Detection of Genetically Active Chemicals Using Various Yeast Systems

et al. 1979

Isolated circular molecules of the yeast plasmid 2-micron DNA were converted to linear molecules with restriction endonuclease Pstl and ligated with T4 DNA ligase to PstI restriction fragments of total yeast DNA. A haploid strain of Saccharomyces cerevisiae carrying a deletion in the his4 locus was transformed with the ligated DNA mixture to histidine prototrophy. One unstable histidine prototrophic transformant was obtained. Grown in the absence of histidine, 70-75 % of the cells were auxotrophic and this number increased in non-selective medium. The histidine auxotrophic variants carried a deletion in the his4 locus which had patterns of complementation and UV-induced mitotic recombination identical to the original his4 deletion. When the transformant was crossed to a his4 strain and sporulated, the unstable histidine prototrophy segregated in a nonmendelian way: All five possible segregations from 0:4 to 4:0 were observed. When strains carrying the transformed character were crossed to a his4 karl strain a low frequency of cytoduction of the unstable histidine prototrophy was observed. Nucleic acid from the transformant was able to transform a strain which carried another deletion in the his4 locus. Treatment of the transformant with ethidium bromide caused an extensive induction of petites without any observable change in the frequency of histidine prototrophic cells. It is concluded that the HIS4 gene function in the transformant is not stably associated with any chromosome. We take the instability as indication that only one or a few copies of the gene conferring the prototrophy are present in the prototrophic cells. The data are consistent with the assumption that the transformant contains a 2-micron DNA in which is inserted a chromosomal DNA region containing the HIS4 gene. A derivative of the transformant with increased stability has been isolated.

Section: Media and Growthmentioning

confidence: 99%

Transformation of yeast without the use of foreign DNA

Kielland‐Brandt

Nilsson-Tillgren

et al. 1979

“…YPG is the same as YPD, except that it contains 20 g/1 of glycerol instead of glucose. Media for the detection of nutritional requirements were minimal media appropriately supplemented with nutrients as described by ZIMMERMANN (15). Antimycin, otigomycin, ethidium bromide, tetracycline, and erythromycin were obtained from Sigma.…”

Section: Mediamentioning

confidence: 99%

A mutant of Saccharomyces cerevisiae temperature sensitive for flocculation. Influence of oxygen and respiratory deficiency on flocculence

Kielland‐Brandt

1978

A flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculence FL04, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Non-flocculent mutants were isolated with a selection procedure based on the slower sedimentation of non-floceulent cells. One closer studied mutant was due to an unlinked suppressor mutation for FL04. This suppressor gene is designated sufl. The gene sufl is neither centromere linked nor linked to his4 or the mating type locus. The gene sufl behaves as a recessive in some diploids and as a dominant in others, illustrating the genetic complexity of the flocculation phenomenon. The mutant was found to be non-flocculent after growth at 30~ whether aerated or not. At 22~ the mutant was flocculent in the absence of aeration during growth, but non-flocculent with aeration or supplementation of the growth medium with ergosterol and unsaturated fatty acids. None of several inhibitors of mitochondrial functions had any effect on the expression of flocculence. A number of petites induced in the mutant strain with ethidium bromide had altered flocculation phenotypes. A method for measuring flocculence using the spectrophotometer is described.

“…Media for the detection of nutritional requirements were minimal medium appropriately supplemented with nutrients as described by ZIMMERMAN (24). Media were solidified with agar (Difco) 20 g-1 -I.…”

Section: Media and Chemicalsmentioning

confidence: 99%

Isolation and characterization of a polypeptide absent from non-flocculent mutants of Saccharomyces cerevisiae

1978

Alkaline cell extracts obtained from whole cells of a flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculence FL04, and a nonflocculent mutant (FLO4,fsul) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mutant lacked a low molecular weight (13,000) polypeptide present in the extract from the parent strain. This polypeptide difference was also observed when five other independently isolated non-flocculent mutants of the parent strain were analyzed. One of these five mutants was characterized genetically and also in this case was non-flocculence shown to be due to an unlinked suppressor mutation of FL04. This suppressor gene is designatedfsu2. Analysis of petites with different flocculation phenotypes further extended the correlation between non-flocculence and the absence of the polypeptide.The polypeptide was isolated by gel filtration in the presence of sodium dodecyl sulfate followed by ionexchange chromatography on DEAE-cellulose. By electrophoretic analysis the purity of the preparation was estimated to be 95%. From amino acid analysis the polypeptide was calculated to consist of 121 residues with a molecular weight of 12,900 daltons.Differential extraction of proteins iodinated in situ by lactoperoxidase suggested an external location of the polypeptide.