Glycolytic genes from Escherichia coli as evolutionary probes

Lyons, Lian (1986) Glycolytic genes from Escherichia coli as evolutionary probes. Masters thesis, Memorial University of Newfoundland.

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Abstract

Comparative studies based on the structure of ribosomes and their components suggested that there are three primary kingdoms: eubacteria, archaebacteria and urkaryotes. However, the validity of this hypothesis is being questioned. In order to resolve the evolutionary relationships among bacteria it is necessary to compare the primary structures of molecules other than those of the translational apparatus. The main objective of this project was to determine whether or not hybrid recombinant plasmids that contain sequences for glycolytic enzymes from Escherichia coli could be used to detect corresponding sequences in preparations of genomic DNA from distantly related organisms. Three plasmids from the Clarke-Carbon E. coli genomic library were chosen for this study. The plasmid pLC 16-4 contains the genes for triose-phosphate isomerase and phosphofructo-kinase, whereas it had been reported that pLC 10-47 and pLC>11-8 both contain the gene for enolase. Restriction endonuclease maps were constructed for these plasmids, and the location of the cloning vector was determined in each case by Southern blotting and probing with Co1 E1. Comparison of the restriction maps of pLC 10-47 and pLC 11-8 indicated that there were no sequences in common in the genomic inserts of these plasmids. Subsequent studies at the protein level showed that only pLC 10-47 contains a gene coding for enolase. Genomic DNA was prepared from representative species of the Enterobacteriaceae, other gram-negative bacteria, gram-positive bacteria, and archaebacteria. The genomic DNA was subjected to Southern blotting and probed with radioactively-labelled pLC10-47 or pLC 16-4. The results from these hybridization studies indicated that genes encoding glycolytic enzymes in E. coli were not able to cross-react with DNA from species that had diverged from E. coli more than 40 million years ago. Therefore, the genes for glycolytic enzymes will not be of use as long range evolutionary probes.

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