Creating aSaccharomyces cerevisiae haploid strain having 21 chromosomes

2003 ◽  
Vol 95 (1) ◽  
pp. 89-94 ◽  
Author(s):  
Donny Widianto ◽  
Eishi Yamamoto ◽  
Minetaka Sugiyama ◽  
Yukio Mukai ◽  
Yoshinobu Kaneko ◽  
...  
Keyword(s):  
Haploid Strain

scholarly journals INDUCTION OF INTRACHROMOSOMAL RECOMBINATION IN YEAST BY INHIBITION OF THYMIDYLATE BIOSYNTHESIS

Genetics ◽  
1986 ◽  
Vol 114 (2) ◽  
pp. 375-392
Author(s):  
B A Kunz ◽  
G R Taylor ◽  
R H Haynes
Keyword(s):  
Gene Conversion ◽  
Dna Sequences ◽  
Dna Hybridization ◽  
Crossing Over ◽  
Repeated Dna ◽  
Yeast Saccharomyces Cerevisiae ◽  
Haploid Strain ◽  
Reciprocal Exchange ◽  
Coli Plasmid ◽  
Antifolate Drugs

ABSTRACT The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

Amylase hyper-producing haploid recombinant strains of Thermomyces lanuginosus obtained by intraspecific protoplast fusion

2000 ◽  
Vol 46 (7) ◽  
pp. 669-673 ◽  
Author(s):  
K Rubinder ◽  
B S Chadha ◽  
S Singh ◽  
H S Saini
Keyword(s):  
Protoplast Fusion ◽  
Culture Filtrate ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Thermomyces Lanuginosus ◽  
Wild Type ◽  
Haploid Strain ◽  
Recombinant Strains ◽  
Specific Activities

Amylase hyper-producing, catabolite-repression-resistant, recombinant strains were produced by intraspecific protoplast fusion of thermophilic fungus Thermomyces lanuginosus strains, using well-characterized, morphological, and 2-deoxy-D-glucose resistant markers. The fusant heterokaryons exhibited enhanced amylase activities as compared to the amylase hyper-producing parental strain (T2). Diploids derived from heterokaryons segregated to stable haploid recombinant strains. In the haploid strain (Tlh 4q), approximately 5-fold higher specific activities of α-amylase and glucoamylase in the culture filtrate were observed as compared to the wild-type strain (W0).Key words: Thermomyces lanuginosus, protoplast fusion, amylase hyper-producing strain, catabolite repression.

scholarly journals Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

1993 ◽  
Vol 13 (10) ◽  
pp. 6012-6023 ◽  
Author(s):  
R Cafferkey ◽  
P R Young ◽  
M M McLaughlin ◽  
D J Bergsma ◽  
Y Koltin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Dna Sequence ◽  
Leucine Zipper ◽  
Sequence Similarity ◽  
Missense Mutations ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Haploid Strain ◽  
Diploid Cells ◽  
Phosphatidylinositol 3

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

scholarly journals Genome-wide prediction of CRISPR/Cas9 targets in Kluyveromyces marxianus and its application to obtain a stable haploid strain

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ming-Hsuan Lee ◽  
Jinn-Jy Lin ◽  
Yu-Ju Lin ◽  
Jui-Jen Chang ◽  
Huei-Mien Ke ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Haploid Strain ◽  
Genome Wide

scholarly journals Isolation, nucleotide sequence analysis, and disruption of the MDH2 gene from Saccharomyces cerevisiae: evidence for three isozymes of yeast malate dehydrogenase.

1991 ◽  
Vol 11 (1) ◽  
pp. 370-380 ◽  
Author(s):  
K I Minard ◽  
L McAlister-Henn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Carbon Source ◽  
Malate Dehydrogenase ◽  
Sodium Dodecyl ◽  
Yeast Cells ◽  
Nucleotide Sequence Analysis ◽  
Haploid Strain

The major nonmitochondrial isozyme of malate dehydrogenase (MDH2) in Saccharomyces cerevisiae cells grown with acetate as a carbon source was purified and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit molecular weight of approximately 42,000. Enzyme assays and an antiserum prepared against the purified protein were used to screen a collection of acetate-nonutilizing (acetate-) yeast mutants, resulting in identification of mutants in one complementation group that lack active or immunoreactive MDH2. Transformation and complementation of the acetate- growth phenotype was used to isolate a plasmid carrying the MDH2 gene from a yeast genomic DNA library. The amino acid sequence derived from complete nucleotide sequence analysis of the isolated gene was found to be extremely similar (49% residue identity) to that of yeast mitochondrial malate dehydrogenase (molecular weight, 33,500) despite the difference in sizes of the two proteins. Disruption of the MDH2 gene in a haploid yeast strain produced a mutant unable to grow on minimal medium with acetate or ethanol as a carbon source. Disruption of the MDH2 gene in a haploid strain also containing a disruption in the chromosomal MDH1 gene encoding the mitochondrial isozyme produced a strain unable to grow with acetate but capable of growth on rich medium with glycerol as a carbon source. The detection of residual malate dehydrogenase activity in the latter strain confirmed the existence of at least three isozymes in yeast cells.

