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. 2015 Nov 21;8(11):e25218.
doi: 10.5812/jjm.25218. eCollection 2015 Nov.

Identification and Sequencing of Candida Krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis

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Free PMC article

Identification and Sequencing of Candida Krusei Aconitate Hydratase Gene Using Rapid Amplification of cDNA Ends Method and Phylogenetic Analysis

Roohollah Fateh et al. Jundishapur J Microbiol. .
Free PMC article

Abstract

Background: The production and development of an effective fungicidal drug requires the identification of an essential fungal protein as a drug target. Aconitase (ACO) is a mitochondrial protein that plays a vital role in tricarboxylic acid (TCA) cycle and thus production of energy within the cell.

Objectives: The current study aimed to sequence Candida krusei ACO gene and determine any amino acid residue differences between human and fungal aconitases to obtain selective inhibition.

Materials and methods: Candida krusei (ATCC: 6258) aconitase gene was determined by 5'Rapid Amplification of cDNA Ends (RACE) method and degenerate Polymerase Chain Reaction (PCR) and analyzed using bioinformatics softwares.

Results: One thousand-four hundred-nineteen nucleotide of C. krusei aconitase gene were clarified and submitted in Genbank as a partial sequence and then taxonomic location of C. krusei was determined by nucleotide and amino acid sequences of this gene. The comparison of nucleotide and amino acid sequences of Candida species ACO genes showed that C. krusei possessed characteristic sequences. No significant differences were observed between C. krusei and human aconitases within the active site amino acid residues.

Conclusions: Results of the current study indicated that aconitase was not a suitable target to design new anti-fungal drugs that selectively block this enzyme.

Keywords: 5’ RACE Method; Aconitate Hydratase; Candida krusei; Degenerate PCR; Phylogenetic Analysis.

Figures

Figure 1.
Figure 1.. Partial Nucleotide Sequence of the Aconitase Gene of Candida krusei
The nucleotide sequences were determined from the cloned PCR amplified Candida krusei aconitase gene.
Figure 2.
Figure 2.. Partial Multiple Alignment of ACO Nucleotide Sequences of Candida Species and Homo sapiens as an Out-Group
Figure 3.
Figure 3.. Partial Multiple Alignment of ACO Amino Acid Sequences of Candida Species and Homo sapiens as an Out-Group
Figure 4.
Figure 4.. Unweighted-Pair-Group Method With Arithmetic Mean Trees of the Fungal Species for the ACO Genes
Unweighted-pair-group method with arithmetic mean trees based on nucleotide (a) and amino acid (b) sequences. The Homo sapiens represents an out-group. Bars exhibit the numbers of nucleotide and amino acid substitutions per nucleotide and amino acid sites.
Figure 5.
Figure 5.. Unweighted-Pair-Group Method With Arithmetic Mean Trees of the Common Candida Species for the ACO Genes
Unweighted-pair-group method with arithmetic mean trees based on nucleotide (a) and amino acid (b) sequences. The Homo sapiens represents an out-group. Bars exhibit the numbers of nucleotide and amino acid substitutions per nucleotide and amino acid sites.
Figure 6.
Figure 6.. Neighbor Joining Trees of the Common Candida Species for the ACO Genes
Neighbor joining trees based on nucleotide (a) and amino acid (b) sequences. The Homo sapiens represents an out-group. Bars exhibit the numbers of nucleotide and amino acid substitutions per nucleotide and amino acid sites.

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