Genetic Modification Stimulated by the Induction of a Site-Specific Break Distant from the Locus of Correction in Haploid and Diploid Yeast Saccharomyces cerevisiae

2014 ◽  
pp. 309-324 ◽  
Author(s):  
Samantha Stuckey ◽  
Francesca Storici
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Modification ◽  
Yeast Saccharomyces Cerevisiae ◽  
Site Specific ◽  
A Site

Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae

1987 ◽  
Vol 7 (6) ◽  
pp. 2087-2096
Author(s):  
B Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Efficient Manner ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Leu2 Gene ◽  
Recombination System ◽  
A Site ◽  
Site Specific Recombination System

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

scholarly journals Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (6) ◽  
pp. 2087-2096 ◽  
Author(s):  
B Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Efficient Manner ◽  
Site Specific ◽  
Site Specific Recombination ◽  
Leu2 Gene ◽  
Recombination System ◽  
A Site ◽  
Site Specific Recombination System

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GALI promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin structure.

The Cloning and Mapping of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 531-540
Author(s):  
Aileen K W Taguchi ◽  
Elton T Young
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Alcohol Dehydrogenase ◽  
Single Gene ◽  
Carbon Sources ◽  
Transcription Unit ◽  
Yeast Cells ◽  
Recessive Mutation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Activating Sequence ◽  
A Site

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

scholarly journals Genomic Considerations for the Modification of Saccharomyces cerevisiae for Biofuel and Metabolite Biosynthesis

2020 ◽  
Vol 8 (3) ◽  
pp. 321 ◽  
Author(s):  
James T. Arnone
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Native Species ◽  
Genetic Manipulation ◽  
Scale Up ◽  
Biosynthetic Pathways ◽  
Yeast Saccharomyces Cerevisiae ◽  
Position Effects ◽  
Industrial Use ◽  
A Site ◽  
Global Population

The growing global population and developing world has put a strain on non-renewable natural resources, such as fuels. The shift to renewable sources will, thus, help meet demands, often through the modification of existing biosynthetic pathways or the introduction of novel pathways into non-native species. There are several useful biosynthetic pathways endogenous to organisms that are not conducive for the scale-up necessary for industrial use. The use of genetic and synthetic biological approaches to engineer these pathways in non-native organisms can help ameliorate these challenges. The budding yeast Saccharomyces cerevisiae offers several advantages for genetic engineering for this purpose due to its widespread use as a model system studied by many researchers. The focus of this review is to present a primer on understanding genomic considerations prior to genetic modification and manipulation of S. cerevisiae. The choice of a site for genetic manipulation can have broad implications on transcription throughout a region and this review will present the current understanding of position effects on transcription.

scholarly journals The Hrq1 helicase stimulates Pso2 translesion nuclease activity to promote DNA inter-strand crosslink repair

2019 ◽  
Author(s):  
Cody M. Rogers ◽  
Chun-Ying Lee ◽  
Samuel Parkins ◽  
Nicholas J. Buehler ◽  
Sabine Wenzel ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage Response ◽  
Nuclease Activity ◽  
Site Specific ◽  
Damage Response ◽  
Physical Barriers ◽  
A Site ◽  
Translesion Polymerases ◽  
Stimulation Of

AbstractDNA inter-strand crosslink (ICL) repair requires a complicated network of DNA damage response pathways. Removal of these lesions is vital as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principle mechanism for ICL repair in metazoans and is coupled to replication. In Saccharomyces cerevisiae, a degenerate FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease that is hypothesized to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked RECQL4, as a novel component of Pso2- mediated ICL repair. Here, we show that Hrq1 stimulates the Pso2 nuclease in a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulates Pso2 translesion nuclease activity through a site- specific ICL in vitro. Stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these dangerous lesions.

scholarly journals A Site-Specific Genetic Modification for Induction of Pluripotency and Subsequent Isolation of Derived Lung Alveolar Epithelial Type II Cells

Stem Cells ◽  
2014 ◽  
Vol 32 (2) ◽  
pp. 402-413 ◽  
Author(s):  
Qing Yan ◽  
Yuan Quan ◽  
Huanhuan Sun ◽  
Xinmiao Peng ◽  
Zhengyun Zou ◽  
...  
Keyword(s):  
Genetic Modification ◽  
Type Ii Cells ◽  
Type Ii ◽  
Site Specific ◽  
Alveolar Epithelial ◽  
A Site

Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

1993 ◽  
Vol 13 (12) ◽  
pp. 7836-7849
Author(s):  
P Russo ◽  
W Z Li ◽  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Site Directed Mutagenesis ◽  
Yeast Cells ◽  
In Vitro Mutagenesis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Upstream Element ◽  
A Site ◽  
Derivatives Of ◽  
Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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