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1
Losson, R., R. P. P. Fuchs i F. Lacroute. "Yeast promoters URA1 and URA3". Journal of Molecular Biology 185, nr 1 (wrzesień 1985): 65–81. http://dx.doi.org/10.1016/0022-2836(85)90183-4.
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Roy, A., F. Exinger i R. Losson. "cis- and trans-acting regulatory elements of the yeast URA3 promoter". Molecular and Cellular Biology 10, nr 10 (październik 1990): 5257–70. http://dx.doi.org/10.1128/mcb.10.10.5257-5270.1990.
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Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.
3
Roy, A., F. Exinger i R. Losson. "cis- and trans-acting regulatory elements of the yeast URA3 promoter." Molecular and Cellular Biology 10, nr 10 (październik 1990): 5257–70. http://dx.doi.org/10.1128/mcb.10.10.5257.
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Expression of the yeast pyrimidine biosynthetic gene, URA3, is induced three- to fivefold in response to uracil starvation, and this regulation is mediated by the transcriptional activator PPR1 (pyrimidine pathway regulator 1). In this study, we have analyzed the regulatory elements of the URA3 promoter by DNase I footprinting, using partially purified yeast cell extracts, by deletion mutagenesis, and by 5'-end mapping of RNA transcripts. Two DNA-binding activities have been detected, and at least four distinct cis-acting regions have been identified. A region rich in poly(dA-dT) serves as an upstream promoter element necessary for the basal level of URA3 expression. A 16-base-pair sequence with dyad symmetry acts acts as a uracil-controlled upstream activating site (UASURA) and shows a specific binding only with cell extracts from strains overproducing PPR1. This in vitro binding does not require dihydroorotic acid, the physiological inducer of URA3. The TATA region appears to be composed of two functionally distinct (constitutive and regulatory) elements. Two G + A-rich regions surrounding this TATA box bind an unidentified factor called GA-binding factor. The 5' copy, GA1, is involved in PPR1 induction and overlaps the constitutive TATA region. The 3' region, GA2, is necessary for maximal expression. Neither of these GA sequences acts as a UAS in a CYC1-lacZ context. The promoters of the unlinked but coordinately regulated URA1 and URA4 genes contain highly conserved copies of the UASURA sequence, which prompted us to investigate the effects of many point mutations within this UASURA sequence on PPR1-dependent binding. In this way, we have identified the most important residues of this binding site and found that a nonsymmetrical change of these bases is sufficient to prevent the specific binding and to suppress the UASURA activity in vivo. In addition, we showed that UASURA contains a constitutive activating element which can stimulate transcription from a heterologous promoter independently of dihydroorotic acid and PPR1.
4
Denis-Duphil, Michèle. "Pyrimidine biosynthesis in Saccharomyces cerevisiae: the ura2 cluster gene, its multifunctional enzyme product, and other structural or regulatory genes involved in de novo UMP synthesis". Biochemistry and Cell Biology 67, nr 9 (1.09.1989): 612–31. http://dx.doi.org/10.1139/o89-094.
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There are six enzymatic steps in the de novo biosynthesis of uridine monophosphate (UMP). In yeast, six structural genes (ura2, ura4, ura1, ura5, ura10, and ura3) and one regulatory gene (PPR1) are involved in this metabolic pathway. Gene ura2 codes for a multifunctional protein that carries the first two enzymatic activities of the pathway, i.e., carbamylphosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Gene ura2 has been cloned and sequenced, revealing the presence of three open reading frames, one of which codes for the multifunctional protein, a polypeptide of 2212 amino acids, with a mRNA of 7 ± 0.3 kilobases. Expression of gene ura2 is regulated at the transcriptional level. As I indicate here, it could also be controlled at the posttranscriptional level since all the consensus sequences for a 1.2-kilobases intron are present in the coding sequence of the open reading frame. The deducted amino acid sequence has allowed the identification of four domains. Starting from the amino terminus of the protein, these are glutamine amido transferase, CPSase, a domain that resembles dihydroorotase (DHOase-like) but does not have DHOase activity, and ATCase. There are also two sites of interest that match known concensus phosphorylation sites; one is located in the distal part of the CPSase domain, the other in the connector region between DHOase-like and ATCase domains. The protein has been purified as a multienzyme aggregate and as a multifunctional protein. The latter form, when isolated from a protease B deficient strain of Saccharomyces cerevisiae, contained mostly polypeptide chains of 220 kilodaltons. Work is currently in progress to determine the site(s) of phosphorylation of this protein in vitro. ATCase activity of both wild-type and protease-deficient strains has been found to be localized in the nucleus. Channeling of carbamyl phosphate, the first intermediate in the pathway, has been demonstrated both in vitro and in permeabilized cells. The other genes of UMP biosynthesis, except for ura5, are regulated by induction of their transcription by the combined action of the product of the ppr1 gene and the inducer, dihydroorotate. Dihydroorotate dehydrogenase activity was found in the cytoplasm. Two isoenzymes of orotate phosphoribosyl transferase have been found, coded for by ura5 and ura10. The products of genes ura10 and ura3 are proposed to participate in the channeling of orotidine monophosphate. The discussion considers the problem posed by the isolation of both multienzyme complexes and multifunctional proteins resulting from the expression of the same cluster genes. I suggest that regulation by processing at the posttranscriptional and posttranslational levels could be regarded as an alternative explanation for these observations, which were previously explained in terms of proteolysis.Key words: yeast, pyrimidines, multifunctional enzyme, phosphorylation, proteolysis.
