An in vivo assay for 2 [mu] plasmid recombination / by Gregg Howe.
| Author/creator | Howe, Gregg author. |
| Other author | Bruschi, Carlo V., degree supervisor. |
| Other author | East Carolina University. Department of Biology. |
| Format | Theses and dissertations |
| Production | 1987. |
| Description | 65 leaves : 13 illustrations ; 28 cm |
| Supplemental Content | Access via ScholarShip |
| Subjects |
| Summary | The 2[mu] plasmid o-f Saccharomyces cerevisiae encodes a site-specific recombinase called FLP which catalyses efficient recombination between two 599 base pair inverted repeats located on the plasmid. The purpose of this study was to develop an in vivo assay for phenotypic detection of FLP-mediated recombination. A tester plasmid was constructed such that the yeast LEU2 and ADE8 genes are flanked on either side by directly oriented copies of the 2[mu].i plasmid repeat sequences. FLP-mediated recombination between these sequences in vivo produces two circular DNA molecules, one containing LEU2 and the 2[mu] origin of replication and the other containing ADE8. The loss of the ADE8-containing circle during subsequent cell divisions, as a result of its inability to replicate autonomously, is detected in this assay as a phenotypic shift from red colonies to white colonies. This system was used to establish that recombination between 2p plasmid repeat sequences occurs at frequencies close to 100% in FLP-r cells. Interestingly, in flp- cells recombination is not fully abolished but rather continues at a frequency of about 15%. Experiments demonstrating that these residual events do not require the FLP recognition sequence suggest that they are mediated by a chromosomally-encoded homologous recombinase. In addition to observing the expected phenotypes, several distinct classes of red and white variegated colonies also occurred. An examination of the recombination products in these cells showed that these patterns are the manifestation of intra- as well as intermolecular recombination. Quantitative measurements of these products indicate that intramolecular recombination is more frequent then intermolecular recombination in vivo. A simple model explaining these findings is proposed. |
| General note | Submitted to the faculty of the Department of Biology. |
| General note | Advisor: Carlo V. Bruschi |
| Dissertation note | M.S. East Carolina University 1987 |
| Bibliography note | Includes bibliographical references (leaves 32-35). |
| Genre/form | dissertations. |
| Genre/form | Academic theses. |
| Genre/form | Academic theses. |
| Genre/form | Thèses et écrits académiques. |
Availability
| Library | Location | Call Number | Status | Item Actions |
|---|---|---|---|---|
| Joyner | University Archives | ASK AT SPECIAL COLLECTIONS DESK | ✔ Available | Request Material |
| Joyner | Microforms | MICROFILM | ✔ Available | |
| Joyner | General Stacks | QH605 .H68X 1987 | ✔ Available | Place Hold |
| Electronic Resources | Access Content Online | ✔ Available |