In this work the abundance, structure, distribution and possible role of the inverted repeat sequence DNA (IR DNA) of R. vannielii was investigated. Approximately 7% of the R. vannielii genome was found to exist as inverted repeat sequences by nuclease Si digestion of heat-denatured, rapidly renatured DNA; this compared with 3% for E. coli K12 DNA. The inverted repeat sequence DNA of R. vannielii formed two size classess a heterogeneous high molecular weight class with a size range of 100-700 base pairs (bp), and a low molecular weight class comprised of fragments of 17 and 27bp. The genomic distribution of these two inverted repeat classes was investigated by DNA/DNA hybridization studies. In vitro 3=P-labelled IR DNA was used to probe Southern blots of EcoRI and Hindi 11 restriction enzyme digests of R. vannieli i DNA. Doth classes of inverted repeat DNA showed hybridization with many bands throughout the restriction digests, suggesting that the sequences were not clustered but were dispersed throughout the genome. Low molecular weight IR DNA however, hybridized to four specific bands in an Haelll digest of R. vannielii DNA suggesting that this enzyme could reveal some repetitive structure in the genome which the other enzymes used could not. Low molecular weight IR DNA was also found to hybridize throughout the high molecular weight IR DNA class, indicating that the two IR DNA classes share some sequences and may be derived from the same chromosomal loci. Competition filter-binding assays, designed to detect protein-DNA interactions, showed that about BX of high molecular weight IR DNA sequences appeared to be bound specifically by R. vannielii protein while low molecular weight IR DNA was not bound. The genomic plasticity of R. vannielii was investigated by hybridization and by a dual-label 1ing method. Although these experiments gave inconclusive results there were indications that sequence rearrangements might occur during R. vannielii swarmer cell differentiation. Attempts to clone IR DNA directly by two methods had only limited success because of difficulties in the identification of IR DNA-containing recombinant plasmids and suggested that cloning of IR DNA should be done by hybridization screening of chromosomal gene libraries. The serine hydrolases of the differentiating swarmer cell were also examined. Using an inhibitor labelling technique up to eight bands could be identified on fluorographs of SDS gradient gels and a number of changes in banding were observed to occur during swarmer cell differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:353069 |
| Date | January 1984 |
| Creators | Russell, George Cameron |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/112048/ |
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