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Horowitz, H., i J. E. Haber. "Identification of autonomously replicating circular subtelomeric Y' elements in Saccharomyces cerevisiae". Molecular and Cellular Biology 5, nr 9 (wrzesień 1985): 2369–80. http://dx.doi.org/10.1128/mcb.5.9.2369-2380.1985.
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We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.
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Horowitz, H., i J. E. Haber. "Identification of autonomously replicating circular subtelomeric Y' elements in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, nr 9 (wrzesień 1985): 2369–80. http://dx.doi.org/10.1128/mcb.5.9.2369.
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We marked a large number of yeast telomeres within their Y' regions by transforming strains with a fragment of Y' DNA into which the URA3 gene had been inserted. A few of the Ura+ transformants obtained were very unstable and were found to contain autonomously replicating URA3-marked circular Y' elements in high copy number. These marked extrachromosomal circles were capable of reintegrating into the chromosome at other telomeric locations. In contrast, most of the Ura+ transformants obtained were quite stable mitotically and were marked at bona fide chromosomal ends. These stable transformants gave rise to mitotically unstable URA3-marked circular Y' elements at a low frequency (up to 2.5%). The likelihood that such excisions and integrations represent a natural process in Saccharomyces cerevisiae is supported by our identification of putative Y' circles in untransformed strains. The transfer of Y' information among telomeres via a circular intermediate may be important for homogenizing the sequences at the ends of yeast chromosomes and for generating the frequent telomeric rearrangements that have been observed in S. cerevisiae.
7
Kiktev, Denis A., Ziwei Sheng, Kirill S. Lobachev i Thomas D. Petes. "GC content elevates mutation and recombination rates in the yeast Saccharomyces cerevisiae". Proceedings of the National Academy of Sciences 115, nr 30 (9.07.2018): E7109—E7118. http://dx.doi.org/10.1073/pnas.1807334115.
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The chromosomes of many eukaryotes have regions of high GC content interspersed with regions of low GC content. In the yeast Saccharomyces cerevisiae, high-GC regions are often associated with high levels of meiotic recombination. In this study, we constructed URA3 genes that differ substantially in their base composition [URA3-AT (31% GC), URA3-WT (43% GC), and URA3-GC (63% GC)] but encode proteins with the same amino acid sequence. The strain with URA3-GC had an approximately sevenfold elevated rate of ura3 mutations compared with the strains with URA3-WT or URA3-AT. About half of these mutations were single-base substitutions and were dependent on the error-prone DNA polymerase ζ. About 30% were deletions or duplications between short (5–10 base) direct repeats resulting from DNA polymerase slippage. The URA3-GC gene also had elevated rates of meiotic and mitotic recombination relative to the URA3-AT or URA3-WT genes. Thus, base composition has a substantial effect on the basic parameters of genome stability and evolution.
8
Voelkel-Meiman, K., i G. S. Roeder. "Gene conversion tracts stimulated by HOT1-promoted transcription are long and continuous." Genetics 126, nr 4 (1.12.1990): 851–67. http://dx.doi.org/10.1093/genetics/126.4.851.
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Abstract The recombination-stimulating sequence, HOT1, corresponds to the promoter of transcription by yeast RNA polymerase I. The effect of HOT1 on mitotic interchromosomal recombination was examined in diploid strains carrying a heterozygous URA3 gene on chromosome III. The frequency of Ura- recombinants was increased 20-fold when HOT1 was inserted into the chromosome III copy marked with URA3, at a location 48 kbp centromere-proximal to URA3. Ura- recombinants were increased only 2-fold when HOT1 and URA3 were on opposite homologues. These results suggest that most HOT1-promoted Ura- recombinants result from gene conversion and that sequences on the HOT1-containing chromosome are preferentially converted. Characterization of Ura- recombinants isolated from strains carrying multiple markers on chromosome III indicates that HOT1-promoted gene conversion tracts are unusually long (often greater than 75 kbp) and almost always continuous. Furthermore, conversion tracts frequently extend to both sides of HOT1. We suggest that HOT1 promotes the formation of a double-strand break which is often followed by exonucleolytic digestion. Repair of the broken chromosome could then result from gap repair or from replicative repair primed only by the centromere-containing chromosomal fragment.
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Puig, Sergi, Amparo Querol, Eladio Barrio i JoséE Pérez-Ortín. "Mitotic Recombination and Genetic Changes inSaccharomyces cerevisiae during Wine Fermentation". Applied and Environmental Microbiology 66, nr 5 (1.05.2000): 2057–61. http://dx.doi.org/10.1128/aem.66.5.2057-2061.2000.
