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  • 1
    ISSN: 0749-503X
    Keywords: Chromosome III ; sequencing ; gene disruption ; ribokinase ; ARS ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report here the DNA sequence of a segment of chromosome III extending over 8·2 kb. The sequence was determined using the random clone strategy followed by oligonucleotide-directed sequencing. The segments contains five long open reading frames, YCR521, 522, 523, 524 and 526, with only short distances between them. YCR523 (333 codons) endodes a ribokinase, a new function for yeast. YCR526 originates inside the MAT cassette, which is in continuity with the present segment, and extends over 358 codons outside of MAT. YCR524 (923 codons) codes for a putative membrane protein. YCR521, 522 and 524, have each been disrupted by insertion of a URA3 cassette and are non-essential genes. An active ARS element is located within YCR523 or its vicinity.
    Additional Material: 8 Ill.
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  • 2
    ISSN: 0749-503X
    Keywords: Methylotrophic yeast ; genetic transformation ; non-homologous integration ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Development of transformation systems for methylotrophic yeasts is the starting point for research aimed at developing molecular genetics of these genera and will be the key to their further successful use in biotechnology. We transformed Pichia methanolica using selector genes ADE2 and LEU2 from Saccharomyces cerevisiae and ADE1 (homologue of S. cerevisiae ADE2 gene) from P. methanolica which was cloned and sequenced in our laboratory (Hiep et al., 1991). Lithium transformation of P. methanolica strains was inefficient with intact plasmids. Linearization of plasmids at a unique restriction site within the ADE1 gene prior to transformation substantially increased its frequency. Transformation with linear ADE1, ADE2 or LEU2 gene fragments was even more effective. Introduced DNA fragments either circularized in vivo, irrespective of the structures of their ends, giving unstable transformants; or integrated at different sites of the host genome. Using this transformation system, we obtained a disruption of the ADE1 gene on the chromosome by inserting the S. cerevisiae LEU2 gene. The disruption mutation ade1::LEU2 was used to study the mechanism of intragenic recombination in P. methanolica.
    Additional Material: 3 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 13 (1997), S. 647-653 
    ISSN: 0749-503X
    Keywords: markers ; gene disruption ; gene replacement ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: One-step gene disruption constructs for disruption of HIS3, LEU2, TRP1 or URA3 with each of the other three markers have been constructed. All of these constructs have been tested and found to effectively convert markers either in gene disruptions or on plasmids. The ‘swapped’ strains allow the unambiguous genetic analysis of synthetic phenotypes with multiple genes, even if the original gene disruptions were made with the same marker. They also allow introduction of multiple plasmids in a single transformant, even if the original plasmids had the same marker, and allow transformation of plasmids into strains containing gene disruptions made with the same marker that is on the plasmids. These ‘marker-swap’ plasmids therefore eliminate the need for much subcloning to change markers. Marker-swapped alleles are acceptably stable mitotically and meiotically for most applications.© 1997 John Wiley & Sons, Ltd.
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  • 4
    ISSN: 0749-503X
    Keywords: yeast functional analysis ; gene disruption ; gap-repair cloning ; selection gene ‘pop-out’ ; I-SceI ; Life Sciences ; Life Sciences (general)
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: New tools are needed for speedy and systematic study of the numerous genes revealed by the sequence of the yeast genome. We have developed a novel transformation strategy, based on ‘split-marker’ recombination, which allows generation of chromosomal deletions and direct gene cloning. For this purpose, pairs of yeast vectors have been constructed which offer a number of advantages for large-scale applications such as one-step cloning of target sequence homologs and combinatorial use. Gene deletions or gap-repair clonings are obtained by cotransformation of yeast by a pair of recombinant plasmids. Gap-repair vectors are based on the URA3 marker. Deletion vectors include the URA3, LYS2 and kanMX selection markers flanked by I-SceI sites, which allow their subsequent elimination from the transformant without the need for counter-selection. The application of the ‘split-marker’ vectors to the analysis of a few open reading frames of chromosome XI is described.
    Additional Material: 7 Ill.
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  • 5
    ISSN: 0749-503X
    Keywords: gene disruption ; functional analysis ; Saccharomyces cerevisiae ; G418-resistance ; sticky-end PCR ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains. © 1998 John Wiley & Sons, Ltd.
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  • 6
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mitochondrial matrix ; homo-oligomeric protein ; Mam33p ; gene disruption ; gC1q-R ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Mam33p (mitochondrial acidic matrix protein) is a soluble protein, located in mitochondria of Saccharomyces cerevisiae. It is synthesized as a precursor with an N-terminal mitochondrial targeting sequence that is processed on import. Mam33p assembles to a homo-oligomeric complex in the mitochondrial matrix. It can bind to the sorting signal of cytochrome b2 that directs this protein into the intermembrane space. Mam33p is encoded by an 801 bp open reading frame. Gene disruption did not result in a significant growth defect. Mam33p exhibits sequence similarity to gC1q-R, a human protein that has been implicated in the binding of complement factor C1q and kininogen. © 1998 John Wiley & Sons, Ltd.
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  • 7
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; gene cloning ; gene disruption ; functional analysis ; chromosome XVI ; translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature-sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non-essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process. © 1998 John Wiley & Sons, Ltd.
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  • 8
    ISSN: 0749-503X
    Keywords: gene disruption ; homologous recombination ; protein A-tagging ; Saccharomyces cerevisiae ; tags ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphyloccocus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain. © 1998 John Wiley & Sons, Ltd.
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  • 9
    ISSN: 0749-503X
    Keywords: Chromosome III ; sequencing ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have determined the nucleotide sequence of a segment of chromosome III contained in the right part of the lambda PM3270 clone, for a total of 8824 bp. This sequence contains an unusual long open reading frame, YCR601, of 6501 bp that encodes for a protein of 2167 amino acids that show no homology with other known proteins. YCR601 was disrupted by internal deletion and insertion of LEU2 gene and is a non-essential gene, however it is transcribed during vegetative growth yielding a polyadenylated mRNA of approximately 7 kb.
    Additional Material: 4 Ill.
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  • 10
    ISSN: 0749-503X
    Keywords: Plasmids ; gene disruption ; selectable cassettes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.
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