Abstract
The Escherichia coli asparagine synthetase B gene (asnB) has been cloned into a temperature-sensitive, low copy plasmid, pOU71, as shown by the complementation of an E. coli asparagine auxotroph, E. coli JE6279. The nucleotide sequence of asnB and the flanking sequences were determined. The proposed coding region for the gene is 1662 nucleotides in length, and the deduced amino acid sequence of the coding region results in a protein that has a molecular weight of 62,666 and contains 554 amino acids. A promoter region is identified based on the transcription start site that was determined by primer extension experiments. Homology studies of the asnB protein sequence with the human asparagine synthetase and E. coli asparagine synthetase A protein show that there is a high degree of homology with only the human asparagine synthetase. A purF type glutamine amide transfer domain was identified upon inspection of the amino-terminal amino acid sequence of the asparagine synthetase B protein.
Highlights
From the *Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588 and the SDepartment and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32611 of Biochemistry
This paper reports the isolation and cloning of the E. coli a.mB gene, the sequencing of the gene, and an examination of the homology of the amino acid sequence of the gene product with the human asparagine synthetase, E. coli ammonia-dependent asparagine synthetase, and the PUFF type glutamine amidotransferase domain of other glutamine amidotransferases
The X781 bacteriophage appeared to be a good source for isolating the E. coli asparagine synthetase B gene (asnB) gene
Summary
Bacterial Strains, Plasmids, and Phage-All strains used were derivatives of E. coli K-12: W3110 (X-, F-, thi), from T. Low copy plasmids with inserts whose gene product complemented the asparagine auxotrophy in E. coli were determined by the growth at 36 “C-of transformed JE6279 cells on minimal medium plates with ampicillin hut without asparagine. Isolation of RNA-E. coli JE6279 and W3110 were grown in minimal media (MS-casamino acids) to an ODs, of 0.6 in the presence or absence of asparagine, and the cells were isolated and the RNA extracted according to the hot sodium dodecyl sulfate-phenol method described by Conway et al [23]. The entire asnA or human asparagine synthetase protein was aligned with the six different translations of the a.snB gene sequence using the IFIND program This program uses the algorithm of Wilber and Lipman [27] for initial identification of homologous regions and generates a refined alignment by the method of Needleman and Wunsch [26]. Homologous segments were aligned as separate fragments as were the inter-
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