Aspergillus nidulans is a filamentous fungus whose cells have highly polarized growth. This requires many genes including hypA, which affects hyphal morphogenesis by promoting tip cell growth and suppressing growth in basal regions. The hypA locus was cloned previously by complementing the hypA1 phenotype. The hypA orthologues in Saccharomyces cerevisiae and Schizosaccharomyces pombe are essential, whereas hypA deletion strains in Aspergillus nidulans are viable but morphologically abnormal.
The A. nidulans hypA locus has two temperature sensitive alleles, hypA1 and hypA6. These alleles were generated independently, but using the same mutagen, and were identified and characterized by different groups. Although the hypA1 strain has been shown to sporulate at restrictive temperature, the hypA6 strain was described as having a restrictive arrest phenotype, which suggested it was a lethal defect and was at odds with the report that hypA was dispensable for growth. To resolve this discrepancy, my study compared the hypA1 and hypA6 strains using morphometry, and compared their genetic lesions. I show that the hypA1 and hypA6 phenotypes are indistinguishable and mutation events in their coding regions are identical.
Both hypA1 and hypA6 strains were able to sporulate at restrictive temperature as shown by scanning electron microscopy. Their cell morphologies were indistinguishable after growth under restrictive conditions, as were their repolarization kinetics when restrictive-grown cells were shifted to permissive temperature.
Sequencing the hypA locus from a hypA6 strain, and comparing it to a hypA1 strain showed that they had identical lesions (G329R), which is consistent with the morphometric analysis. Unexpectedly, the hypA1 and hypA6 strains shared additional non-conservative amino acid substitutions, K885F and E932K, with their wildtype parent A28 compared to the strain used for complementing hypA1. The repair of these two amino acid changes can not rescue hypA1 or hypA6 phenotype at 42C.
The S. cerevisiae homologue of hypA protein is TRS120p, which is involved in endomembrane trafficking. FM4-64 endomembrane staining of hypA1 and hypA6 strains showed they had aberrant endomembrane arrays at restrictive versus permissive temperature, which recovered the wildtype pattern after a return to permissive conditions. This is consistent with the similarities between the hypA and TRS120 sequences.
The disparity between the published descriptions of the hypA1 and hypA6 restrictive phenotypes has been resolved, and the allele renamed hypA1/A6.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-08292003-142249 |
| Date | 03 September 2003 |
| Creators | Sha, Yu |
| Contributors | Kaminskyj, Susan G. W., Hemmingsen, Sean M., Chivers, Douglas P., Buchanan, Fiona C., Wei, Yangdou |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-08292003-142249/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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