Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene

1986 ◽  
Vol 6 (7) ◽  
pp. 2638-2645 ◽  
Author(s):  
S S Wang ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxidase Activity ◽  
Single Copy ◽  
Yeast Genome ◽  
Mrna Levels ◽  
Proline Oxidase ◽  
S1 Nuclease ◽  
Genomic Libraries ◽  
Proline Utilization ◽  
Bona Fide

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

scholarly journals Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT1 gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2638-2645 ◽  
Author(s):  
S S Wang ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxidase Activity ◽  
Single Copy ◽  
Yeast Genome ◽  
Mrna Levels ◽  
Proline Oxidase ◽  
S1 Nuclease ◽  
Genomic Libraries ◽  
Proline Utilization ◽  
Bona Fide

The PUT1 gene was isolated by functional complementation of a put1 (proline oxidase-deficient) mutation in Saccharomyces cerevisiae. Three independent clones with overlapping inserts of 6.8, 10.5, and 11 kilobases (kb) were isolated from S. cerevisiae genomic libraries in YEp24 (2 micron) and YCp50 (CEN) plasmids. The identity of the PUT1 gene was determined by a gene disruption technique, and Southern hybridization and genetic analyses confirmed that the bona fide gene had been cloned. Plasmids containing the PUT1 gene restored regulated levels of proline oxidase activity to put1 recipient strains. The PUT1 DNA was present in a single copy in the yeast genome and encoded a transcript of ca. 1.5 kb. S1 nuclease protection experiments were used to determine the direction of transcription of the PUT1 message and to localize its 5' and 3' termini within a subcloned 3-kb DNA fragment. Approximately 50-fold more PUT1-specific mRNA was detected in induced (proline-grown) cells than in uninduced (ammonia-grown) cells. A yeast strain carrying the previously identified put3 regulatory mutation that caused constitutive levels of proline oxidase activity was found to have sevenfold elevated PUT1 mRNA levels under noninducing conditions. The absence of a functional electron transport system in vegetative petite (rho-) strains interfered with their ability to use proline as a nitrogen source. Although these strains were Put- and made no detectable proline oxidase activity, PUT1 message was detected under inducing conditions. The PUT1 gene was mapped distal to the GAL2 gene on chromosome XII by tetrad analysis.

Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene

1983 ◽  
Vol 3 (10) ◽  
pp. 1846-1856
Author(s):  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Fold Increase ◽  
Activity Levels ◽  
Mrna Levels ◽  
S1 Nuclease ◽  
Proline Utilization ◽  
Cloned Gene ◽  
His3 Gene ◽  
Specific Mrna

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment. When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII.

scholarly journals Proline utilization in Saccharomyces cerevisiae: analysis of the cloned PUT2 gene.

1983 ◽  
Vol 3 (10) ◽  
pp. 1846-1856 ◽  
Author(s):  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Recombinant Dna ◽  
Fold Increase ◽  
Activity Levels ◽  
Mrna Levels ◽  
S1 Nuclease ◽  
Proline Utilization ◽  
Cloned Gene ◽  
His3 Gene ◽  
Specific Mrna

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (delta 1-pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the put2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in S1 nuclease protection experiments to locate the gene to a 3-kilobase HindIII fragment. When delta 1-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2 mu-containing vector was used to map the PUT2 gene to chromosome VIII.

scholarly journals Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution.

Genetics ◽  
1991 ◽  
Vol 127 (4) ◽  
pp. 681-698 ◽  
Author(s):  
A J Link ◽  
M V Olson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Physical Map ◽  
Single Copy ◽  
Restriction Endonucleases ◽  
Yeast Genome ◽  
Hybridization Probes ◽  
Copy Dna ◽  
Single Copy Dna

Abstract A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

scholarly journals Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435 ◽  
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

Specific transcripts are elevated in Saccharomyces cerevisiae in response to DNA damage

1984 ◽  
Vol 4 (11) ◽  
pp. 2356-2363
Author(s):  
T McClanahan ◽  
K McEntee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Southern Hybridization ◽  
Single Copy ◽  
Strand Break ◽  
Yeast Genome ◽  
Dna Strand Break ◽  
Isolation And Characterization ◽  
Dna Strand

Differential hybridization has been used to identify genes in Saccharomyces cerevisiae displaying increased transcript levels after treatment of cells with UV irradiation or with the mutagen/carcinogen 4-nitroquinoline-1-oxide (NQO). We describe the isolation and characterization of four DNA damage responsive genes obtained from screening ca. 9,000 yeast genomic clones. Two of these clones, lambda 78A and pBR178C, contain repetitive elements in the yeast genome as shown by Southern hybridization analysis. Although the genomic hybridization pattern is distinct for each of these two clones, both of these sequences hybridize to large polyadenylated transcripts ca. 5 kilobases in length. Two other DNA damage responsive sequences, pBRA2 and pBR3016B, are single-copy genes and hybridize to 0.5- and 3.2-kilobase transcripts, respectively. Kinetic analysis of the 0.5-kilobase transcript homologous to pBRA2 indicates that the level of this RNA increases more than 15-fold within 20 min after exposure to 4-nitroquinoline-1-oxide. Moreover, the level of this transcript is significantly elevated in cells containing the rad52-1 mutation which are deficient in DNA strand break repair and gene conversion. These results provide some of the first evidence that DNA damage stimulates transcription of specific genes in eucaryotic cells.

