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.
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.
Ohkuma
M
M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Muraoka
S
S
Hwang
C W
CW
Ohta
A
A
Takagi
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002175:Candida / Q000235:genetics*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D009964:Orotidine-5'-Phosphate Decarboxylase