The Morphological Features of Adaptation of the Yeast of the Genus Saccharomyces to Extreme Values of Glucose and Ethanol

2020 ◽  
pp. 95-101
Author(s):  
Elvira A. Islammagomedova ◽  
Eslanda A. Khalilova ◽  
Svetlana Ts. Kotenko ◽  
Aida A. Abakarova ◽  
Dinara A. Aliverdieva
Keyword(s):  
Extreme Values ◽  
Surface Profile ◽  
Morphological Properties ◽  
Ethanol Stress ◽  
Extreme Conditions ◽  
Haploid Strain ◽  
Food Biotechnology ◽  
Round Shape ◽  
Granular Cytoplasm ◽  
Selection Of

The influence of extreme values of glucose and ethanol on the morphological properties of the yeast of the genus Saccharomyces was studied. The ability of strains S. cerevisiae Y-503, S. cerevisiae DAW-3a, and S. oviformis M-12X to adapt to stress was found. The dependence of the functional state and morphological properties of the studied strains on ploidy was shown. It was established that the cell forms of S. cerevisiae Y-503 and S. oviformis M-12X were diverse, the cells of the haploid strain S. cerevisiae DAW-3a were characterized by a round shape and smaller sizes compared to the polyploid strain Y-503 and diploid M-12X. Adaptation to different concentrations of glucose was accompanied by a change in the shape of the cells Y-503 and DAW-3a, while the sizes of cells were practically un-changed. Under conditions of simultaneous osmotic and ethanol stress in the cells of the studied strains, the presence of granular cytoplasm was revealed, which is associated with the accumulation of reserve nutrients. A change in the shape of the cells, form, surface, profile, color, size of colonies was detected. The study of the properties of the yeast of the genus Saccharomyces in the process of adaptation to extreme conditions may serve as the basis for the selection of strains that are promising for use in food biotechnology.

scholarly journals Mitochondrial instability in a strain ofSaccharomyces cerevisiae

1978 ◽  
Vol 32 (2) ◽  
pp. 171-182 ◽  
Author(s):  
I. H. Evans ◽  
D. Wilkie
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Glucose Repression ◽  
Point Mutations ◽  
Nuclear Gene ◽  
Fermentable Sugars ◽  
Glucose Medium ◽  
Haploid Strain ◽  
Respiratory Impairment ◽  
Mutator Effect

SUMMARYA haploid strain ofSaccharomyces cerevisiaehas been described which, on glucose medium, segregates vegetatively a high frequency of mutants with different degrees of respiratory impairment. The range of mutants seemingly encompasses both non-revertible ρ- petites and revertible point mutations resembling leakymit- mutations. The segregants have aberrant cytochrome contents and reduced growth capabilities on fermentable sugars other than glucose; these defects apparently correlate with the degree of respiratory impairment. Genetic analysis of this mutator phenomenon has implicated a nuclear gene which appears to show specificity of interaction with the mitochondrial genome as well as a requirement for glucose repression. The mutator effect seems to extend also to the loci in mitochondrial DNA for resistance to the antibiotics erythromycin and oligomycin.

scholarly journals AN ENDOMITOTIC EFFECT OF A CELL CYCLE MUTATION OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1981 ◽  
Vol 97 (3-4) ◽  
pp. 551-562 ◽  
Author(s):  
David Schild ◽  
Honnavara N Ananthaswamy ◽  
Robert K Mortimer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Nuclear Division ◽  
Pedigree Analysis ◽  
Visual Observation ◽  
Temperature Sensitive ◽  
Cell Stage ◽  
Haploid Strain ◽  
A Cell ◽  
Dna Measurements

ABSTRACT A recessive temperature-sensitive mutation of Saccharomyces cerevisiae has been isolated and shown to cause an increase in ploidy in both haploids and diploids. Genetic analysis revealed that the strain carrying the mutation was an aa diploid, although MNNG mutagenesis had been done on an a haploid strain. When the mutant strain was crossed with an aa diploid and the resultant tetraploid sporulated, some of the meiotic progeny of this tetraploid were themselves tetraploid, as shown by both genetic analysis and DNA measurements, instead of diploid as expected of tetraploid meiosis. The ability of these tetraploids to continue to produce tetraploid meiotic progeny was followed for four generations. Homothallism was excluded as a cause of the increase in ploidy; visual pedigree analysis of spore clones to about the 32-cell stage failed to reveal any zygotes, and haploids that diploidized retained their mating type. An extra round of meiotic DNA synthesis was also considered and excluded. It was found that tetraploidization was independent of sporulation temperature, but was dependent on the temperature of germination and the growth of the spores. Increase in ploidy occurred when the spores were germinated and grown at 30°, but did not occur at 23°. Two cycles of sporulation and growth at 23° resulted in haploids, which were shown to diploidize within 24 hr when grown at 30°. Visual observation of the haploid cells incubated at 36° revealed a celldivisioncycle phenotype characteristic of mutations that affect nuclear division; complementation analysis demonstrated that the mutation, cdc31-2, is allelic to cdc31-1, a mutation isolated by HARTWEeLL et al.(1973) and characterized as causing a temperature-sensitive arrest during late nuclear division. The segregation of cdc31-2 in heterozygous diploids was 2:2 and characteristic of a noncentromere-linked gene.

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