| Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636. | |
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MedLine Citation:
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PMID: 8682799 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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An Escherichia coli clone expressing activity on DNase agar was obtained by cloning chromosomal DNA of Aeromonas hydrophila JMP636 into plasmid pUC19. Examination (of the clone's nuclease activity on a sodium dodecyl sulfate (SDS)-polyacrylamide gel containing DNA as a substrate revealed an activity band at approximately 100 kDa. Subsequently, subcloning localized the gene, designated nucH, to a 3.6-kb DNA fragment (pJP9521). Southern blotting of the nucH gene against chromosomal DNA of JMP636 confirmed that it had originated from this strain and demonstrated that it was present in a single copy, although additional faint bands were also detected. Analysis of the subclone using in vivo transcription and translation revealed only a single polypeptide of approximately 110 kDa. Sequencing of pJP9521 predicted an open reading frame of 3,213 bp encoding a protein of 1,070 amino acids and having a molecular mass of 114 kDa. Comparison of the deduced nucleotide sequence and the NucH predicted protein sequence with relevant databases indicated that no known homologs have previously been identified. A signal sequence was predicted from these data, and cellular fractionation of a nucH clone in E. coli indicated that the protein was able to be processed to the periplasm. An activity similar in size was detected in an extracellular protein sample of JMP636, while inactivation of the nucH gene resulted in loss of this activity band. By native SDS-polyacrylamide gel electrophoresis, NucH substrate specificity, cofactor requirements, and sensitivity to denaturing agents were assessed. |
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Authors:
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H N Dodd; J M Pemberton |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Journal of bacteriology Volume: 178 ISSN: 0021-9193 ISO Abbreviation: J. Bacteriol. Publication Date: 1996 Jul |
Date Detail:
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Created Date: 1996-08-20 Completed Date: 1996-08-20 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 2985120R Medline TA: J Bacteriol Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 3926-33 Citation Subset: IM |
Affiliation:
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Department of Microbiology, University of Queensland, Australia. |
| Data Bank Information | |
Bank Name/Acc. No.:
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GENBANK/L76304 |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Aeromonas hydrophila
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enzymology*,
genetics Amino Acid Sequence Bacterial Proteins / genetics*, metabolism Base Sequence Cell Fractionation Chromosomes, Bacterial Cloning, Molecular DNA, Bacterial Deoxyribonucleases / genetics*, metabolism Gene Dosage Molecular Sequence Data Phenotype Protein Biosynthesis Transcription, Genetic |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/DNA, Bacterial; EC 3.1.-/Deoxyribonucleases; EC 3.1.-/NucH protein, Aeromonas hydrophila |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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