Orthogonal‐field‐alternation gel electrophoresis banding patterns of DNA from yeasts

Jonge, Peter de; Jongh, F.C.M. De; Meijers, Ruud W. J.; Steensma, H. Yde; Scheffers, W. A.

doi:10.1002/yea.320020307

Cited by 153 publications

(64 citation statements)

References 25 publications

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“…His-, Arg-, Met-, and Met-Thrmutants were isolated by Poulter and Hanrahan (26) (6) and are compatible with the observation of Magee et al (21). It should be noted that various C. albicans isolates may differ in their chromosomal patterns.…”

Section: Discussionsupporting

confidence: 82%

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph

et al. 1990

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Genetic studies were done with Candida albicans CBS 562. Various auxotrophs were isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. SAG5 (his4C), a stable histidine auxotroph defective in histidinol dehydrogenase activity, was characterized and chosen for further molecular studies. Therefore, the C. albicans HIS4 gene was isolated. The gene was obtained from a genomic library of the wild-type strain, which was constructed in plasmid YEp24. The HIS4 gene was isolated by transformation of a Saccharomyces cerevisiae strain that carried a his4 mutation. The isolated C. albicans HIS4 gene complemented S. cerevisiae his4A, his4B, his4C, and his4ABC mutant strains, which indicates that the clone contains the entire HIS4 gene. The gene was isolated on plasmid pSTC7, whose physical map was constructed with BamHI, SalI, and EcoRV restriction endonucleases, locating the HIS4 gene on a 14-kilobase-pair DNA fragment. Hybridization experiments with HIS4 and C. albicans genomic DNA showed correspondence between the restriction patterns of the gene with that of the chromosomal DNA, indicating that the gene originates from C. albicans and appears in a single copy. Chromosomes of C. albicans CBS562 and four other strains were resolved by orthogonal-field alteration gel electrophoresis. The electrokaryotyping results showed heterogeneity in chromosomal sizes. The electrokaryotyping of CBS 562 showed a resolution of six chromosomal bands, three of which seemed to be doublets. The C. albicans HIS4 gene was located on the largest resolvable chromosome in all of the strains.

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Section: Discussionsupporting

confidence: 82%

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph

et al. 1990

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“…Chromosomal DNA molecules were prepared according to a modified protocol of de Jonge et al (1986). The EDTA concentration was reduced to 0.125 M and the agarose plugs were stored in 10 mM Tris/HCl, 1 mM EDTA at 4°C.…”

Section: Pulsed-field Gel Electrophoresismentioning

confidence: 99%

Characterization of Saccharomyces cerevisiae mutants lacking the E1α subunit of the pyruvate dehydrogenase complex

Wenzel

Berg

Visser

et al. 1992

European Journal of Biochemistry

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Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDAl gene. To this end, the PDAl gene encoding the E1α subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDAl gene abolished production of the E1α subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh− mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho°], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDAl gene. Explanations for these phenotypes are discussed.

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“…C. neoformans MP415 and the acapsular mutant 309 (ATCC 52817) (9) 6,000 x g for 5 min. The supernatant was placed on ice for 10 min, and 1 ml of 5 M potassium acetate was added.…”

Section: Methodsmentioning

confidence: 99%

Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans

Restrepo

Barbour

1989

J Bacteriol

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Cryptococcus neoformans is an important pathogenic fungus that has been classified as a basidiomycete. Little is known of the molecular genetics of this fungal pathogen. To begin such studies, we devised a procedure for extraction of DNA from cryptococci; this method involved the use of the cell wall-active enzyme NovoZym 234. Using cloned rDNA of Saccharomyces cerevisiae as a probe, we identified homologous restriction fragments in a Southern blot of digested C. neoformans DNA. An 8.6-kilobase HindIII fragment that hybridized with the yeast rDNA probe was ligated with the vector pBR322 and cloned into Escherichia coli. When the fragment was used as a probe, it hybridized to the 18S and 25S rRNAs of C. neoformans in Northern (RNA) blots of native and denatured RNA. It bound at high stringency only weakly to the rRNAs of the ascomycete S. cerevisiae. The locations of the genes for 5/5.8S, 18S, and 25S subunits in the cloned fragment were identified with labeled rRNA of these different types.

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Orthogonal‐field‐alternation gel electrophoresis banding patterns of DNA from yeasts

Cited by 153 publications

References 25 publications

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph

Isolation of the Candida albicans histidinol dehydrogenase (HIS4) gene and characterization of a histidine auxotroph

Characterization of Saccharomyces cerevisiae mutants lacking the E1α subunit of the pyruvate dehydrogenase complex

Cloning of 18S and 25S rDNAs from the pathogenic fungus Cryptococcus neoformans

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