PMID:3514575
| Citation |
Spring, KJ, Jerlström, PG, Burns, DM and Beacham, IR (1986) L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product. J. Bacteriol. 166:135-42 |
|---|---|
| Abstract |
Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments. Therefore, the genes encoding asparaginases I and II are highly sequence related. |
| Links | |
| Keywords |
Asparaginase/analysis; Asparaginase/biosynthesis; Asparaginase/genetics; Asparagine/analogs & derivatives; Asparagine/pharmacology; Cross Reactions; DNA, Bacterial/analysis; DNA, Recombinant/isolation & purification; Escherichia coli/enzymology; Escherichia coli/genetics; Genes, Bacterial; Mutation; Plasmids; Recombination, Genetic; Sequence Homology, Nucleic Acid |
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