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Cloning and analysis of an actin-encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum
1University of Southern Mississippi, Department of Biological Sciences, Hattiesburg 39406-5018, USA.
Abstract:
We have cloned an actin-encoding cDNA from the dimorphic fungus Histoplasma capsulatum, an important pathogen of humans. The predicted amino acid sequence as well as the general codon pattern of Histoplasma actin revealed the highest degree of similarity to the actin of the filamentous ascomycete Aspergillus nidulans. Southern blot analysis determined that actin was encoded by a single copy in the Histoplasma genome. Northern blot analysis showed a single 1700 nt transcript in yeast and mould cells as well as in cells undergoing the temperature induced mould-to-yeast conversion. Actin mRNA levels normalized to 18 S rRNA were found to be equivalent in all the stages examined, except for a sharp four-fold transient decrease 4 h into the mould-to-yeast conversion. These data suggest that actin mRNA would not be a suitable internal marker for expression studies during Histoplasma mould-to-yeast morphogenesis.
Insights
Researchers cloned actin from the dimorphic fungus Histoplasma capsulatum, finding it similar to Aspergillus nidulans actin. Actin mRNA levels are generally stable but show a transient decrease during mould-to-yeast conversion.
Area of Science:
- Molecular biology
- Mycology
- Pathogen research
Background:
- Histoplasma capsulatum is a dimorphic fungus and a significant human pathogen.
- Actin is a crucial cytoskeletal protein conserved across eukaryotes.
- Understanding gene expression during fungal morphogenesis is vital for pathogen control.
Purpose of the Study:
- To clone and characterize the actin gene from Histoplasma capsulatum.
- To investigate the expression patterns of Histoplasma actin mRNA during its dimorphic transition.
- To evaluate the suitability of actin mRNA as an internal control for gene expression studies in H. capsulatum.
Main Methods:
- Cloning of actin-encoding cDNA from H. capsulatum.
- Bioinformatic analysis of predicted amino acid sequence and codon usage.
- Southern blot analysis to determine gene copy number.
- Northern blot analysis to assess transcript levels during different growth phases and temperature-induced conversion.
Main Results:
- The cloned H. capsulatum actin cDNA showed high similarity to Aspergillus nidulans actin.
- Actin is encoded by a single gene copy in the H. capsulatum genome.
- A single 1700 nt actin transcript was detected in yeast and mould forms.
- Actin mRNA levels remained relatively constant except for a four-fold transient decrease at 4 hours during mould-to-yeast conversion.
Conclusions:
- The H. capsulatum actin gene is conserved and present as a single copy.
- Actin mRNA expression is largely stable during dimorphism but exhibits a transient dip.
- Actin mRNA is not a reliable internal marker for gene expression studies during H. capsulatum morphogenesis.