Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

Journal Article · Thu Dec 01 04:00:00 EST 1988 · Molecular and Cellular Biology; (USA)

DOI:https://doi.org/10.1128/MCB.8.12.5417· OSTI ID:5560758

Banks, G R; Taylor, S Y ^[1]

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1. National Institute for Medical Research, London (England)

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

• OSTI ID: 5560758
• Journal Information: Molecular and Cellular Biology; (USA), Journal Name: Molecular and Cellular Biology; (USA) Vol. 8:12; ISSN MCEBD; ISSN 0270-7306
• Country of Publication: United States
• Language: English

Related Subjects

560130* -- Radiation Effects on Microorganisms
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
BACTERIA
CHROMOSOMES
CLONING
DNA HYBRIDIZATION
DNA-CLONING
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
EUMYCOTA
FUNGI
GENES
HYBRIDIZATION
HYDROLASES
MICROORGANISMS
MUTATIONS
PARASITES
PLANTS
RADIATIONS
RADIOSENSITIVITY
SACCHAROMYCES
SACCHAROMYCES CEREVISIAE
TRANSCRIPTION
ULTRAVIOLET RADIATION
USTILAGO
YEASTS