Transformation of yeast by a replicating hybrid plasmid

Beggs, Jean D.

doi:10.1038/275104a0

Cited by 1,312 publications

(446 citation statements)

References 36 publications

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“…This interspecific complementation method might have succeeded because of the use of high-copy number plasmid vectors, which allowed sufficient expression of yeast genes in E. coli. S. cerevisiae genes were cloned by the complementation of S. cerevisiae mutants with S. cerevisiae plasmid libraries (Beggs, 1978;Hinnen et al, 1978;Hsiao and Carbon, 1979) and later genomic DNA libraries consisting of DNA fragments randomly inserted into yeast vectors using E. coli as a cloning host have been used for complementation cloning (Lundblad, 2001;Rose and Broach, 1991). Therefore, the cloning of genes into plasmid vectors was a common tool for gene manipulations in yeast as well as other species (Bergkamp et al, 1993;Janssen and Chen, 1998).…”

Section: Identification Of K Marxianus Auxotrophic Mutants With Genementioning

confidence: 99%

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

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Section: Identification Of K Marxianus Auxotrophic Mutants With Genementioning

confidence: 99%

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yarimizu

Nonklang

Nakamura

et al. 2013

Yeast

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“…Cells were incubated with YRp7 DNA in a 10mM Tris' HCI solution, pH 7.4, containing (1) 40% PEG 4,000, (2) 40% PEG. 4,000 and 50mM CaCI 2 , and (3) 50 mM CaCI 2 • The numbers of Trp + colonies were 12, 113 and 160 per plate under conditions (1), (2) and (3), respectively. Thus, we could.…”

Section: Effects Of Some Other Factorsmentioning

confidence: 99%

Yeast Transformation without the Spheroplasting Process

Iimura

Gotoh

Ouchi

et al. 1983

Agricultural and Biological Chemistry

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“…Cloning of the COX15-The wild-type COX15 gene was cloned by transformation of E412/L1 and N7-211/L1 with a yeast genomic library by the method of Beggs (24). The library used for the transformation was constructed from partial Sau3A fragments of nuclear DNA (averaging 7-10 kb) from the respiratory competent haploid strain S. cerevisiae D273-10B/A1.…”

Section: Methodsmentioning

confidence: 99%

COX15 Codes for a Mitochondrial Protein Essential for the Assembly of Yeast Cytochrome Oxidase

Muroff

Jin

et al. 1997

Journal of Biological Chemistry

104

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The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of a pet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme a biosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15 has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase in cox15 mutants, indicating that the carboxylterminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombe and Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms.The completion of the Saccharomyces cerevisiae genome sequence (1) has spawned new large scale projects designed to unravel the functions of the numerous unknown reading frames. A substantial fraction of the total information in yeast chromosomal DNA is comprised of PET genes that are essential for the biogenesis of respiratory competent mitochondria. Mutations in these genes were shown in the early 1950s (2, 3) to affect the ability of yeast to respire. Renewed efforts to mutationally saturate for this class of genes (4, 5) have helped to expand our knowledge of the extent and nature of the contribution made by the nucleus toward maintenance of respiring mitochondria.Biochemical studies of pet mutants 1 have revealed that the assembly of respiratory chain enzymes is governed by an unexpectedly large number of genes. For example, some three dozen complementation groups have been reported to consist of mutants displaying a selective deficiency in cytochrome oxidase (4, 6). In addition to mutations in the structural genes, these strains are also affected in: 1) processing of the mitochondrial cytochrome oxidase pre-mRNAs (7-9), 2) translation of the resultant mRNAs (10, 11), 3) heme a biosynthesis (12), 4) copper import and transfer to the apoenzyme (13, 14), and 5) as yet poorly understood events in the pathway leading to the functional enzyme (15-17). To learn more about the assembly of this heteroligomeric membrane complex, we have continued to screen for, and to analyze pet mutants with lesions in cytochrome oxidase. In this article, we report the properties of mutants from complementation group G4 whose cytochrome oxidase d...

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Transformation of yeast by a replicating hybrid plasmid

Cited by 1,312 publications

References 36 publications

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

Yeast Transformation without the Spheroplasting Process

COX15 Codes for a Mitochondrial Protein Essential for the Assembly of Yeast Cytochrome Oxidase

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