Deoxyribonucleic acid restriction/modification systems and gene transfer strategies in Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1
Morrison, Mark
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Permalink
https://hdl.handle.net/2142/21717
Description
Title
Deoxyribonucleic acid restriction/modification systems and gene transfer strategies in Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1
Author(s)
Morrison, Mark
Issue Date
1991
Doctoral Committee Chair(s)
Mackie, Roderick I.
Department of Study
Animal Sciences
Discipline
Animal Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Agriculture, Animal Culture and Nutrition
Language
eng
Abstract
One goal critical to the use of recombinant DNA technologies with rumen bacteria is the establishment of a stable DNA transfer system. Research to date has shown that Ruminococcus can be classified as a genus resistant to transformation, and that electroporation may offer the only means to introduce foreign DNA. This thesis aims to elucidate and solve limitations to the utilization of electroporation with Ruminococcus albus 8 and Ruminococcus flavefaciens FD-1. The limited degradation of DNA by restriction enzymes and confirmation of plasmid uptake were given priority, although the consequences of incompatible plasmid replicons and failure in the expression of selectable markers cannot be overlooked. Fluorescent labelled dextrans were used in place of plasmid DNA and indicated that electroporation resulted in the uptake of macromolecules. Type-II endonuclease activities were present in most strains of Ruminococcus tested. However, the cell-free extract of R. flavefaciens FD-1 did not provide a simple DNA protection strategy, so the restriction/modification systems of R. albus 8 and R. flavefaciens FD-1 were studied in more detail. Plasmids derived from a dam proficient Escherichia coli background are protected against the Type-IIS restriction enzyme of R. albus 8. Adenine methylation by M.TaqI and a methylase from Chlorella will inhibit DNA cleavage by rflFI and rflFII, respectively. The initial electroporation experiments utilizing methylated DNA were unsuccessful in yielding phenotypic transformants of R. albus 8. Plasmid DNA isolated directly from Ruminococcus would be a valuable tool in addressing some of the problems still affecting gene transfer. The plasmid pBAW301, present in R. flavefaciens strain R13c2, was isolated and is sufficiently small to facilitate construction of potential shuttle vectors as well as broad host range, chimeric plasmids. Knowledge of DNA modification in Ruminococcus has been obtained. Other species possessing stable plasmids and gene/s encoding antibiotic resistance have also been identified. Greater emphasis can now be placed on the electroporation technique itself, as well as a wider range of selective markers. Ruminococcus spp. require further investigation if model genetic systems are to be developed and some of the proposed goals of molecular biology research for this genus are to be fully realized.
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