One way to approach the question of the evolution of bacteria and bacteriophages is by examining their DNA sequence homology. The electron microscope is a tool that allows the detection and measurement of regions of single- and double-stranded DNA. By using the techniques of DNA heteroduplex formation and DNA spreading I was able to use the electron microscope to seek regions of DNA sequence homology between various pairs of bacteriophages within the lambdoid family and between the tna and tar genes in the Escherichia coli L coli chromosome. Various phage DNA's were annealed in the presence of formamide and the resulting heteroduplex molecules were examined by electron microscopy (Davis and Davidson, 1968; Westmoreland et al, 1969; Davis et al, 1970). Regions of homology in such heteroduplexes appeared as double-stranded segments; regions of nonhomology gave two separate single-stranded segments. Regions of homology and nonhomology were mapped in heteroduplexes made between the DNA's of the various lambdoid coliphages and between A-transducing phages carrying the tr and tna genes of E. coli. This technique was also used to determine the sizes of various DNA molecules and DNA segments. The sizes of the following DNA molecules and DNA fragments were determined: the double-stranded DNA of plasmid pSC101 (19.0 + 0.2%A (s.d.; n = 25)); the single-stranded DNA of bacteriophage M13 (12.7%a); the DNA of bacteriophage A (49,150 ± 730 base pairs (s.d.; n = 25)); the DNA of bacteriophage PA2 (99.6 + 2.0%A (s.d.; n - 19)); the DNA of bacteriophage 424 (96.4 +•1.3%A (s.d.; n = 20)); the HindIII restriction fragment carrying the tna gene of E. coli (12.0 + 0.5%A (s.d.; n = 15)); the HindIII restriction fragment carrying the trpA, trpB and trpC genes of E. coli (11.1 ± 0.4%A (s.d.; n = 38)); the immunity region of a immX) (8.0 + 0.7%A (s.d.; n = 15)); the immunity region of bacteriophage 21 imm21) (3.6 ± 0.4%A (s.d.; n = 15)) and; the nin region of A (5.4 ± O.4%A (s.d.; n = 15)). These values are expressed as the mean ± standard deviation. The number of base pairs comprising A DNA was deduced from the two spreadings A with pSC101 and pSC101 with $X174 (double-stranded); the size of $X174 was determined to be 5386 bases (Sanger et al, 1978). Other DNA segments within the heteroduplexes were also measured. The following heteroduplexes between pairs of lambdoid phages were analyzed x/424, A/PA2, 434/424, 434/21, 434/PA2, 424/PA2 and 21/PA2. The total amount of homology between each pair was 48.3%, 56.7%, 45.4%, 42.7%, 27.8%, 56.6%, 53.3% and 37.4%, respectively. My results indicate that it is likely that the processes envisaged in the modular hypothesis and in the ancestral chromosome hypothesis were important in the evolution of the lambdoid phages. Lambda-transducing phages carrying the trpABC and tna bacterial fragments,generated by restriction of E. coli and A DNA's (with the endonuclease HindIII), followed by ligation and selection for complementation by these genes, were allowed to form heteroduplexes using various denaturing and spreading conditions. The following heteroduplexes were analyzed: NH540/Atna immX c1857 nin+; AtrpABC1/AtrpABCr; A/AtrpABC1; A/AtrpABCr; AtrpABC1/Atna imml cI857 nin-F and; AtrpABC/Atna ion 1857 nin+. The DNA fragments containing the trpABC and tna genes were found to share no more than 58% homology by using the least denaturing conditions possible.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:659841 |
| Date | January 1980 |
| Creators | Myers, Rhonda Jan |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15470 |
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