Forensic DNA typing.
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DNA Fingerprinting↗
Biomedical subjects
R A Bever
Publications and source records attributed to R A Bever.
Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene.
The elastase structural gene (lasB) from Pseudomonas aeruginosa PAO1 has been previously cloned on an 8-kilobase (kb) DNA fragment. The lasB gene, cloned in both orientations in pUC18, produced elastase in Escherichia coli, indicating that its promoter and translation initiation sites are functional in E. coli. Deletion analysis further defined the location of the lasB gene to a 3.0-kb EcoRI-KpnI fragment (pRB1803). Elastase prepared from E. coli TB1 (pRB1803) corresponded in molecular weight to mature P. aeruginosa extracellular elastase (33,000). The lasB gene directed the synthesis of 54- and 50-kilodalton (kDa) proteins in a bacterial cell-free transcription-translation system. The 33-, 50-, and 54-kDa proteins reacted with elastase-specific antiserum. To further characterize the lasB gene, the nucleotide sequence of the 3.0-kb EcoRI-KpnI fragment was determined. This DNA fragment contained a 1,491-base-pair open reading frame encoding 498 amino acids, corresponding to a predicted molecular weight of 53,600. The deduced amino acid sequence contained a putative signal sequence followed by a large polypeptide which preceded the mature 33-kDa elastase protein. Three zinc ligands and an active site were predicted for the mature elastase on the basis of its amino acid sequence homology with Bacillus thermoproteolyticus thermolysin.
Amino Acid Sequence↗
Cloning and characterization of elastase genes from Pseudomonas aeruginosa.
A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.
Antigens, Bacterial↗