Transcriptional regulation of ribosomal proteins during a nutritional upshift in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (7) ◽  
pp. 2429-2435
Author(s):  
D M Donovan ◽  
N J Pearson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Proteins ◽  
Protein Gene ◽  
Relative Rate ◽  
Ribosomal Protein Gene ◽  
Single Copy ◽  
Protein Product ◽  
Yeast Genome ◽  
Base Pairs ◽  
Beta Galactosidase

The relative rates of synthesis of Saccharomyces cerevisiae ribosomal proteins increase coordinately during a nutritional upshift. We constructed a gene fusion which contained 528 base pairs of sequence upstream from and including the TATA box of ribosomal protein gene rp55-1 (S16A-1) fused to a CYC1-lacZ fusion. This fusion was integrated in single copy at the rp55-1 locus in the yeast genome. During a nutritional upshift, in which glucose was added to cells growing in an ethanol-based medium, we found that the increase in the relative rate of synthesis of the beta-galactosidase protein product followed the same kinetics as the change in relative rates of synthesis of several ribosomal proteins measured in the same experiment. This demonstrates that the nontranscribed sequences upstream from the rp55-1 gene, which are present in the fusion, are sufficient to mediate the change in rates of synthesis characteristic of ribosomal proteins under these conditions. The results also suggest that a change in transcription rates is mainly responsible for the increase in relative rates of synthesis of ribosomal proteins during a nutritional upshift in S. cerevisiae.

Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae

1984 ◽  
Vol 4 (11) ◽  
pp. 2396-2405
Author(s):  
R L Last ◽  
J B Stavenhagen ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Copy ◽  
Yeast Genome ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Mrna Accumulation ◽  
Isolation And Characterization ◽  
Polyadenylated Rna

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

Characterization of two abundantly expressed constitutive genes of Neurospora crassa

1987 ◽  
Vol 33 (3) ◽  
pp. 199-204
Author(s):  
Ronald L. Frank ◽  
George A. Marzluf
Keyword(s):  
Neurospora Crassa ◽  
Dna Sequences ◽  
Blot Analysis ◽  
Detailed Comparison ◽  
Single Copy ◽  
Conclusive Evidence ◽  
Dot Blot ◽  
S1 Nuclease ◽  
Genomic Libraries ◽  
Nuclease Mapping

Two abundantly expressed, constitutive genes of Neurospora crassa were isolated during differential screening of Neurospora genomic libraries. The coding regions of these two genes, designated RLF1 and RLF3, were identified by hybridization of the cloned DNA sequences with cDNA probes made from polyadenylated RNA. The RLF3 gene was carried on a 15-kilobase Neurospora BamHI DNA fragment present in a lambda 1059 recombinant; a 2-kilobase restriction fragment that contains RLF3 was subcloned into plasmid pBR322 prior to further characterization. Southern blot analysis revealed that both RLF1 and RLF3 are single copy genes. Northern blot analysis and S1 nuclease mapping demonstrated that RLF1 is transcribed to yield a 1.6-kilobase RNA, whereas RLF3 appears to give rise to two distinct sized RNA species of 1.0 and 1.6 kilobases. RNA dot blot analysis provided conclusive evidence that both of these genes are constitutively expressed. These constitutive genes will be valuable to provide a detailed comparison with the 5′ flanking regions of regulated genes.

scholarly journals The Formation of Neochromosomes during Experimental Evolution in the Yeast Saccharomyces cerevisiae

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1678
Author(s):  
Agnès Thierry ◽  
Varun Khanna ◽  
Bernard Dujon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Single Copy ◽  
Trna Synthetase ◽  
Repeated Sequences ◽  
Yeast Genome ◽  
Template Switching ◽  
Yeast Saccharomyces Cerevisiae ◽  
Engineered Saccharomyces Cerevisiae ◽  
Diploid Cells

Novel, large-scale structural mutations were previously discovered during the cultivation of engineered Saccharomyces cerevisiae strains in which essential tRNA synthetase genes were replaced by their orthologs from the distantly related yeast Yarrowia lipolytica. Among those were internal segmental amplifications forming giant chromosomes as well as complex segmental rearrangements associated with massive amplifications at an unselected short locus. The formation of such novel structures, whose stability is high enough to propagate over multiple generations, involved short repeated sequences dispersed in the genome (as expected), but also novel junctions between unrelated sequences likely triggered by accidental template switching within replication forks. Using the same evolutionary protocol, we now describe yet another type of major structural mutation in the yeast genome, the formation of neochromosomes, with functional centromeres and telomeres, made of extra copies of very long chromosomal segments ligated together in novel arrangements. The novel junctions occurred between short repeated sequences dispersed in the genome. They first resulted in the formation of an instable neochromosome present in a single copy in the diploid cells, followed by its replacement by a shorter, partially palindromic neochromosome present in two copies, whose stability eventually increased the chromosome number of the diploid strains harboring it.

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