Molecular genetic characterization of a highpH sensitive strain of fission yeast, Schizosaccharomyces pombe

• Main Author: Saleki, Reza
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-1997
• Subjects: Genetics; Molecular biology
• ISBN: 9780612205833; 0612205835

A strain of Schizosaccharomyces pombe carrying a disrupted $\rm Na\sp+/H\sp+$ antiporter (sod2::sup3-5) gene, in addition to the common auxotrophic mutations ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Genetic and physiological characterization of this strain revealed that high pH sensitivity was due to the uracil and leucine auxotrophies and was dependent on the presence of a rich source of nitrogen such as $\rm NH\sb4\sp+$ in the medium. Reversion analysis of the high pH sensitive strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of these strains, called J227, was conducted. Two independently segregating mutations named pub1-1 (protein ubiquitin ligase) and elp3-1 (elongated at low pH) were identified as extragenic suppressors of high pH sensitivity in $\rm NH\sb4\sp+$-containing medium. The pub1 and elp3 gene products seem to be involved in $\rm NH\sb4\sp+$-induced inactivation of uracil and leucine permeases. Unlike wild type cells, J227 cells (pub1-1 elp3-1) are elongated and incapable of colony formation at external pH 3.5. Genetic dissection of this strain indicated that growth inhibition at pH 3.5 was caused by pub1-1 mutation. The cell elongation phenotype is mainly caused by elp3-1 mutation but is extended as a result of synergistic interactions between the two mutations at pH 3.5. A genomic clone of pub1 was isolated by complementation of the low pH sensitivity phenotype in the J227 background. The pub1 gene encodes a 767 amino acid-protein that is interrupted by 3 introns near its N terminus. The predicted pub1 protein contains a C2 domain near its N terminus followed by three repeats of the WW domain and a putative protein ubiquitin ligase catalytic domain at its carboxyl terminus. Sequences with strong homology to pub1 have been identified in S. cerevisiae, human, mouse and rat. These proteins belong to a conserved family of E3 type protein ubiquitin ligases. Attempts have been made to clone the elp3 gene by visual selection for reversal of the cell elongation phenotype at pH 3.5 in an elp3-1 background. Also, insertional mutagenesis is used to target the elp3 sequence in a pub1-1 background and relies on positive selection for leucine auxotrophic cells capable of growth at pH 6.8 in $\rm NH\sb4\sp+$-containing medium.