Integrative transformation of the zygomycete Rhizomucor pusillus by homologous recombination.
For development of a homologous transformation system for the zygomycete fungus, Rhizomucor pusillus, the isopropylmalate isomerase (leuA) gene was cloned from R. pusillus IFO 4578 by the DNA-probing method with the leuA sequence of Mucor circinelloides as probe. The nucleotide sequence revealed that leuA of R. pusillus encoded a 755-amino-acid protein of 82.5 kDa with no intron. The leuA gene on pUC19 (plasmid pRPLeu10) was introduced by polyethyleneglycol-assisted transformation into protoplasts of a leuA- mutant of R. pusillus that was obtained by UV mutagenesis. Transformation under optimal conditions yielded 20 Leu+ transformants (micrograms pRPLeu10 DNA)-1 (1 x 10(6) viable protoplasts)-1. Blot analysis of DNA from the transformants showed that the pRPLeu10 sequence was integrated into the genome by homologous recombination at the leuA locus.
Wada
M
M
Department of Biotechnology, University of Tokyo, Japan.
Beppu
T
T
Horinouchi
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D007535:Isomerases / Q000235:genetics*; D008969:Molecular Sequence Data; D009090:Mucorales / Q000235:genetics*; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic
C032241:isopropylmalate isomerase; D007535:Isomerases