Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector.

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Rearrangements occurring adjacent to a single Ty1 yeast retrotransposon in the presence and absence of full-length Ty1 transcription.

Genetics ◽

10.1093/genetics/131.4.833 ◽

1992 ◽

Vol 131 (4) ◽

pp. 833-850

Author(s):

P R Sutton ◽

S W Liebman

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Transposable Element ◽

Gene Conversion ◽

Genetic Background ◽

Repetitive Sequences ◽

Full Length ◽

Yeast Genome ◽

Yeast Saccharomyces Cerevisiae ◽

Ty1 Retrotransposon

Abstract The structures of two unusual deletions from the yeast Saccharomyces cerevisiae are described. These deletions extend from a single Ty1 retrotransposon to an endpoint near a repetitive tRNA(Gly) gene. The deletions suggest that unique sequences flanked by two nonidentical repetitive sequences, or bordered on only one side by a transposable element, have the potential to be mobilized in the yeast genome. Models for the formation of these two unusual deletions were tested by isolating and analyzing 32 additional unusual deletions of the CYC1 region that extend from a single Ty1 retrotransposon. Unlike the most common class of deletions recovered in this region, these deletions are not attributable solely to homologous recombination among repetitive Ty1 or delta elements. They arose by two distinct mechanisms. In an SPT8 genetic background, most unusual deletions arose by transposition of a Ty1 element to a position adjacent to a tRNA(Gly) gene followed by Ty1-Ty1 recombination. In an spt8 strain, where full-length Ty1 transcription and, therefore, transposition are reduced, most deletions were due to gene conversion of a 7-kb chromosomal interval flanked by a Ty1 element and a tRNA(Gly) gene.

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Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

Journal of Fungi ◽

10.3390/jof7070520 ◽

2021 ◽

Vol 7 (7) ◽

pp. 520

Author(s):

Jianmin Fu ◽

Nohelli E. Brockman ◽

Brian L. Wickes

Keyword(s):

Agrobacterium Tumefaciens ◽

Yeast Cell ◽

Transformation Efficiency ◽

Transformation Frequency ◽

Yeast Cells ◽

Agar Concentration ◽

Genetic Tool ◽

Number Of Factors ◽

Cryptococcus Spp ◽

Transformation Systems

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

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

Genetics ◽

10.1093/genetics/127.4.681 ◽

1991 ◽

Vol 127 (4) ◽

pp. 681-698 ◽

Cited By ~ 6

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.

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A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5130-5143.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5130-5143 ◽

Cited By ~ 32

Author(s):

Christine Soustelle ◽

Laurence Vernis ◽

Karine Fréon ◽

Anne Reynaud-Angelin ◽

Roland Chanet ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Nonhomologous End Joining ◽

End Joining ◽

Recombination Mechanism ◽

Growth Defects ◽

Protein Conjugates ◽

Synthetically Lethal ◽

Mutant Cells ◽

Insight Into

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

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Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae

Mutagenesis ◽

10.1093/mutage/ges069 ◽

2013 ◽

Vol 28 (2) ◽

pp. 187-195 ◽

Cited By ~ 9

Author(s):

L. Spugnesi ◽

C. Balia ◽

A. Collavoli ◽

E. Falaschi ◽

V. Quercioli ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Homologous Recombination ◽

Nuclear Foci

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Synergistic action of the Saccharomyces cerevisiae homologous recombination factors Rad54 and Rad51 in chromatin remodeling

DNA Repair ◽

10.1016/j.dnarep.2007.04.012 ◽

2007 ◽

Vol 6 (10) ◽

pp. 1496-1506 ◽

Cited By ~ 17

Author(s):

YoungHo Kwon ◽

Peter Chi ◽

Dong Hyun Roh ◽

Hannah Klein ◽

Patrick Sung

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Chromatin Remodeling ◽

Synergistic Action

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2429 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2429-2435 ◽

Cited By ~ 34

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.

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CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

Life ◽

10.3390/life11010013 ◽

2020 ◽

Vol 11 (1) ◽

pp. 13

Author(s):

João Rainha ◽

Joana L. Rodrigues ◽

Lígia R. Rodrigues

Keyword(s):

Saccharomyces Cerevisiae ◽

Yeast Genome ◽

Specific Method ◽

Complex Processes ◽

Biological Studies ◽

Precise Tool ◽

Long Time ◽

Gene Knockouts ◽

Heterologous Pathway ◽

A Current

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

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An examination of quinone toxicity using the yeast Saccharomyces cerevisiae model system

Toxicology ◽

10.1016/j.tox.2004.04.016 ◽

2004 ◽

Vol 201 (1-3) ◽

pp. 185-196 ◽

Cited By ~ 65

Author(s):

Chester E Rodriguez ◽

Masaru Shinyashiki ◽

John Froines ◽

Rong Chun Yu ◽

Jon M Fukuto ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Model System ◽

Yeast Saccharomyces Cerevisiae ◽

Quinone Toxicity

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Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.2887-2893.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 2887-2893

Author(s):

M Neitz ◽

J Carbon

Keyword(s):

Saccharomyces Cerevisiae ◽

Restriction Fragment ◽

Dna Sequences ◽

Yeast Genome ◽

Ura3 Gene ◽

Dna Restriction ◽

Meiotic Analyses ◽

The Right ◽

In Cis

A functional centromere located on a small DNA restriction fragment from Saccharomyces cerevisiae was identified as CEN14 by integrating centromere-adjacent DNA plus the URA3 gene by homologous recombination into the yeast genome and then by localizing the URA3 gene to chromosome XIV by standard tetrad analysis. DNA sequence analysis revealed that CEN14 possesses sequences (elements I, II, and III) that are characteristic of other yeast centromeres. Mitotic and meiotic analyses indicated that the CEN14 function resides on a 259-base-pair (bp) RsaI-EcoRV restriction fragment, containing sequences that extend only 27 bp to the right of the element I to III region. In conjunction with previous findings on CEN3 and CEN11, these results indicate that the specific DNA sequences required in cis for yeast centromere function are contained within a region about 150 bp in length.

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