Identifying Mutations in Duplicated Functions in Saccharomyces cerevisiae: Recessive Mutations in HMG-CoA Reductase Genes

Abstract

The two yeast genes for 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase, HMGl and HMG2, each encode a functional isozyme. Although cells bearing null mutations in both genes are inviable, cells bearing a null mutation in either gene are viable. This paper describes a method of screening for recessive mutations in the HMGl gene, the gene encoding the majority of HMG-CoA reductase activity in the cell. This method should be applicable to the isolation of mutations in other cases of duplicated genes or duplicated functions in yeast. Four recessive point mutations were recovered in HMGl. These mutations exhibited intragenic complementation: one allele is in one complementation group and three alleles are in a second complementation group. Assays of HMGCoA reductase activity indicated that the point mutations destroy most if not all of the activity encoded by HMGl. Intragenic complementation occurred with partial restoration of enzymatic activity. HMGI was mapped to the left arm of chromosome XIII near SUP79, and HMG2 was mapped to the right arm of chromosome XII near SST2. A slight deleterious effect of a null mutation in either HMG-CoA reductase gene was detected by a co-cultivation experiment involving the wild-type strain and the two single mutants.

Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

“…While mmm1-1 cells can grow at the permissive temperature on nonfermentable medium, we find that mmm1-1 mmm2-1 cells cannot. Mutants that are incompatible with each other, so-called synthetic lethal mutants, often identify proteins that interact or whose gene products act in the same pathway (Basson et al, 1987;Bender and Pringle, 1991). A number of observations suggest that the Mmm2 and Mmm1 proteins both play a role in mitochondrial shape and mtDNA nucleoid structure: (1) lack of either protein results in a dramatic loss of mitochondrial shape and mtDNA organization, (2) high levels of MMM2 partially rescue the morphology defect of mmm1⌬ cells (unpublished data), (3) both proteins are located in the mitochondrial outer membrane, (4) Mmm2p and Mmm1p are each located in small particles on the mitochondrial surface, next to a subset of mtDNA nucleoids, (5) a significant fraction of the Mmm2p-containing dots colocalize with Mmm1p-containing structures, and (6) Mmm2p and Mmm1p are required for each other's distribution in the cell.…”

Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae

“…The s/a mutants were isolated using a synthetic lethal strategy based on selection against the LYS2 and URA3 genes (Basson et al, 1987). DDY 262 (Table I) contains a nearly complete disruption of the ABP1 gene (extending from an XhoI site 227-bp upstream of the start codon to a PvulI site 246-bp 1.…”

“…The strategy that we used to isolate mutations that require ABP1 relies on the ability to select against the URA3 and LYS2 genes with 5-FOA and t~-aminoadipate, respectively (Boeke et al, 1984;Chattoo and Sherman, 1979), and on the fact that in the absence of positive selection, centromerebased plasmids are lost from a small percentage of the cells that form a colony (Basson et al, 1987). The starting haploid strain, DDY 262 (Table I), contains a complete disruption of ABP1 (see Materials and Methods).…”

Structural and Functional Conservation Between Yeast and Human 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases, the Rate-Limiting Enzyme of Sterol Biosynthesis

“…The methods and parameters used are outlined in the legend to Fig. 4. The secondary structures of the human and hamster proteins are predicted to be virtually identical, reflecting their high degree of sequence similarity: only 32 nonconservative amino acid replacements exist between the two proteins (10,36).…”