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Cloning and sequence analysis of an H(+)-ATPase-encoding gene from the human dimorphic pathogen Histoplasma
1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, OH 45267-0524.
Abstract:
A gene related to the PMA1 gene from Saccharomyces cerevisiae was isolated from the pathogenic human dimorphic fungus, Histoplasma capsulatum, using fungal-specific oligodeoxyribonucleotide (oligo) probes. This gene has been given the name Hc-PMA1. The structural organization of Hc-PMA1 consists of three exons (375, 2329 and 44 bp) and two introns (115 and 116 bp). The nucleotide sequence predicts an H(+)-ATPase-related protein of 916 amino acids (aa). Comparison of the deduced aa sequence to that of Neurospora crassa and S. cerevisiae (PMA1) plasma membrane H(+)-ATPases showed a greater similarity to that from N. crassa (85% identity). Furthermore, the two introns in the Hc-PMA1 gene interrupt the coding region in the precise locations determined for two of the four N. crassa Nc-PMA introns. H. capsulatum intron 1 contains two repeat motifs, d(TA)16 and d(TG)10, each potentially capable of forming non-B DNA structures. Northern analysis of H. capsulatum total RNA indicated that the Hc-PMA1-specific mRNA is approx. 3.3 kb in size, in agreement with the predicted size of the gene.
Insights
Researchers identified the Hc-PMA1 gene in Histoplasma capsulatum, a human fungal pathogen. This gene encodes a protein similar to H(+)-ATPases found in other fungi, offering insights into fungal biology.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- Histoplasma capsulatum is a dimorphic fungus causing histoplasmosis.
- Plasma membrane H(+)-ATPases are crucial for cellular function in eukaryotes.
- Understanding fungal-specific genes aids in developing targeted therapies.
Purpose of the Study:
- To isolate and characterize a gene homologous to Saccharomyces cerevisiae PMA1 in Histoplasma capsulatum.
- To analyze the structural organization and sequence of the Hc-PMA1 gene.
- To investigate the evolutionary relationship of Hc-PMA1 with other H(+)-ATPases.
Main Methods:
- Fungal-specific oligodeoxyribonucleotide probes were used for gene isolation.
- Nucleotide sequencing was performed to determine gene structure and predict protein sequence.
- Sequence homology comparisons were made with known H(+)-ATPases.
- Northern blot analysis was conducted to determine mRNA size.
Main Results:
- The Hc-PMA1 gene was successfully isolated from H. capsulatum.
- Hc-PMA1 comprises three exons and two introns, predicting a 916 amino acid H(+)-ATPase-related protein.
- The deduced amino acid sequence showed 85% identity to Neurospora crassa PMA1.
- Intron positions in Hc-PMA1 precisely matched those in N. crassa Nc-PMA.
- Northern analysis confirmed an Hc-PMA1 mRNA size of approximately 3.3 kb.
Conclusions:
- The Hc-PMA1 gene represents a functional H(+)-ATPase in Histoplasma capsulatum.
- The high sequence similarity to N. crassa suggests conserved function and evolutionary linkage.
- Intron characteristics may provide insights into gene regulation and evolution in fungi.