Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae.
A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 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 T1 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 T1. 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 T1.
Fujii
T
T
National Research Institute of Brewing, Tokyo, Japan.
Yamaoka
H
H
Gomi
K
K
Kitamoto
K
K
Kumagai
C
C
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D006163:Ribonuclease T1 / Q000235:genetics*; D012441:Saccharomyces cerevisiae
D018076:DNA, Complementary; D004271:DNA, Fungal; D006163:Ribonuclease T1