Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636

Dodd, Helen N. and Pemberton, John M. (1996) Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636. Journal of Bacteriology, 178 13: 3926-3933.

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Author Dodd, Helen N.
Pemberton, John M.
Title Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636
Formatted title
Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636
Journal name Journal of Bacteriology   Check publisher's open access policy
ISSN 0021-9193
1098-5530
Publication date 1996-07
Sub-type Article (original research)
Open Access Status File (Publisher version)
Volume 178
Issue 13
Start page 3926
End page 3933
Total pages 8
Place of publication Washington, DC, United States
Publisher American Society for Microbiology
Language eng
Formatted abstract
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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Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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