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Updated: Jun 18, 2026
Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast
Published on: May 15, 2018
Reverse genetics through random mutagenesis in Histoplasma capsulatum
Brian H Youseff1, Julie A Dougherty, Chad A Rappleye
1Departments of Microbiology and Internal Medicine, The Center for Microbial Interface Biology, Ohio State University, Columbus, OH 43210, USA. youseff.1@osu.edu
Background:
The dimorphic fungal pathogen Histoplasma capsulatum causes respiratory and systemic disease in humans and other mammals. Progress in understanding the mechanisms underlying the biology and the pathogenesis of Histoplasma has been hindered by a shortage of methodologies for mutating a gene of interest.
Results:
We describe a reverse genetics process that combines the random mutagenesis of Agrobacterium-mediated transformation with screening techniques to identify targeted gene disruptions in a collection of insertion mutants. Isolation of the desired mutant is accomplished by arraying individual clones from a pool and employing a PCR-addressing method. Application of this procedure facilitated the isolation of a cbp1 mutant in a North American type 2 strain, a Histoplasma strain recalcitrant to gene knock-outs through homologous recombination. Optimization of cryopreservation conditions allows pools of mutants to be banked for later analysis and recovery of targeted mutants.
Conclusion:
This methodology improves our ability to isolate mutants in targeted genes, thereby facilitating the molecular genetic analysis of Histoplasma biology. The procedures described are widely applicable to many fungal systems and will be of particular interest to those for which homologous recombination techniques are inefficient or do not currently exist.
Insights
Researchers developed a new reverse genetics method to efficiently create gene mutations in Histoplasma capsulatum. This breakthrough aids in understanding fungal pathogens and developing new treatments.
Area of Science:
- Mycology
- Pathogen Biology
- Molecular Genetics
Background:
- Histoplasma capsulatum is a dimorphic fungus causing disease in mammals.
- Understanding its biology and pathogenesis is limited by a lack of gene mutation tools.
Purpose of the Study:
- To develop and validate a novel reverse genetics methodology for Histoplasma capsulatum.
- To overcome limitations of existing gene knockout techniques in this fungal pathogen.
Main Methods:
- Agrobacterium-mediated transformation with random mutagenesis.
- Screening techniques including PCR-addressing for targeted gene disruption.
- Optimization of cryopreservation for mutant library banking.
Main Results:
- Successful isolation of a cbp1 mutant in a recalcitrant Histoplasma strain.
- Demonstration of a functional reverse genetics system for gene targeting.
- Established cryopreservation protocols for mutant recovery.
Conclusions:
- The new methodology significantly enhances the ability to isolate targeted gene mutants in Histoplasma.
- This approach facilitates molecular genetic analysis of fungal biology and pathogenesis.
- The technique is broadly applicable to other fungi, especially those with inefficient homologous recombination.
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