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ABSTRACT Natural strains of Saccharomyces cerevisiae are prototrophic homothallic yeasts that sporulate poorly, are often heterozygous, and may be aneuploid. This genomic constitution may confer selective advantages in some environments. Different mechanisms of recombination, such as meiosis or mitotic rearrangement of chromosomes, have been proposed for wine strains. We studied the stability of the URA3 locus of a URA3/ura3 wine yeast in consecutive grape must fermentations. ura3/ura3homozygotes were detected at a rate of 1 × 10−5 to 3 × 10−5 per generation, and mitotic rearrangements for chromosomes VIII and XII appeared after 30 mitotic divisions. We used the karyotype as a meiotic marker and determined that sporulation was not involved in this process. Thus, we propose a hypothesis for the genome changes in wine yeasts during vinification. This putative mechanism involves mitotic recombination between homologous sequences and does not necessarily imply meiosis.
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Huang, Hanhua, Joo Yun Hong, Carol L. Burck i Susan W. Liebman. "Host Genes That Affect the Target-Site Distribution of the Yeast Retrotransposon Ty1". Genetics 151, nr 4 (1.04.1999): 1393–407. http://dx.doi.org/10.1093/genetics/151.4.1393.
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Abstract We report here a simple genetic system for investigating factors affecting Ty1 target-site preference within an RNAP II transcribed gene. The target in this system is a functional fusion of the regulatable MET3 promoter with the URA3 gene. We found that the simultaneous inactivation of Hir3 (a histone transcription regulator) and Cac3 (a subunit of the chromatin assembly factor I), which was previously shown by us to increase the Ty1 transposition rate, eliminated the normally observed bias for Ty1 elements to insert into the 5′ vs. 3′ regions of the MET3-URA3 and CAN1 genes. The double cac3 hir3 mutation also caused the production of a short transcript from the MET3-URA3 fusion under both repressed and derepressed conditions. In a hir3Δ single-mutant strain, the Ty1 target-site distribution into MET3-URA3 was altered only when transposition occurred while the MET3-URA3 fusion was actively transcribed. In contrast, transcription of the MET3-URA3 fusion did not alter the Ty1 target-site distribution in wild-type or other mutant strains. Deletion of RAD6 was shown to alter the Ty1 target-site preference in the MET3-URA3 fusion and the LYS2 gene. These data, together with previous studies of Ty1 integration positions at CAN1 and SUP4, indicate that the rad6 effect on Ty1 target-site selection is not gene specific.
11
Sikorski, R. S., i P. Hieter. "A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae." Genetics 122, nr 1 (1.05.1989): 19–27. http://dx.doi.org/10.1093/genetics/122.1.19.
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Abstract A series of yeast shuttle vectors and host strains has been created to allow more efficient manipulation of DNA in Saccharomyces cerevisiae. Transplacement vectors were constructed and used to derive yeast strains containing nonreverting his3, trp1, leu2 and ura3 mutations. A set of YCp and YIp vectors (pRS series) was then made based on the backbone of the multipurpose plasmid pBLUESCRIPT. These pRS vectors are all uniform in structure and differ only in the yeast selectable marker gene used (HIS3, TRP1, LEU2 and URA3). They possess all of the attributes of pBLUESCRIPT and several yeast-specific features as well. Using a pRS vector, one can perform most standard DNA manipulations in the same plasmid that is introduced into yeast.
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Alani, Eric, i Nancy Kleckner. "A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast". Genetics 117, nr 1 (1.09.1987): 5–12. http://dx.doi.org/10.1093/genetics/117.1.5.
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ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.
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Garcia-Ruiz, Hernan, i Paul Ahlquist. "Inducible Yeast System for Viral RNA Recombination Reveals Requirement for an RNA Replication Signal on Both Parental RNAs". Journal of Virology 80, nr 17 (1.09.2006): 8316–28. http://dx.doi.org/10.1128/jvi.01790-05.
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ABSTRACT To facilitate RNA recombination studies, we tested whether Saccharomyces cerevisiae, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BMV RNA replication proteins 1a and 2apol were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Δ3′ lacked the coat protein gene and negative-strand RNA promoter. B3Δ5′ lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable URA3 gene. After 12 to 72 h of induction, B3Δ3′ and B3Δ5′ transcription was repressed and Ura+ yeast cells were selected. All Ura+ cells contained recombinant RNA3 replicons expressing URA3. Most replicons arose by intermolecular homologous recombination between B3Δ3′ and B3Δ5′. Such recombinants were isolated only when 1a and 2apol were expressed and after transient transcription of both B3Δ3′ and B3Δ5′, showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3Δ5′, independently of B3Δ3′, by 5′ truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Δ3′-B3Δ5′ recombination occurred only when both parental RNAs bore a functional, cis-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.
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Alani, Eric, Liang Cao i Nancy Kleckner. "A Method for Gene Disruption That Allows Repeated Use of URA3 Selection in the Construction of Multiply Disrupted Yeast Strains". Genetics 116, nr 4 (1.08.1987): 541–45. http://dx.doi.org/10.1093/genetics/116.4.541.
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Abstract In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura+. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10-4) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura+ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.
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Ishikawa, Masayuki, Michael Janda i Paul Ahlquist. "The 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus RNA replicons in yeast but retained over 1045-fold amplification". Journal of General Virology 81, nr 9 (1.09.2000): 2307–11. http://dx.doi.org/10.1099/0022-1317-81-9-2307.
