Cloning of theC-URA3gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering ofCandida maltosa
✍ Scribed by Moriya Ohkuma; Shin-ichiro Muraoka; Chel Won Hwang; Akinori Ohta; Masamichi Takagi
- Publisher
- Springer-Verlag
- Year
- 1993
- Tongue
- English
- Weight
- 686 KB
- Volume
- 23
- Category
- Article
- ISSN
- 0172-8083
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✦ Synopsis
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.