| dc.contributor.author | Mosher, Roy Henry. | en_US |
| dc.date.accessioned | 2014-10-21T12:33:27Z | |
| dc.date.available | 1993 | |
| dc.date.issued | 1993 | en_US |
| dc.identifier.other | AAINN93711 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/55389 | |
| dc.description | A chloramphenicol resistance determinant from S. venezuelae ISP5230 genomic DNA, originally cloned as a 6.5-kb SstI-SstI fragment in the recombinant plasmid pJV3, was localized by in vitro deletion to a 2.4-kb KpnI-SstI DNA fragment in the recombinant plasmid pJV4. When subcloned in the phagemid vectors pTZ18R and pTZ19R, neither the 2.4-kb nor 6.5-kb fragment conferred chloramphenicol resistance on Escherichia coli. | en_US |
| dc.description | DNA sequencing and nucleotide sequence analysis of the 2.4-kb fragment, predicted the presence of three complete and one incomplete open reading frames. The largest open reading frame, orf-4, encoded a hydrophobic polypeptide that showed significant sequence similarity to the putative chloramphenicol-efflux proteins of Streptomyces lividans and Rhodococcus fascians. Located immediately downstream of orf-4 was an open reading frame (orf-3) encoding a polypeptide (Orf3) that showed limited but significant sequence similarity to a number of proteins that required nucleotide co-factors. The discovery of a conserved ATP/GTP-binding site motif near the amino-terminus of Orf3, suggested a biological process such as a phosphotransferase reaction requiring a high-energy co-factor. A comparison of the derived amino acid sequences for orf-1 and orf-2 with those in current databases showed no significant similarities. | en_US |
| dc.description | Streptomyces lividans transformants RM3 and RM4, carrying plasmids pJV3 and pJV4 respectively, rapidly metabolized chloramphenicol to one predominant product. Structure analysis based on $\sp1$H-NMR and $\sp $C-NMR spectra of the purified product showed the substance to be related to chloramphenicol but, with an electron-withdrawing group attached to carbon-1$\sp\prime$. $\sp{31}$P-NMR and mass spectroscopy established that the compound was chloramphenicol-1$\sp\prime$-phosphate. The results strongly suggest that orf-3 encodes a chloramphenicol kinase activity that detoxifies chloramphenicol by O-phosphorylation at carbon-1$\sp\prime$. In a bioassay using the chloramphenicol-hypersensitive Micrococcus luteus, chloramphenicol-1$\sp\prime$-phosphate showed negligible antibiotic activity when compared to chloramphenicol. | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 1993. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Molecular. | en_US |
| dc.subject | Biology, Microbiology. | en_US |
| dc.title | DNA sequencing and characterization of a chloramphenicol resistance gene from Streptomyces venezuelae. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
