Molecular cloning and characterization of the Streptomyces hygroscopicus alpha-amylase gene.
We have isolated and sequenced a gene (amy) coding for alpha-amylase (EC 3.2.1.1.) from the Streptomyces hygroscopicus genome (H. Hidaka, Y. Koaze, K. Yoshida, T. Niwa, T. Shomura, and T. Niida, Die Stärke 26:413-416, 1974). Amylase was purified to obtain amino acid sequence information which was used to synthesize oligonucleotide probes. amy-containing Escherichia coli cosmids identified by hybridization did not express amylase activity. Subcloning experiments indicated that amy could be expressed from the lac promoter in E. coli or from its own promoter in S. lividans. The amy nucleotide sequence indicated that it coded for a protein of 52 kilodaltons (478 amino acids). Secreted alpha-amylase contained amino- and carboxy-terminal as well as internal amino acid sequences which were consistent with the nucleotide sequence. The 30-residue leader sequence showed similarities to those found in other procaryotes. The DNA sequence 5' to the amy structural gene contained a sequence complementary to the 3'-terminal sequence of 16S rRNA of S. lividans (M. J. Bibb and S. N. Cohen, Mol. Gen. Genet. 187:265-277, 1982). The transcriptional start points of amy were determined by mung bean nuclease mapping, but the promoter of amy was not similar to the consensus sequence found in other procaryotes.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D009693:Nucleic Acid Hybridization; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics*; D000516:alpha-Amylases / Q000235:genetics*
D004262:DNA Restriction Enzymes; D000516:alpha-Amylases