Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique |
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Authors: | Victoria Levterova Stefan Panaiotov Nadia Brankova Kristin Tankova |
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Institution: | (1) Laboratory of Molecular Microbiology, National Center of Infectious and Parasitic Diseases, Janko Sakasov 26, 1504 Sofia, Bulgaria; |
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Abstract: | Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a
challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in
the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism
(cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important
fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants.
Resistant mutants Candida albicans
FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture
medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA
overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes
under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and
reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing
insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could
be tested for typing of the genome response of any organism to physical or chemical stress factors. |
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