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Alternative oxidase expression in Neurospora crassa
Lesley L Tanton1, Cheryl E Nargang, Katherine E Kessler
1Department of Biological Sciences, University of Alberta, Edmonton, T6G 2E9, Alberta, Canada
Fungal Genetics and Biology : FG & B
|June 5, 2003
Summary
The alternative oxidase (AOD) in Neurospora crassa is regulated post-transcriptionally. While transcription increases upon induction, protein levels are controlled, suggesting complex gene expression mechanisms.
Area of Science:
- Mitochondrial respiration
- Fungal gene expression
- Enzyme regulation
Background:
- Neurospora crassa possesses a KCN-insensitive alternative oxidase (AOD) encoded by the aod-1 gene, crucial when the main electron transport chain is inhibited.
- The induction mechanism for this alternative oxidase remains largely uncharacterized.
Purpose of the Study:
- To investigate the regulatory mechanisms controlling the induction of the alternative oxidase (AOD) in Neurospora crassa.
- To identify potential cis-regulatory elements and explore transcriptional and post-transcriptional control of the aod-1 gene.
Main Methods:
- Comparative sequence analysis of the aod-1 upstream region across fungal species.
- Electrophoretic mobility shift assays (EMSA) to assess transcription factor binding.
- Nuclear run-on assays to measure gene transcription rates.
- Western blot analysis to detect AOD protein levels.
- Northern blot analysis to examine aod-1 and aod-3 gene expression.
Main Results:
- A conserved cyclic AMP responsive element (CRE) was identified upstream of the aod-1 gene in multiple fungal species.
- EMSA confirmed specific binding of a nuclear factor to the CRE, but its removal did not abolish normal regulation.
- Nuclear run-on assays revealed increased aod-1 gene transcription upon antimycin A induction.
- Western blots showed a lack of detectable AOD1 protein in uninduced cells despite occasional aod-1 mRNA presence, indicating post-transcriptional regulation.
- A second potential alternative oxidase gene, aod-3, was identified but showed no detectable expression.
Conclusions:
- The regulation of alternative oxidase expression in Neurospora crassa involves both transcriptional activation and post-transcriptional control mechanisms.
- The identified CRE sequence may not be essential for the primary induction pathway, suggesting alternative regulatory elements or pathways.
- Post-transcriptional events significantly influence the final protein levels of alternative oxidase, highlighting the complexity of its expression regulation.