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In yeast expressing the RNA replication proteins encoded by brome mosaic virus (BMV), B3URA3, a BMV RNA3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene URA3, was replicated and maintained in 85–95% of progeny at each cell division. Transmission of the B3URA3 RNA replicon from mother to daughter yeast did not require the 3a gene. Nevertheless, even after passaging for 165 cycles of RNA replication and yeast cell division, each of 40 independent Ura+ colonies tested retained B3URA3 RNAs whose electrophoretic mobilities and accumulation levels were indistinguishable from those of the original B3URA3. These and other results suggest that unselected genes in many positive-strand RNA virus replicons can be stably retained if the presence of the gene does not confer a selective disadvantage in RNA replication.
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Neitz, M., i J. Carbon. "Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae". Molecular and Cellular Biology 5, nr 11 (listopad 1985): 2887–93. http://dx.doi.org/10.1128/mcb.5.11.2887-2893.1985.
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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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Neitz, M., i J. Carbon. "Identification and characterization of the centromere from chromosome XIV in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, nr 11 (listopad 1985): 2887–93. http://dx.doi.org/10.1128/mcb.5.11.2887.
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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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Cormack, Brendan P., i Stanley Falkow. "Efficient Homologous and Illegitimate Recombination in the Opportunistic Yeast Pathogen Candida glabrata". Genetics 151, nr 3 (1.03.1999): 979–87. http://dx.doi.org/10.1093/genetics/151.3.979.
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Abstract The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.
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Cook, Jeffry R., Stuart L. Emanuel i Sidney Pestka. "Yeast artificial chromosome fragmentation vectors that utilize URA3 selection". Genetic Analysis: Biomolecular Engineering 10, nr 5 (styczeń 1993): 109–12. http://dx.doi.org/10.1016/1050-3862(93)90033-f.
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McNabb, David S., Robin Reed i Robert A. Marciniak. "Dual Luciferase Assay System for Rapid Assessment of Gene Expression in Saccharomyces cerevisiae". Eukaryotic Cell 4, nr 9 (wrzesień 2005): 1539–49. http://dx.doi.org/10.1128/ec.4.9.1539-1549.2005.
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ABSTRACT A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multicloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.
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Erickson, J. R., i M. Johnston. "Direct cloning of yeast genes from an ordered set of lambda clones in Saccharomyces cerevisiae by recombination in vivo." Genetics 134, nr 1 (1.05.1993): 151–57. http://dx.doi.org/10.1093/genetics/134.1.151.
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Abstract We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Escherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura+ transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.
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Huisman, Olivier, Wendy Raymond, Kai-Uwe Froehlich, Patrick Errada, Nancy Kleckner, David Botstein i M. Andrew Hoyt. "A Tn 10-lacZ-kanR-URA3 Gene Fusion Transposon for Insertion Mutagenesis and Fusion Analysis of Yeast and Bacterial Genes". Genetics 116, nr 2 (1.06.1987): 191–99. http://dx.doi.org/10.1093/genetics/116.2.191.
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ABSTRACT We describe here a new variant of transposon Tn 10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn 10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn 10-LUK system has several important advantages. (1) Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. (2) Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. (3) The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. (4) Under favorable circumstances, usable lacZ protein fusions are created. (5) Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.
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Jinks-Robertson, Sue, Shariq Sayeed i Tamara Murphy. "Meiotic Crossing Over Between Nonhomologous Chromosomes Affects Chromosome Segregation in Yeast". Genetics 146, nr 1 (1.05.1997): 69–78. http://dx.doi.org/10.1093/genetics/146.1.69.
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Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover eventS have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura+ random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.
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Yarger, J. G., G. Armilei i M. C. Gorman. "Transcription terminator-like element within a Saccharomyces cerevisiae promoter region". Molecular and Cellular Biology 6, nr 4 (kwiecień 1986): 1095–101. http://dx.doi.org/10.1128/mcb.6.4.1095-1101.1986.
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We analyzed a cloned fragment of the yeast URA3 promoter region that contains a sequence of DNA capable of functioning as a highly efficient transcription terminator. BAL 31 deletions have shown the signal for the transcription termination activity is less than or equal to 110 base pairs and resides between bases 45 and 155 upstream of the URA3 primary ATG codon at base 227. In our in vivo assay system, the DNA fragment is able to terminate transcripts very efficiently in either orientation. The terminated transcripts bind to oligodeoxythymidylate cellulose columns and promote the synthesis of full-length cDNAs, suggesting that the transcripts are polyadenylated. The 110-base-pair region contains no sequence resembling terminator consensus sequences described by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell, 28:563-573, 1982) or Henikoff and Cohen (S. Henikoff and E.H. Cohen, Mol. Cell. Biol., 4:1515-1520, 1984). We discuss the possible physiological relevance of this sequence to bona fide termination of transcription and to URA3 regulation in Saccharomyces cerevisiae.
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Yarger, J. G., G. Armilei i M. C. Gorman. "Transcription terminator-like element within a Saccharomyces cerevisiae promoter region." Molecular and Cellular Biology 6, nr 4 (kwiecień 1986): 1095–101. http://dx.doi.org/10.1128/mcb.6.4.1095.
