RRC ID 74976
Author Kimura M, Kamakura T, Tao QZ, Kaneko I, Yamaguchi I.
Title Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae.
Journal Mol Gen Genet
Abstract Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequence of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nucleotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These result promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.
Volume 242(2)
Pages 121-9
Published 1994-1-1
DOI 10.1007/BF00391004
PMID 8159161
MeSH Amino Acid Sequence Aminohydrolases / genetics* Aspergillus / drug effects Aspergillus / enzymology* Aspergillus / genetics* Bacillus cereus / enzymology Bacillus cereus / genetics Base Sequence Cloning, Molecular Codon / genetics DNA, Fungal / genetics Drug Resistance, Microbial / genetics Escherichia coli / genetics Genes, Bacterial Genes, Fungal* Genetic Markers Mitosporic Fungi / enzymology Mitosporic Fungi / genetics Molecular Sequence Data Nucleosides / pharmacology Plasmids / genetics Schizosaccharomyces / enzymology Schizosaccharomyces / genetics Sequence Homology, Amino Acid Transformation, Genetic
DNA material pBSA712 (RDB19904) pBF101 (RDB19905 )