A search of the Saccharomyces genome data base revealed an open reading frame of 2039 amino acids with homology to L-type calcium channels. Northern blots probed with a 540 bp PCR product of the ORF showed a transcript of 6.1 kb. Two procedures were used to disrupt the gene: the ORF was truncated by an integrative disruption after the third pore motif, or deleted in the first three pore domains using a one-step disruption construct. In most strains tested, the disruptions gave no apparent phenotype when tested under a variety of conditions. However, conspicuous phenotypes were seen in the strain YEL161-2A, a strain super-sensitive to alpha-mating factor (sst1). In most respects, truncation gave a less severe phenotype than deletion, suggesting that the truncated gene retains partial function. Calcium uptake during normal growth, as well as the increased calcium uptake in response to mating factor, were reduced progressively by the truncation and deletion respectively. Growth rate and cell viability were reduced, cell size heterogeneity increased, and recovery from mating factor arrest was delayed and abnormal. The cells became sensitive to MnCl$ sb2.$ The phenotype resulting from gene truncation was alleviated by a high-calcium medium, and exacerbated by low calcium. Complementation of the deleted strain by a Yep13 plasmid containing BAR1 (SST1) restored normal growth and viability. However, somewhat paradoxically, deletion of the putative calcium channel gene in another sst1 strain (SY1159) showed no phenotype.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.27302 |
| Date | January 1996 |
| Creators | Cho, John Myung-Jae. |
| Contributors | Poole, Ronald J. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001561768, proquestno: MQ29677, Theses scanned by UMI/ProQuest. |
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