Pełny tekst źródłaStreszczenie:
We analyzed a cloned fragment of the yeast URA3 promoter region that contains a sequence of DNA capable of functioning as a highly efficient transcription terminator. BAL 31 deletions have shown the signal for the transcription termination activity is less than or equal to 110 base pairs and resides between bases 45 and 155 upstream of the URA3 primary ATG codon at base 227. In our in vivo assay system, the DNA fragment is able to terminate transcripts very efficiently in either orientation. The terminated transcripts bind to oligodeoxythymidylate cellulose columns and promote the synthesis of full-length cDNAs, suggesting that the transcripts are polyadenylated. The 110-base-pair region contains no sequence resembling terminator consensus sequences described by Zaret and Sherman (K.S. Zaret and F. Sherman, Cell, 28:563-573, 1982) or Henikoff and Cohen (S. Henikoff and E.H. Cohen, Mol. Cell. Biol., 4:1515-1520, 1984). We discuss the possible physiological relevance of this sequence to bona fide termination of transcription and to URA3 regulation in Saccharomyces cerevisiae.
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Tian, Xuelei, Xin Xu i Wei Xiao. "Novel Method for Genomic Promoter Shuffling by Using Recyclable Cassettes". Applied and Environmental Microbiology 79, nr 22 (6.09.2013): 7042–47. http://dx.doi.org/10.1128/aem.02159-13.
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ABSTRACTGenetic elements of interest can be introduced into theSaccharomyces cerevisiaegenome via homologous recombination. The current method is to link such an element to a selectable marker gene to be integrated into the target locus. However, the marker gene in this method cannot be reused, which limits repeated manipulation of the yeast genome. An alternative method is to utilize a counterselectable gene, such asURA3, with flanking tandem repeats. After integration,URA3along with one copy of the repeat can be popped out via internal recombination, leaving behind one copy of the unwanted repeat. Here we describe a novel concept of genetic element shuffling in which the tandem repeats are made of the desired genetic element, so that after integration and popping out, only one copy of the element remains at the desired locus to function. As a proof of principle, we constructed three recyclable cassettes (PPGK1-URA3-PPGK1,PGAL1-URA3-PGAL1, andPtetO7-URA3-PtetO7) and integrated them upstream of an engineered chromosomalPHIS3-mCherry-Myclocus. After promoter shuffling, themCherry-Mycgene was regulated precisely as anticipated.
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Weng, Yi-shin, i Jac A. Nickoloff. "Nonselective URA3 Colony-Color Assay in Yeast ade1 or ade2 Mutants". BioTechniques 23, nr 2 (sierpień 1997): 237–42. http://dx.doi.org/10.2144/97232bm13.
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Singhal, Anupriya. "Characterizing the Fitness Effects of Mutations in the Yeast URA3 Gene". Biophysical Journal 96, nr 3 (luty 2009): 332a. http://dx.doi.org/10.1016/j.bpj.2008.12.1672.
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Solow, Steven P., Larissa Lezina i Paul M. Lieberman. "Phosphorylation of TFIIA Stimulates TATA Binding Protein-TATA Interaction and Contributes to Maximal Transcription and Viability in Yeast". Molecular and Cellular Biology 19, nr 4 (1.04.1999): 2846–52. http://dx.doi.org/10.1128/mcb.19.4.2846.
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ABSTRACT Posttranslational modification of general transcription factors may be an important mechanism for global gene regulation. The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential for high-level transcription mediated by various activators. Modulation of the TFIIA-TBP interaction is a likely target of transcriptional regulation. We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylated in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA complex and is required for high-level transcription. Alanine substitution of serine residues 220, 225, and 232 completely eliminated in vivo phosphorylation of Toa1, although no single amino acid substitution of these serine residues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold more efficient in forming a stable complex with TBP and TATA DNA. Dephosphorylation of yeast-derived TFIIA reduced DNA binding activity, and recombinant TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing thetoa1 S220/225/232A showed reduced high-level transcriptional activity at the URA1, URA3, andHIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined with an alanine substitution mutation at W285, which disrupts the TFIIA-TBP interface. Phosphorylation of TFIIA could therefore be an important mechanism of transcription modulation, since it stimulates TFIIA-TBP association, enhances high-level transcription, and contributes to yeast viability.
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Friel, Damien, Nenmaura Maria Gomez Pessoa, Micheline Vandenbol i M. Haïssam Jijakli. "Separate and Combined Disruptions of Two Exo-β-1,3-Glucanase Genes Decrease the Efficiency of Pichia anomala (Strain K) Biocontrol Against Botrytis cinerea on Apple". Molecular Plant-Microbe Interactions® 20, nr 4 (kwiecień 2007): 371–79. http://dx.doi.org/10.1094/mpmi-20-4-0371.
