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.
Department of Biotechnology, University of Tokyo, Japan.
N
N
Takaya
K
K
Yanai
H
H
Horiuchi
A
A
Ohta
M
M
Takagi
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