- Collection:
- Atlanta University and Clark Atlanta University Theses and Dissertations
- Title:
- Isolation and characterization of membrane-associated forms of penicillinase plasmid (pi524) DNA in staphylococcus aureus., 1986
- Creator:
- Latinwo, Lekan Mustapha
- Date of Original:
- 1986-05-01
- Subject:
- Degrees, Academic
Dissertations, Academic - Location:
- United States, Georgia, Fulton County, Atlanta, 33.749, -84.38798
- Medium:
- theses
dissertations - Type:
- Text
- Format:
- application/pdf
- Description:
- The investigation of bacterial plasmid DNA maintenance requires an understanding of the relationship of these molecules to other cellular components. Our studies on the relationship of penicillinase plasmid (pI524) DNA with its host bacterial (Staphylococcus aureus) membrane have shown that the membrane-associated forms of this plasmid could be isolated from exponentially grown cells lysed on sucrose gradient. The identity of the plasmid pi524 DNA regions as covalently closed circular (CCC) duplex monomer DNA molecules was confirmed by both CsCl-ethidium bromide density centrifugation and agarose gel electrophoresis. Analysis of the putative plasmid-membrane complexes isolated on clear lysate on sucrose were performed on various density gradients. The results indicated that approximately 23% of plasmid pI524 DNA was stably associated to bacterial cell membrane fractions, and suggested that one of the three copies of this plasmid per cell was complexed to the cellular membrane. An analysis of the protein patterns of the complexes on sodium dodecyl sulfate-polyacrylamide gels shows similarity in protein patterns of complexes to purified membranes of Su aureus. Studies on effects of various enzymes, e.g., ribonuclease and protease, as well as antibiotics, rifampicin and chloramphenicol on complexing have suggested the possible involvement of protein(s) over RNA in mediating the complexing of this plasmid to its cell membrane. Nuclease activity associated with this complex was observed when purified CCC molecules of pi524 or lambda DNA were incubated with the complexes in the presence of Mg2+ ions. Degradation of these molecules was shown both by agarose gel electrophoresis and by sedimentation in sucrose gradient analysis. In vitro membrane binding studies have shown that purified covalently closed circular of plasmid p1524 DNA could be found complexing to membrane when incubated in the presence of crude (membrane) extracts. Exposure of these complexes to high concentrations of salt (KC1, NaCl, or CsCl) resulted in a separation of membrane-associated forms when compared to salt free samples. This indicates the presence of two possible types of DNA-membrane binding sites i.e., salt sensitive (unstable binding) and salt insensitive (stable complexing). Whether the association of this plasmid to cell membrane serves to fulfill the various functions involved in plasmid maintenance needs further studies. However, data from these studies support the in vivo binding of pI524 to its cell membrane and suggest that this complexing is mediated by proteins.
Degree type: dissertation
Degree name: Doctor of Philosophy (PhD)
Department: Department of Biology
Advisor: Archibold, Errol - Metadata URL:
- http://hdl.handle.net/20.500.12322/cau.td:1986_latinwo_lekan_mustapha.pdf
- Holding Institution:
- Atlanta University Center Robert W. Woodruff Library
- Rights:
-
