CLONING AND NUCLEOTIDE-SEQUENCE OF THE RIBONUCLEASE T-1 GENE (RNTA) FROM ASPERGILLUS-ORYZAE AND ITS EXPRESSION IN SACCHAROMYCES-CEREVISIAE AND ASPERGILLUS-ORYZAE

A genomic DNA encoding ribonuclease (RNase) T-1 from A spergillus oryzae was cloned using a synthetic oligonucleotide probe, The cloned gene (designated rntA) encoded functional RNase T-1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T-1 activity (over 200 times) than a transformant with a vector, A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene, Nucleotide sequencing analysis of both DNAs found that RNase T-1 had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence. The A. oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T-1. On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose. These results suggests that A. oryzae may have a protection mechanism from RNase T-1,