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In vivo generation of linear plasmids with addition of telomeric sequences by Histoplasma capsulatum
1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Histoplasma capsulatum is a dimorphic pathogenic fungus that is a major cause of respiratory and systemic mycosis. We previously developed a transformation system for Histoplasma and demonstrated chromosomal integration of transforming plasmid sequences. In this study, we describe another Histoplasma mechanism for maintaining transforming DNA i.e. the generation of modified, multicopy linear plasmids carrying DNA from the transforming Escherichia coli plasmid. Under selective conditions, these linear plasmids were stable and capable of retransforming Histoplasma without further modification. In vivo modification of the transforming DNA included duplication of plasmid sequence and telomeric addition at the termini of linear DNA. Apparently Histoplasma telomerase, like that of other organisms such as humans and Tetrahymena, is able to act on non-telomeric substrates. The terminus of a Histoplasma linear plasmid was cloned and shown to contain multiple repeats of GGGTTA, the telomeric repeat unit also found in vertebrates, trypanosomes, and slime moulds.
Insights
Histoplasma capsulatum can maintain foreign DNA through novel linear plasmids. These modified plasmids, generated in vivo, are stable and can retransform the fungus, revealing telomerase activity on non-telomeric DNA.
Area of Science:
- Mycology
- Molecular Biology
- Genetics
Background:
- Histoplasma capsulatum is a dimorphic fungus causing significant human mycoses.
- Previous work established a transformation system with chromosomal integration in Histoplasma.
- Understanding DNA maintenance mechanisms is crucial for fungal genetics.
Purpose of the Study:
- To investigate alternative mechanisms of transforming DNA maintenance in Histoplasma capsulatum.
- To characterize the nature and stability of foreign DNA constructs within the fungus.
- To explore the role of telomerase in processing non-native DNA.
Main Methods:
- Introduction of Escherichia coli plasmid DNA into Histoplasma capsulatum.
- Selection for transformed cells under specific conditions.
- Molecular analysis of maintained DNA, including sequencing and characterization of linear plasmids.
- Cloning and analysis of DNA termini from linear plasmids.
Main Results:
- Histoplasma generated modified, multicopy linear plasmids containing sequences from the transforming E. coli plasmid.
- These linear plasmids were stable under selective pressure and capable of retransformation.
- In vivo modification involved DNA duplication and telomeric addition at termini.
- The telomeric repeat sequence GGGTTA was identified at the termini, suggesting telomerase activity on non-telomeric substrates.
Conclusions:
- Histoplasma capsulatum possesses a mechanism for maintaining transforming DNA via stable, multicopy linear plasmids.
- The fungus's telomerase can process non-telomeric DNA substrates, adding telomeric repeats.
- This finding offers new insights into fungal DNA replication and genome stability.