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The modes of action of the antagonistic yeast Pichia anomala (strain K) have been studied; however, thus far, there has been no clear demonstration of the involvement of exo-β-1,3-glucanase in determining the level of protection against Botrytis cinerea afforded by this biocontrol agent on apple. In the present study, the exo-β-1,3-glucanase-encoding genes PAEXG1 and PAEXG2, previously sequenced from the strain K genome, were separately and sequentially disrupted. Transfer of the URA3-Blaster technique to strain K, allowing multiple use of URA3 marker gene, first was validated by efficient inactivation of the PaTRP1 gene and recovery of a double auxotrophic strain (uracil and tryptophan). The PAEXG1 and PAEXG2 genes then were inactivated separately and sequentially with the unique URA3 marker gene. The resulting mutant strains showed a significantly reduced efficiency of biocontrol of B. cinerea when applied to wounded apple fruit, the calculated protection level dropping from 71% (parental strain) to 8% (mutated strain) under some experimental conditions. This suggests that exo-β-1,3-glucanases play a role in the biological control of B. cinerea on apple. Furthermore, biological control experiments carried out in this study underline the complexity of the host-antagonist-pathogen interaction. Two experimental parameters (yeast inoculum concentration and physiological stage of the fruit) were found to influence dramatically the protection level. Results also suggest that, under some conditions, the contribution of exo-β-1,3-glucanase to biological control may be masked by other modes of action, such as competition.
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Chopra, Rohini, Vishva Mitra Sharma i K. Ganesan. "Elevated Growth of Saccharomyces cerevisiae ATH1 Null Mutants on Glucose Is an Artifact of Nonmatching Auxotrophies of Mutant and Reference Strains". Applied and Environmental Microbiology 65, nr 5 (1.05.1999): 2267–68. http://dx.doi.org/10.1128/aem.65.5.2267-2268.1999.
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ABSTRACT Yeast strains disrupted for ATH1, which encodes vacuolar acid trehalase, have been reported to grow to higher cell densities than reference strains. We showed that the increase in cell density is due to the URA3 gene introduced as a part of the disruption and concluded that the misinterpretation is a result of not using a control strain with matching auxotrophic markers.
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Jerome, J. F., i J. A. Jaehning. "mRNA transcription in nuclei isolated from Saccharomyces cerevisiae". Molecular and Cellular Biology 6, nr 5 (maj 1986): 1633–39. http://dx.doi.org/10.1128/mcb.6.5.1633-1639.1986.
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We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.
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Jerome, J. F., i J. A. Jaehning. "mRNA transcription in nuclei isolated from Saccharomyces cerevisiae." Molecular and Cellular Biology 6, nr 5 (maj 1986): 1633–39. http://dx.doi.org/10.1128/mcb.6.5.1633.
Pełny tekst źródłaStreszczenie:
We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.
34
Irniger, S., C. M. Egli i G. H. Braus. "Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae". Molecular and Cellular Biology 11, nr 6 (czerwiec 1991): 3060–69. http://dx.doi.org/10.1128/mcb.11.6.3060-3069.1991.
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This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.
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Irniger, S., C. M. Egli i G. H. Braus. "Different classes of polyadenylation sites in the yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 11, nr 6 (czerwiec 1991): 3060–69. http://dx.doi.org/10.1128/mcb.11.6.3060.
Pełny tekst źródłaStreszczenie:
This report provides an analysis of the function of polyadenylation sites from six different genes of the yeast Saccharomyces cerevisiae. These sites were tested for their ability to turn off read-through transcription into the URA3 gene in vivo when inserted into an ACT-URA3 fusion gene. The 3' ends of all polyadenylation sites inserted into the test system in their natural configuration are identical to the 3' ends of the chromosomal genes. We identified two classes of polyadenylation sites: (i) efficient sites (originating from the genes GCN4 and PHO5) that were functional in a strict orientation-dependent manner and (ii) bidirectional sites (derived from ARO4, TRP1, and TRP4) that had a distinctly reduced efficiency. The ADH1 polyadenylation site was efficient and bidirectional and was shown to be a combination of two polyadenylation sites of two convergently transcribed genes. Sequence comparison revealed that all efficient unidirectional polyadenylation sites contain the sequence TTTTTAT, whereas all bidirectional sites have the tripartite sequence TAG...TA (T)GT...TTT. Both sequence elements have previously been proposed to be involved in 3' end formation. Site-directed point mutagenesis of the TTTTTAT sequence had no effect, whereas mutations within the tripartite sequence caused a reduced efficiency for 3' end formation. The tripartite sequence alone, however, is not sufficient for 3' end formation, but it might be part of a signal sequence in the bidirectional class of yeast polyadenylation sites. Our findings support the assumption that there are at least two different mechanisms with different sequence elements directing 3' end formation in yeast.
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Judd, S. R., i T. D. Petes. "Physical lengths of meiotic and mitotic gene conversion tracts in Saccharomyces cerevisiae." Genetics 118, nr 3 (1.03.1988): 401–10. http://dx.doi.org/10.1093/genetics/118.3.401.
