Nucleosome structure and repair of N-methylpurines in the GAL1-10 genes of Saccharomyces cerevisiae

Nucleosome structure and repair of N-methylpurines were analyzed at nucleotide resolution in the divergent GAL1-10 genes of intact yeast cells, encompassing their common upstream-activating sequence. In glucose cultures where genes are repressed, nucleosomes with fixed positions exist in regions adjacent to the upstream-activating sequence, and the variability of nucleosome positioning sharply increases with increasing distance from this sequence. Galactose induction causes nucleosome disruption throughout the region analyzed, with those nucleosomes close to the upstream-activating sequence being most striking. In glucose cultures, a strong correlation between N-methylpurine repair and nucleosome positioning was seen in nucleosomes with fixed positions, where slow and fast repair occurred in nucleosome core and linker DNA, respectively. Galactose induction enhanced N-methylpurine repair in both strands of nucleosome core DNA, being most dramatic in the clearly disrupted, fixed nucleosomes. Furthermore, N-methylpurines are repaired primarily by the Mag1-initiated base excision repair pathway, and nucleotide excision repair contributes little to repair of these lesions. Finally, N-methylpurine repair is significantly affected by nearest-neighbor nucleotides, where fast and slow repair occurred in sites between pyrimidines and purines, respectively. These results indicate that nucleosome positioning and DNA sequence significantly modulate Mag1-initiated base excision repair in intact yeast cells.     

Allelism of pleiotropic drug resistance in Saccharomyces cerevisiae

Allelism of pleiotropic drug resistant (pdr) mutants was evaluated by complementation tests, linkage to chromosome-VII centromere markers and response to a partial suppressor (sur). Complementation tests were confounded by incomplete dominance and somatic segregation. Phenotypic suppression by sur was observed for all mutant and wild type alleles and thus could not be used to distinguish alleles. Five different alleles were tentatively identified by their close linkage to leul; 88 tetrads from three factor crosses produced the following linkages--leul (4.7) pdrl (17.0) trp5. Resistance of DRI 9/T7, a [cir o] strain of French origin, was not inherited as an allele of pdr but was controlled by a different pleiotropic centromere linked gene. An evaluation of published data suggest that antl, AMYl, till, cyh3, BOR2, and axe1 may be alleles of pdr. Thus pdr appears to be an allele that influences permeability to many inhibitors.     

GAL3 gene product is required for maintenance of the induced state of the GAL cluster genes in Saccharomyces cerevisiae.

The activities of the first three enzymes for galactose catabolism normally become detectable within 15 min after the addition of galactose into a culture of the yeast Saccharomyces cerevisiae. In S. cerevisiae with a recessive mutation termed gal3, a longer-than-normal lag is observed before the appearance of the enzyme activities (O. Winge and C. Roberts, C. R. Trav. Lab. Carlsberg Ser. Physiol. 24:263-315, 1948). I isolated two S. cerevisiae mutants with temperature-sensitive defects in the GAL3 gene. Temperature shift experiments with one of those mutants led to the conclusion that the GAL3 function is required not only for the initiation of enzyme induction but also for the maintenance of the induced state in galactose-nonfermenting S. cerevisiae because of a defect in any of the genes for the galactose-catabolizing enzymes, such as gal1 or gal10. In contrast, the GAL3 function is phenotypically dispensable in galactose-metabolizing S. cerevisiae. Thus, the normal catabolism of galactose can substitute for the GAL3 function.     

Mutations in GAL2 or GAL4 alleviate catabolite repression produced by galactose in Saccharomyces cerevisiae

Galactose does not allow growth of pyruvate carboxylase mutants in media with ammonium as a nitrogen source, and inhibits growth of strains defective in phosphoglyceromutase in ethanol–glycerol mixtures. Starting with pyc1, pyc2, and gpm1 strains, we isolated mutants that eliminated those galactose effects. The mutations were recessive and were named dgr1-1 and dgr2-1. Strains bearing those mutations in an otherwise wild-type background grew slower than the wild type in rich galactose media, and their growth was dependent on respiration. Galactose repression of several enzymes was relieved in the mutants. Biochemical and genetic evidence showed that dgr1-1 was allelic with GAL2 and dgr2-1 with GAL4. The results indicate that the rate of galactose consumption is critical to cause catabolite repression.     

PET genes of Saccharomyces cerevisiae.

We describe a collection of nuclear respiratory-defective mutants (pet mutants) of Saccharomyces cerevisiae consisting of 215 complementation groups. This set of mutants probably represents a substantial fraction of the total genetic information of the nucleus required for the maintenance of functional mitochondria in S. cerevisiae. The biochemical lesions of mutants in approximately 50 complementation groups have been related to single enzymes or biosynthetic pathways, and the corresponding wild-type genes have been cloned and their structures have been determined. The genes defined by an additional 20 complementation groups were identified by allelism tests with mutants characterized in other laboratories. Mutants representative of the remaining complementation groups have been assigned to one of the following five phenotypic classes: (i) deficiency in cytochrome oxidase, (ii) deficiency in coenzyme QH2-cytochrome c reductase, (iii) deficiency in mitochondrial ATPase, (iv) absence of mitochondrial protein synthesis, and (v) normal composition of respiratory-chain complexes and of oligomycin-sensitive ATPase. In addition to the genes identified through biochemical and genetic analyses of the pet mutants, we have cataloged PET genes not matched to complementation groups in the mutant collection and other genes whose products function in the mitochondria but are not necessary for respiration. Together, this information provides an up-to-date list of the known genes coding for mitochondrial constituents and for proteins whose expression is vital for the respiratory competence of S. cerevisiae.     

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.