Date of Award
Doctor of Philosophy
Zoology and Genetics
Alan G. Atherly
Partial physical maps of each of the three replicons of Rhizobium meliloti were constructed using restriction enzymes PacI, SpeI, and AseI. Using transverse alternating field gel electrophoresis (TAFE) in combination with very long pulse times, low agarose concentrations, and low field gradients, or in combination with PacI restriction enzyme digestions, the three replicons of R. meliloti 1021 were separated. Three bands were separated from undigested genomic DNA samples using TAFE; Southern hybridizations with representative cloned genes from each replicon revealed that each band corresponded to one of the replicons. The three bands were excised, digested with SpeI, and separated using field inversion gel electrophoresis (FIGE). A total of 35 SpeI fragments were generated; 19, 10, and 6 SpeI fragments were assigned to the chromosome, pSym-a, and pSym-b, respectively. PacI digestion of total genomic samples produced four fragments. Southern hybridizations with replicon-specific gene probes showed that pSym-b contains two PacI sites, while the chromosome and pSym-a each contain one PacI site;Pulsed-field gel electrophoresis techniques were also applied to the physical mapping of the soybean genome. Techniques for the isolation of high molecular weight DNA in embedded soybean protoplasts, the use of statistical methods to identify restriction enzymes for generating large DNA fragments, and the conditions for separating large DNA fragments using transverse alternating-field electrophoresis (TAFE) and an electrophoretic device (ED) were presented. Complete digestion was confirmed by Southern hybridization using single copy clones of nodule-specific proteins (nodulins) expressed during symbiosis. Identical hybridization patterns with two nodulin clones, pNod15 and pNod24, for four restriction enzymes suggest linkage between these nodulins. These data are being used to generate a physical map of the nodulin region(s) of the soybean genome.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Rhonda Jean Honeycutt
Honeycutt, Rhonda Jean, "Physical mapping of Rhizobium meliloti and Glycine max L. (Merr.) using pulsed-field gel electrophoresis " (1991). Retrospective Theses and Dissertations. 9533.