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Abstract Physical lengths of gene conversion tracts for meiotic and mitotic conversions were examined, using the same diploid yeast strain in all experiments. This strain is heterozygous for a mutation in the URA3 gene as well as closely linked restriction site markers. In cells that had a gene conversion event at the URA3 locus, it was determined by Southern analysis which of the flanking heterozygous restriction sites had co-converted. It was found that mitotic conversion tracts were longer on the average than meiotic tracts. About half of the tracts generated by spontaneous mitotic gene conversion included heterozygous markers 4.2 kb apart; none of the meiotic conversions included these markers. Stimulation of mitotic gene conversion by ultraviolet light or methylmethanesulfonate had no obvious effect on the size or distribution of the tracts. Almost all conversion tracts were continuous.
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Clark, A. B., C. C. Dykstra i A. Sugino. "Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha". Molecular and Cellular Biology 11, nr 5 (maj 1991): 2576–82. http://dx.doi.org/10.1128/mcb.11.5.2576-2582.1991.
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DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.
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Jinks-Robertson, S., M. Michelitch i S. Ramcharan. "Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae". Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 3937–50. http://dx.doi.org/10.1128/mcb.13.7.3937-3950.1993.
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An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination. The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats). While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length. The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome. An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.
39
Clark, A. B., C. C. Dykstra i A. Sugino. "Isolation, DNA sequence, and regulation of a Saccharomyces cerevisiae gene that encodes DNA strand transfer protein alpha." Molecular and Cellular Biology 11, nr 5 (maj 1991): 2576–82. http://dx.doi.org/10.1128/mcb.11.5.2576.
Pełny tekst źródłaStreszczenie:
DNA strand transfer protein alpha (STP alpha) from meiotic Saccharomyces cerevisiae cells promotes homologous pairing of DNA without any nucleotide cofactor in the presence of yeast single-stranded DNA binding protein. This gene (DNA strand transferase 1, DST1) encodes a 309-amino-acid protein with a predicted molecular mass of 34,800 Da. The STP alpha protein level is constant in both mitotic and meiotic cells, but during meiosis the polypeptide is activated by an unknown mechanism, resulting in a large increase in its specific activity. A dst1::URA3/dst1::URA3 mutant grows normally in mitotic media; however, meiotic cells exhibit a greatly reduced induction of both DNA strand transfer activity and intragenic recombination between his1 heteroalleles. Spore viability is normal. These results suggest that DST1 is required for much of the observed induction of homologous recombination in S. cerevisiae during meiosis but not for normal sporulation.
40
Jinks-Robertson, S., M. Michelitch i S. Ramcharan. "Substrate length requirements for efficient mitotic recombination in Saccharomyces cerevisiae." Molecular and Cellular Biology 13, nr 7 (lipiec 1993): 3937–50. http://dx.doi.org/10.1128/mcb.13.7.3937.
Pełny tekst źródłaStreszczenie:
An ectopic recombination system using ura3 heteroalleles varying in size from 80 to 960 bp has been used to examine the effect of substrate length on spontaneous mitotic recombination. The ura3 heteroalleles were positioned either on nonhomologous chromosomes (heterochromosomal repeats) or as direct or inverted repeats on the same chromosome (intrachromosomal repeats). While the intrachromosomal events occur at rates at least 2 orders of magnitude greater than the corresponding heterochromosomal events, the recombination rate for each type of repeat considered separately exhibits a linear dependence on substrate length. The linear relationships allow estimation of the corresponding minimal efficient processing segments, which are approximately 250 bp regardless of the relative positions of the repeats in the yeast genome. An examination of the distribution of recombination events into simple gene conversion versus crossover events indicates that reciprocal exchange is more sensitive to substrate size than is gene conversion.
41
Silva, Fernando de Godoi, Daiane Dias Lopes, Ronald E. Hector, Maikon Thiago do Nascimento, Tatiana de Ávila Miguel, Emília Kiyomi Kuroda, Gisele Maria de Andrade de Nóbrega, Ken-Ichi Harada i Elisa Yoko Hirooka. "Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9". Microorganisms 11, nr 3 (24.02.2023): 575. http://dx.doi.org/10.3390/microorganisms11030575.
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Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.
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Galvão, Teca Calcagno, i Víctor de Lorenzo. "Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain". Applied and Environmental Microbiology 71, nr 2 (luty 2005): 883–92. http://dx.doi.org/10.1128/aem.71.2.883-892.2005.
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ABSTRACT A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.
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Johnson, S. P., i J. R. Warner. "Unusual enhancer function in yeast rRNA transcription". Molecular and Cellular Biology 9, nr 11 (listopad 1989): 4986–93. http://dx.doi.org/10.1128/mcb.9.11.4986-4993.1989.
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The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidence that transcription of one of these genes may not be independent of transcription of others. In particular, in the yeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of the transcription initiation site is at least partly within the upstream transcription unit. To ask more directly about the relationship of the tandemness of these genes to their transcription, we have constructed a minirepeat containing two identifiable test genes, with or without enhancer(s). On integration into the URA3 locus, these genes were transcribed by RNA polymerase I. A single enhancer effectively stimulated transcription of both genes by 10- to 30-fold, even when it was located upstream of both or downstream of both. Two enhancers had roughly additive effects. These results suggest a model of enhancer function in tandemly repeated genes.
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Johnson, S. P., i J. R. Warner. "Unusual enhancer function in yeast rRNA transcription." Molecular and Cellular Biology 9, nr 11 (listopad 1989): 4986–93. http://dx.doi.org/10.1128/mcb.9.11.4986.
