Abstract
The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5′-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.
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Communicated by A. Hinnen
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Ohkuma, M., Muraoka, Si., Hwang, C.W. et al. Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa . Curr Genet 23, 205–210 (1993). https://doi.org/10.1007/BF00351497
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DOI: https://doi.org/10.1007/BF00351497