scholarly journals Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene.

1986 ◽  
Vol 6 (8) ◽  
pp. 2828-2838 ◽  
Author(s):  
D A Barnes ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Gene ◽  
Minimal Medium ◽  
Genetic Manipulation ◽  
Gene Replacement ◽  
Complete Medium ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Chromosome Ii ◽  
In Cells

The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems.

Genetic manipulation of Saccharomyces cerevisiae by use of the LYS2 gene

1986 ◽  
Vol 6 (8) ◽  
pp. 2828-2838
Author(s):  
D A Barnes ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Gene ◽  
Minimal Medium ◽  
Genetic Manipulation ◽  
Gene Replacement ◽  
Complete Medium ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Chromosome Ii ◽  
In Cells

The structural gene for alpha-aminoadipate reductase (LYS2) was isolated from a Saccharomyces cerevisiae genomic DNA library by complementation of a lys2 mutant. Both genetic and biochemical criteria confirmed that the DNA obtained corresponds to the LYS2 locus on chromosome II. Subcloning and deletion analysis showed that a functional LYS2 gene is contained within a 4.6-kilobase (kb) EcoRI-HindIII fragment of the original insert, and the slightly larger EcoRI-ClaI segment (4.8 kb) was used to construct a series of cloning vehicles, including integrating, episomal, replicative, and centromeric vectors. The cloned DNA was also used to generate a genomic deletion that lacks all LYS2 coding sequences on chromosome II. The level of the LYS2 transcript (4.2 kb) was 10-fold higher in cells grown on minimal medium than in cells grown on complete medium and was not repressed by the presence of lysine alone. Gene disruption, gene replacement, and promoter analysis of the major alpha-factor structural gene (MF alpha 1) were performed to illustrate the utility of the LYS2 gene for the genetic manipulation of yeasts. Because all fungi synthesize lysine via the alpha-aminoadipate pathway, the techniques developed here for using the S. cerevisiae LYS2 gene should be directly applicable to other fungal systems.

scholarly journals Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 739-752 ◽  
Author(s):  
C A Woolford ◽  
C K Dixon ◽  
M F Manolson ◽  
R Wright ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Open Reading Frame ◽  
Relative Molecular Mass ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library

Abstract pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

scholarly journals Characterisation of a novel endophyte NRPS gene and its role in endophyte-grass symbioses

2007 ◽  
Vol 13 ◽  
pp. 491-493
Author(s):  
H. Harzer ◽  
R.D. Johnson ◽  
S. Rasmussen ◽  
C.R. Voisey ◽  
L.J. Johnson
Keyword(s):  
Fungal Endophytes ◽  
Gene Clusters ◽  
Gene Replacement ◽  
In Planta ◽  
Dna Library ◽  
Peptide Synthetase ◽  
Genomic Dna Library ◽  
Targeted Gene Replacement ◽  
Fungal Secondary Metabolite

Symbiotic grass associations with fungal endophytes (genera Neotyphodium and Epichloë) display enhanced fitness as well as prolonged field persistence over their endophyte free equivalents. Perennial ryegrass, an important agronomic grass, is typically associated with the N. lolii endophyte. The endophyte lives within the intercellular spaces without inducing any symptoms in the plant. The aim of this study is to elucidate the biosynthetic function of fungal secondary metabolite gene clusters. Non-ribosomal peptide synthetase genes (NRPSs) of unknown function were targeted, as these genes are commonly associated with the production of bioactive peptides some of which are ecologically important. Some novel endophyte NRPS genes have been identified using a degenerate PCR screen; one of these, NRPS5 will be discussed here. Clones were obtained by screening a fosmid Epichloë festucae genomic DNA library and we are currently determining gene function by using targeted gene replacement followed by an assessment in vitro and in planta using metabolomics and appropriate bioassay screens. Keywords: endophyte, NRPS, secondary metabolism

The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein

1988 ◽  
Vol 8 (3) ◽  
pp. 1282-1289
Author(s):  
W Haggren ◽  
D Kolodrubetz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Mobility ◽  
Hmg Proteins ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Library ◽  
Associated Proteins ◽  
Genomic Dna Library

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

scholarly journals The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases.

