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Behaviour of a replicating mitochondrial DNA sequence from Aspergillus amstelodami in Saccharomyces cerevisiae and
R K Beri1, E L Lewis, G Turner
1Department of Microbiology, Medical School, University of Bristol, UK.
Abstract:
An amplified sequence of mitochondrial DNA from a ragged (rgd) mutant of Aspergillus amstelodami has been shown to exist in multimeric circular form, suggesting that it is excised from the genome and can exist independently of it. This sequence has replicative (ARS) activity in Saccharomyces cerevisiae, and a subfragment responsible for this activity has been identified and sequenced. A homologous sequence from Aspergillus nidulans mtDNA also has ARS activity in S. cerevisiae. Both A. amstelodami and A. nidulans ARS elements have been incorporated into the integrative transformation vector pDJB1 and the derived vectors used to transform A. nidulans. Inclusion of the A. nidulans ARS element enhanced the transformation frequency 5-fold relative to pDJB1. No increase in transformation frequency was evident with the ARS element from A. amstelodami. The stability of transformants was variable but in comparison to pDJB1, ARS-containing plasmids were mitotically unstable in A. nidulans. Although plasmid DNAs could be rescued in Escherichia coli from undigested transformant DNA, no freely replicating plasmids were detected by Southern hybridisation.
Insights
Researchers identified autonomously replicating sequences (ARS) in Aspergillus mitochondrial DNA. These ARS elements, when integrated into a vector, enhanced transformation frequency in Aspergillus nidulans, though plasmids remained mitotically unstable.
Area of Science:
- Molecular Biology
- Genetics
- Mycology
Background:
- Mitochondrial DNA (mtDNA) can be amplified and exist independently of the main genome.
- Autonomously Replicating Sequences (ARS) are crucial for independent DNA replication.
- Understanding ARS elements in fungi like Aspergillus is key for genetic manipulation.
Purpose of the Study:
- To investigate the ARS activity of amplified mtDNA sequences from Aspergillus amstelodami.
- To assess the functional ARS homology in Aspergillus nidulans mtDNA.
- To evaluate the utility of these ARS elements in fungal transformation vectors.
Main Methods:
- Isolation and characterization of amplified mtDNA sequences from Aspergillus amstelodami.
- Functional assay of ARS activity in Saccharomyces cerevisiae.
- Subcloning, sequencing, and integration of ARS elements into the integrative transformation vector pDJB1 for Aspergillus nidulans transformation.
Main Results:
- An amplified Aspergillus amstelodami mtDNA sequence exhibited ARS activity, with a functional subfragment identified and sequenced.
- A homologous sequence from Aspergillus nidulans mtDNA also demonstrated ARS activity in Saccharomyces cerevisiae.
- Integration of the Aspergillus nidulans ARS element increased transformation frequency fivefold in Aspergillus nidulans, while the Aspergillus amstelodami ARS showed no significant enhancement. ARS-containing plasmids exhibited mitotic instability.
Conclusions:
- Fungal mtDNA contains functional ARS elements that can be leveraged for genetic studies.
- The Aspergillus nidulans ARS element shows potential for enhancing transformation efficiency in its homologous species.
- Further research is needed to improve the mitotic stability of ARS-containing plasmids in Aspergillus.