Cloning and characterization of the Rhizopus niveus leu1 gene and its use for homologous transformation.
The Rhizopus niveus leul gene was cloned using a 2-kb HindIII fragment of the leuA gene of Mucor circinelloides as a hybridization probe. The coding region of this gene comprises 1890 bp that encode a putative protein of 630 amino acids. The predicted amino acid sequence is similar to those of other alpha-isopropylmalate isomerases (alpha-IPMIs), showing 79.4%, 71.7%, and 60.6% identity with the Mucor circinelloides LeuA, Phycomyces blaskesleeanus Leu1, and Ustilago maydis Leu1, respectively. The vector pRN1, which contains a 3.7-kb BamHI-SacI fragment of the R. niveus leul gene, could complement leucine auxotrophy of R. niveus M-37 (leu1-), suggesting that the gene codes for alpha-IPMI of R. niveus. Both the transformation frequency and mitotic stability with pRN1 were higher than those with leuA gene-containing vectors. It was found by using the leul gene that the autonomously replicating sequence (ARS) of P. blakesleeanus partially functions as an ARS in R. niveus.
Takaya
N
N
Department of Biotechnology, University of Tokyo, Japan.
Yanai
K
K
Horiuchi
H
H
Ohta
A
A
Takagi
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010800:Phycomyces / Q000235:genetics; D012233:Rhizopus / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic
D004271:DNA, Fungal