1986 ◽  
Vol 6 (7) ◽  
pp. 2500-2510 ◽  
Author(s):  
C A Woolford ◽  
L B Daniels ◽  
F J Park ◽  
E W Jones ◽  
J N Van Arsdell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Predicted Amino Acid Sequence ◽  
Posttranslational Regulation ◽  
Aspartyl Protease ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Pep4 Gene ◽  
Precursor Molecules ◽  
Recombination Experiments

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

scholarly journals BFR1, a multicopy suppressor of brefeldin A-induced lethality, is implicated in secretion and nuclear segregation in Saccharomyces cerevisiae.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 423-437 ◽  
Author(s):  
C L Jackson ◽  
F Képès
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Brefeldin A ◽  
Secretory Proteins ◽  
Multicopy Suppressor ◽  
Dna Library ◽  
Golgi Membranes ◽  
Multicopy Suppressors ◽  
Genomic Dna Library

Abstract Brefeldin A (BFA) blocks protein transport out of the Golgi apparatus and causes disassembly of this organelle in mammalian cells. The primary effect of BFA is the release of the non-clathrin coat from Golgi membranes and vesicles. We sought to elucidate the mechanism of BFA action using a genetic approach in Saccharomyces cerevisiae. When an erg6 S. cerevisiae strain is treated with BFA, cell growth is arrested, cells lose viability and secretory proteins are accumulated in the endoplasmic reticulum (ER) and early Golgi compartments. We demonstrate that the mutant sec21 (defective in the S. cerevisiae homolog of gamma-COP, a non-clathrin coat protein) is supersensitive to BFA. Hence BFA probably affects the same processes in S. cerevisiae as in mammalian cells. We used a multicopy genomic DNA library to search for multicopy suppressors of BFA-induced lethality. We identified one such gene, BFR1, that, in addition, partially suppresses the growth and secretion defects of the ER-to-Golgi secretion mutant sec17. A bfr1-delta 1::URA3 deletion strain is viable, but has defects in cell morphology and nuclear segregation, and the mutation accentuates the growth and secretion defects of a sec21 mutant.

The PEP4 gene encodes an aspartyl protease implicated in the posttranslational regulation of Saccharomyces cerevisiae vacuolar hydrolases

1986 ◽  
Vol 6 (7) ◽  
pp. 2500-2510
Author(s):  
C A Woolford ◽  
L B Daniels ◽  
F J Park ◽  
E W Jones ◽  
J N Van Arsdell ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Predicted Amino Acid Sequence ◽  
Posttranslational Regulation ◽  
Aspartyl Protease ◽  
Dna Library ◽  
Genomic Dna Library ◽  
Pep4 Gene ◽  
Precursor Molecules ◽  
Recombination Experiments

pep4 mutants of Saccharomyces cerevisiae accumulate inactive precursors of vacuolar hydrolases. The PEP4 gene was isolated from a genomic DNA library by complementation of the pep4-3 mutation. Deletion analysis localized the complementing activity to a 1.5-kilobase pair EcoRI-XhoI restriction enzyme fragment. This fragment was used to identify an 1,800-nucleotide mRNA capable of directing the synthesis of a 44,000-dalton polypeptide. Southern blot analysis of yeast genomic DNA showed that the PEP4 gene is unique; however, several related sequences exist in yeasts. Tetrad analysis and mitotic recombination experiments localized the PEP4 gene proximal to GAL4 on chromosome XVI. Analysis of the DNA sequence indicated that PEP4 encodes a polypeptide with extensive homology to the aspartyl protease family. A comparison of the PEP4 predicted amino acid sequence with the yeast protease A protein sequence revealed that the two genes are, in fact, identical (see also Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986). Based on our observations, we propose a model whereby inactive precursor molecules produced from the PEP4 gene self-activate within the yeast vacuole and subsequently activate other vacuolar hydrolases.

scholarly journals Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome.