Pełny tekst źródłaStreszczenie:
The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidence that transcription of one of these genes may not be independent of transcription of others. In particular, in the yeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of the transcription initiation site is at least partly within the upstream transcription unit. To ask more directly about the relationship of the tandemness of these genes to their transcription, we have constructed a minirepeat containing two identifiable test genes, with or without enhancer(s). On integration into the URA3 locus, these genes were transcribed by RNA polymerase I. A single enhancer effectively stimulated transcription of both genes by 10- to 30-fold, even when it was located upstream of both or downstream of both. Two enhancers had roughly additive effects. These results suggest a model of enhancer function in tandemly repeated genes.
45
Picologlou, S., N. Brown i S. W. Liebman. "Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition". Molecular and Cellular Biology 10, nr 3 (marzec 1990): 1017–22. http://dx.doi.org/10.1128/mcb.10.3.1017-1022.1990.
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The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.
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Picologlou, S., N. Brown i S. W. Liebman. "Mutations in RAD6, a yeast gene encoding a ubiquitin-conjugating enzyme, stimulate retrotransposition." Molecular and Cellular Biology 10, nr 3 (marzec 1990): 1017–22. http://dx.doi.org/10.1128/mcb.10.3.1017.
Pełny tekst źródłaStreszczenie:
The Saccharomyces cerevisiae DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme which polyubiquitinates histones in vitro. Here we show that mutations in rad6 increase the frequency of transposition of the retrotransposon Ty into the CAN1 and URA3 loci. Using isogenic RAD6 and rad6 strains, we measured a more than 100-fold increase in the spontaneous rate of retrotransposition due to rad6, although there was no increase in the Ty message level. This is the first time that a mutation in a host gene has been shown to result in an increased rate of retrotransposition.
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Horsfall, Wendy H., i Ronald E. Pearlman. "Micronuclear DNA sequences from Tetrahymena do not confer mitotic stability on ARS plasmids in Saccharomyces". Genome 30, nr 5 (1.10.1988): 690–96. http://dx.doi.org/10.1139/g88-116.
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Genomic libraries containing micronuclear DNA sequences from Tetrahymena thermophila have been constructed in a vector containing ARS1, SUP11, and ura3 sequences from the yeast Saccharomyces cerevisiae. When transformed into a strain of S. cerevisiae carrying a suppressible ochre mutation in the ade2 gene, viable transformants are obtained only if the transforming plasmid is maintained at a copy number of one or two per cell. Mitotic segregation of the plasmid is easily assessed in a colour assay of transformants. Using this assay system, we showed that micronuclear DNA from Tetrahymena does not contain sequences that confer mitotic stability on yeast ARS-containing plasmids; i.e., sequences that function analogously to yeast centromere sequences. One transformant was analyzed that carries Tetrahymena sequences that maintain the copy number of the ARS plasmid at one or two per cell. However, these sequences do not confer mitotic stability on the transformants and they confer a phenotype in this assay similar to that of the REP3 gene of the yeast 2 μm plasmid.Key words: mitotic stability, centromere, Tetrahymena, Saccharomyces.
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Suzuki, Y., Y. Nogi, A. Abe i T. Fukasawa. "GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae". Molecular and Cellular Biology 8, nr 11 (listopad 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991-4999.1988.
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Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.
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Suzuki, Y., Y. Nogi, A. Abe i T. Fukasawa. "GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae." Molecular and Cellular Biology 8, nr 11 (listopad 1988): 4991–99. http://dx.doi.org/10.1128/mcb.8.11.4991.
Pełny tekst źródłaStreszczenie:
Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.
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Jedrusik, Monika A., i Ekkehard Schulze. "Telomeric Position Effect Variegation in Saccharomyces cerevisiae by Caenorhabditis elegans Linker Histones Suggests a Mechanistic Connection between Germ Line and Telomeric Silencing". Molecular and Cellular Biology 23, nr 10 (15.05.2003): 3681–91. http://dx.doi.org/10.1128/mcb.23.10.3681-3691.2003.
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ABSTRACT Linker histones are nonessential for the life of single-celled eukaryotes. Linker histones, however, can be important components of specific developmental programs in multicellular animals and plants. For Caenorhabditis elegans a single linker histone variant (H1.1) is essential in a chromatin silencing process which is crucial for the proliferation and differentiation of the hermaphrodite germ line. In this study we analyzed the whole linker histone complement of C. elegans by telomeric position effect variegation in budding yeast. In this assay an indicator gene (URA3) placed close to the repressive telomeric chromatin structure is subject to epigenetically inherited gene inactivation. Just one out of seven C. elegans linker histones (H1.1) was able to enhance the telomeric position effect in budding yeast. Since these results reflect the biological function of H1.1 in C. elegans, we suggest that chromatin silencing in C. elegans is governed by molecular mechanisms related to the telomere-dependent silencing in budding yeast. We confirmed this hypothesis by testing C. elegans homologs of three yeast genes which are established modifiers of the yeast telomeric chromatin structure (SIR2, SET1, and RAD17) for their influence on repeat-dependent transgene silencing for C. elegans.