Abstract
ArsA protein is the catalytic component of the bacteria plasmid R773-encoded ArsAB pump that is in involved in As3+ detoxification. Homologues of the ArsA protein are found in nearly all organisms but the biological functions of these homolog proteins are still unclear. The ArsA homologue in S. cerevisiae is encoded by the ORF YDL100c.
Initial studies show that deletion of YDL100c in S. cerevisiae was not lethal and had no effect on As3+ sensitivity at 30¢J. However, the disrupted strain (KO strain) is unable to grow at 40¢J and shows increased sensitivity to Co2+,Zn2+,As3+ and Sb3+ at 37¢J by spotting assay. In this study, a plasmid (YEp352) carrying the YDL100c under the control of its endogenous promoter was used to study the induction of YDL100c under various stress conditions. The data show that the expression of Ydl100cp increased 30 % at 37¢J compared to that at 30¢J, and the expression can be induced by low dosage of Zn2+, Ni2+, Sb3+ and neutral to alkaline pH. Overall, temperature is the best inducer for Ydl100cp expression.
Besides, searching Ydl100cp in Internet yeast two hybrid database and YDL100c promoter sequence analysis database suggest the following experiments and results:¡]1¡^2D gel electrophoresis assay to demonstrate different protein patterns between WT and KO strain under nonpermissive temperature. ¡]2¡^Flow cytometry data indicate most of KO strain cells growth arrest at G2/M phase in nonpermissive temperature. ¡]3¡^Microscopic data reveal KO stain cells grew very densely and showed cluster phenotype at nonpermissive temperature. When Congo red was used to stain cell wall¡Ait was found that these cluster cells is actually one cell. Although the cell wall between mother and daughter cell can form cleavage furrow, the formation is not complete and cell can¡¦t separate into two individuals. Consequently, the cells grow densely with cluster form and mega-polynuclear cells. It suggests Ydl100cp is induced and plays a role in cell cycle under nonpermissive temperature. The function of Ydl100cp may be a late mitosis cyclin-like protein or cyclin dependent kinase inhibitor that controls several downstream genes related to cell wall formation, maintenance, and structure. Because KO strain does not have Ydl100cp, it shows different growth patterns compared to WT strain when grow at nonpermissive temperature.
Initial studies suggest that YDL100c is involved in general responses because KO strain shows sensitivity to a broad range of metals. However, based on the results have, it is possible that Ydl100cp is involved in cell wall structure, formation and maintenance. Under nonpermission temperature cell wall of KO strain had defect that led to defect in ion transport structure. Therefore cell can remove not only can not poison metals especially Zn2+, Ni2+, Co2+, arsenite and antimonite metals right away but these metals can also pass cell wall into cytoplasm that causes KO strain reveals sensitivity to metals.
To sum up the results, the expression of Ydl100cp can be induced under nonpremssive temperature to decrease mega-polynuclear cells formation and control downstream genes for cell wall formation, maintenance and structure. Therefore yeast cells can survive at nonpermissive temperature instead to be killed.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0729102-122254 |
| Date | 29 July 2002 |
| Creators | Hung, Shih-Ya |
| Contributors | Ching-meu Hsu, David Chao, Z. H. Liu |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0729102-122254 |
| Rights | unrestricted, Copyright information available at source archive |
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