1986 ◽  
Vol 6 (12) ◽  
pp. 4516-4525 ◽  
Author(s):  
K G Coleman ◽  
H Y Steensma ◽  
D B Kaback ◽  
J R Pringle
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Cloning ◽  
Genomic Dna ◽  
Shuttle Vector ◽  
Blot Hybridization ◽  
Yeast Genome ◽  
Dna Library ◽  
Isolation And Characterization ◽  
Genomic Dna Library ◽  
Chromosome I

Molecular cloning techniques were used to isolate and characterize the DNA including and surrounding the CDC24 and PYK1 genes on the left arm of chromosome I of the yeast Saccharomyces cerevisiae. A plasmid that complemented a temperature-sensitive cdc24 mutation was isolated from a yeast genomic DNA library in a shuttle vector. Plasmids containing pyk1-complementing DNA were obtained from other investigators. Several lines of evidence (including one-step gene replacement experiments) demonstrated that the complementing plasmids contained the bona fide CDC24 and PYK1 genes. These sequences were then used to isolate additional DNA from chromosome I by probing a yeast genomic DNA library in a lambda vector. A total of 28 kilobases (kb) of contiguous DNA surrounding the CDC24 and PYK1 genes was isolated, and a restriction map was determined. Electron microscopy of R-loop-containing DNA and RNA blot hybridization analyses indicated that an 18-kb segment contained at least seven transcribed regions, only three of which corresponded to previously known genes (CDC24, PYK1, and CYC3). Southern blot hybridization experiments suggested that none of the genes in this region was duplicated elsewhere in the yeast genome. The centers of CDC24 and PYK1 were only approximately 7.5 kb apart, although the genetic map distance between them is approximately 13 centimorgans. As previous studies with S. cerevisiae have indicated that 1 centimorgan generally corresponds to approximately 3 kb, the region between CDC24 and PYK1 appears to undergo meiotic recombination at an unusually high frequency.

scholarly journals The Saccharomyces cerevisiae ACP2 gene encodes an essential HMG1-like protein.

1988 ◽  
Vol 8 (3) ◽  
pp. 1282-1289 ◽  
Author(s):  
W Haggren ◽  
D Kolodrubetz
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
High Mobility ◽  
Hmg Proteins ◽  
Gene Encoding ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Library ◽  
Associated Proteins ◽  
Genomic Dna Library

The high-mobility-group (HMG) proteins, a group of nonhistone chromatin-associated proteins, have been extensively characterized in higher eucaryotic cells. To test the biological function of an HMG protein, we have cloned and mutagenized a gene encoding an HMG-like protein from the yeast Saccharomyces cerevisiae. A yeast genomic DNA library was screened with an oligonucleotide designed to hybridize to any yeast gene containing an amino acid sequence conserved in several higher eucaryotic HMG proteins. DNA sequencing and Northern (RNA) blot analysis revealed that one gene, called ACP2 (acidic protein 2), synthesizes a poly(A)+ RNA in S. cerevisiae which encodes a 27,000-molecular-weight protein whose amino acid sequence is homologous to those of calf HMG1 and HMG2 and trout HMGT proteins. Standard procedures were used to construct a diploid yeast strain in which one copy of the ACP2 gene was mutated by replacement with the URA3 gene. When this diploid was sporulated and dissected, only half of the spores were viable. About half of the nonviable spores proceeded through two or three cell divisions and then stopped dividing; the rest did not germinate at all. None of the viable spores contained the mutant ACP2 gene, thus proving that the protein encoded by ACP2 is required for cell viability. The results presented here demonstrate that an HMG-like protein has an essential physiological function.

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