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Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity
Published on: April 13, 2012
Molecular cloning, characterization and differential expression of Cdc42 in Fonsecaea monophora
Zhi Xie1, Peiying Feng, Junming Zhang
1Department of Dermatology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, 107 West Yanjiang Road, Guangzhou 510120, China.
Abstract:
The cell divisions cycle 42 (Cdc42) gene has been characterized in the fungi, such as Candida albicans, Penicillium marneffei, and Wangiella (Exophiala) dermatitidis, which plays important roles during growth and development. The partial cDNA sequence of Cdc42 of Fonsecaea monophora (F. monophora), designated FmCdc42, was obtained using degenerate primers based on the conserved domain of the other fungi Cdc42. Then the complete cDNA sequence of FmCdc42 was obtained by 5' and 3' RACE. The full-length cDNA is 1,510 bp in size which had an open reading frame (ORF) of 582 bp, encoding 193 amino acid residues. The predicted molecular mass of FmCdc42 is 21.5 kDa with an estimated theoretical isoelectric point of 5.67. The deduced amino acid sequence of FmCdc42 shows 99% identity to that of Wangiella (Exophiala) dermatitidis. 5 exons and 4 introns are identified within the 1,617 bp FmCdc42 genomic DNA sequence of F. monophora. The ORF could be subcloned into the pCDNA6/myc-His B expression vector. The recombinant protein about 27.5 kD infusion protein had high expression level in Vero cells with SDS-PAGE and Western blot analysis. Quantitative real time RT-PCR revealed that FmCdc42 was the highest expression in the sclerotic bodies' stage compared with that in the mycelia and conidia stages, which indicated that the FmCdc42 may be involved in formation of F. monophora sclerotic bodies.
Insights
The cell division cycle 42 (Cdc42) gene in Fonsecaea monophora, FmCdc42, was sequenced and characterized. Its expression is highest during the sclerotic bodies stage, suggesting a role in their formation.
Area of Science:
- Molecular Biology
- Mycology
- Genetics
Background:
- The cell division cycle 42 (Cdc42) gene is crucial for fungal growth and development.
- Previous studies have characterized Cdc42 in various fungi, including Candida albicans and Wangiella (Exophiala) dermatitidis.
Purpose of the Study:
- To isolate and characterize the Cdc42 gene from Fonsecaea monophora (FmCdc42).
- To investigate the expression pattern of FmCdc42 during different developmental stages of F. monophora.
Main Methods:
- Degenerate primers and 5'/3' RACE were used to obtain the full-length FmCdc42 cDNA sequence.
- Genomic DNA sequencing identified 5 exons and 4 introns.
- Recombinant protein expression in Vero cells was analyzed by SDS-PAGE and Western blot.
- Quantitative real-time RT-PCR was employed to determine gene expression levels.
Main Results:
- The full-length FmCdc42 cDNA is 1,510 bp, encoding 193 amino acids with a predicted molecular mass of 21.5 kDa.
- The deduced amino acid sequence showed 99% identity to that of W. dermatitidis Cdc42.
- FmCdc42 exhibited the highest expression in the sclerotic bodies stage compared to mycelia and conidia stages.
Conclusions:
- The FmCdc42 gene has been successfully cloned and characterized.
- FmCdc42 expression is developmentally regulated in F. monophora.
- The findings suggest FmCdc42 plays a role in the formation of F. monophora sclerotic bodies.
