Date of Award
Doctor of Philosophy
Alan G. Atherly
The migration properties of circular, covalently closed (ccc) and linear DNAs were studied in pulsed-field gel electrophoresis as a model system for resolving large plasmids in the Rhizobiaceae. The effects of varying pulse time and agarose concentration on the electrophoretic mobility cccDNAs, ranging from 2 kilobase pairs (kb) to 16 kb, was investigated. We used both field inversion gel electrophoresis (FIGE) and electrophoresis in contour-clamped homogeneous electric fields (CHEF); both ccc and linear molecules had a minimum mobility as a function of pulse time in a CHEF apparatus. Linear and cccDNAs of the same size were affected differently by pulse time. The electrophoretic mobility of cccDNAs was pulse-time dependent in both electrophoretic systems;We used pulsed-field gel electrophoresis (PFG) and statistical analysis of Rhizobiaceae DNA sequences from GENBANK to identify rare-cutting restriction endonucleases. By using FIGE and CHEF, the genome sizes of Rhizobium meliloti and Bradyrhizobium japonicum were calculated. Comparisons were made between the genomes of free-living and bacteroid forms. The stability of genomic fingerprints produced by rare-cutting enzymes was studied on B. japonicum DNA plugs. It seems likely that large-scale DNA rearrangements do not occur during B. japonicum bacteroid development, after dedifferentiation, or by maintenance on rich media;FIGE and digestion with rare-cutting restriction enzymes were used to compare the genomes of B. japonicum field isolates from the 123 serocluster, as well as serotype strains USDA 123, USDA 127, and USDA 129. Genomic fingerprints (or field inversion gel electrophoretic types (FIGETs)), produced by four restriction enzymes, showed a high degree of diversity in the sizes of restriction fragments. Isolates could be classified according to FIGETs, and this classification was correlated with a serological classification. Hybridization with a nifHD gene probe to FIGE gels of B. japonicum DNA revealed that restriction fragment length polymorphisms (RFLPs) existed within members of serocluster 123 and provided further evidence of the relatedness between members of serogroups 123 and 127.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Bruno Walther S. Sobral
Sobral, Bruno Walther S., "Pulsed-field gel electrophoresis studies of Rhizobiaceae " (1989). Retrospective Theses and Dissertations. 9181.