 1969; SHERMAN et al. 1978), were scored on a synthetic complete medium or on a synthetic medium containing 1% casamino acids. The nonsense suppressor SUPI6, which was previously denoted SUQ5 (COX 1965; LIEBMAN, STEWART and SHERMAN 1975), was scored by suppression of the lysl-I marker. Strains carrying the rad1 gene, which causes UV sensitivity (RESNICK 1969), were scored by irradiating spots of cell suspensions with 30 J m-2. The iso-2-cytochrome c mutants used in this investigation are listed in Table 1. Since all of the strains used for pedigree analysis contained cycl mutant genes, which cause deficiency of iso-1-cytochrome c, the CYC7 genotypes could be determined from the levels of iso-2-cytochrome c that were estimated by low-temperature (-190") spectroscopic examinations of intact cells (SHERMAN and SLONIMSKI 1964). Derepressed cells for spectral examinations were prepared by growing strains as narrow lines transversing the nutrient plates (SHERMAN et al. 1974). The meiotic segregants derived from CYC7f/CYC7-l strains, which are heterozygous for the translocation, were examined directly, while the meiotic segregants derived from CYC7-I/cyc7-1-50 strains, which are homozygous for the translocation but heterozygous for alteration in the structural gene (Table l), were first converted to pderivatives before examination of the spectra. The pcyc7-1-50 strains lack iso-2-cytochrome c, while the pCYC7-I strains contain the same high level as the p+ CYC7-1 strains (DOWNIE, STEWART and SHERMAN TABLE 1 Designation of iso-%cytochrome c mutants used in genetic analyses Chromosomal Structural Level of Symbol constitution gene iso-2-cytochrome c CYC7+ normal normal 5% CYC7-I translocation normal 150% cyc7-1-50 translocation missense 150%inp+; <5%inpcyc7-1-1 translocation nonsense 0% OVERPRODUCTION O F YEAST ISO-2-CYTOCHROME C 69 1 1977). The pstrains were conveniently prepared by spotting cells suspensions onto a nutrient medium containing ethidium bromide (1 % Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose and 40 mg/l ethidium bromide).