Enzymatic construction of bacteriophage øx174 mutants with increased genome sizes / by Perry Walker Russell.
| Author/creator | Russell, Perry Walker author. |
| Other author | Müller, U. R. (Uwe R.), degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 1983. |
| Description | v, 73 leaves : illustrations ; 28 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | The icosahedral bacteriophage ØX174 is a single-stranded DNA virus with a genome containing 5,386 nucleotides. It codes for 10 genes and has four intergenic or intercistronic regions which are transcribed but not translated. One of these regions located between the two major capsid component genes J and F has been proven non-essential. Insertion of additional nucleotides into a portion of this region did not disrupt normal phage growth. The described work made use of this information to investigate how much DNA could be inserted into the ØX174 genome before phage viability was lost. This was attempted experimentally using modern recombinant DNA methodology to construct ØX174 genomes with increasing genome sizes in vitro and introducing these into the appropriate host cell. Before this could be accomplished, it was necessary to devise a biochemical scheme for the construction and isolation of such mutants. The scheme involved site-specific mutagenesis of the J-F intercistronic region of a previously constructed insertion mutant of ØX174. Cleavage of a unique site in the J-F region of this mutant with a restriction endonuclease allowed for insertion of DNA fragments of varying sizes by an enzyme which ligates DNA ends together. Successful insertions are a relatively rare event in this reaction, but since the original unique restriction site is lost in these cases, the desired mutant genomes could be enzymatically selected for. By using the appropriate combination of restriction enzymes, genomes without an insert (i.e. parental) were fragmented and rendered non-infective. Subsequent infection of E. coif host cells resulted in propagation of mutant bacteriophages which could be isolated and studied. This scheme was used in three separate insertion experiments resulting in several mutants with genomes larger than any previously constructed ØX174 phage. The largest of these contains an insert which increases its genome approximately 340 nucleotides above that of wt ØX174. The conclusions drawn from this study are as follows: a) The genome of ØX174 can be enlarged by at least 6.3% without loss of viability, b) The maximum number of nucleotides which can be inserted remains to be determined but appears to be less than 550 (10.2% of the genome), c) The insertion mutants obtained appear to grow less efficiently than the parental strain, d) The system of overlapping genes in the single-stranded icosahedral DNA phages must have evolved due to factors other than physical constraints in packaging the DNA into the phage head. |
| General note | "Presented to the faculty of the Department of Biology ... in partial fulfillment of the requirements for the degree Master of Sciences in Biology." |
| General note | Advisor: Uwe R. Muller |
| Dissertation note | M.A. East Carolina University 1983 |
| Bibliography note | Includes bibliographical references (leaves 65-73). |
| Genre/form | Academic theses. |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |
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