The objective of this project is to ask what kinds of DNA rearrangement cause changes in genome structure over time. To answer this question, physical maps of the chromosomes of two closely related isolates of H. halobium will be constructed and compared. This archaebacterium was chosen because of the interest in its evolutionary status and published data suggesting that stable and hypervariable domains are interspersed within the chromosome. Strains NRC-1 and S9 will be studied because of evidence that their genomes have changed substantially since they diverged 18 years ago. Two methods will be combined to obtain a chromosomal map for each strain. The first is low resolution restriction mapping of uncloned chromosomal DNA digested with enzymes that cut infrequently. The resulting large restriction fragments will be separated by pulsed-field gel electrophoresis. Southern blotting with previously clones genes and probes overlapping the ends of the large restriction fragments (junction clones) will determine which of these fragments are adjacent. All available cloned genes will be located within the map of both strains to assess the possible linkage of functionally related genes, such as those for the four opsins. On the basis of this map domains that appear to differ between the two strains will be identified. These will then be subject to more detailed restriction mapping by rapid chromosome walking. The variable restriction fragments will be cloned and random clones subject to detailed restriction mapping. The data will be digitized and processed by computer programs which identify significant overlaps between clones and allow the determination of composite restriction maps corresponding to portions of the genome. Additional Southern blots will be used to verify the deduced history of genome rearrangement over time. Comparison of the chromosomal maps for the two strains will show the types of mutation that lead to the evolution of the H. halobium genome.
| Hackett, N R; Bobovnikova, Y; Heyrovska, N (1994) Conservation of chromosomal arrangement among three strains of the genetically unstable archaeon Halobacterium salinarium. J Bacteriol 176:7711-8 |
