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Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

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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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References

  1. Poretsky, R. S., Hewson, I., Sun, S., Allen, A. E., Zehr, J. P., & Moran, M. A. (2009). Comparative day/night metatranscriptomic analysis of microbial communities in the North Pacific subtropical gyre. Environmental Microbiology, 11, 1358–1375.

    Article  CAS  Google Scholar 

  2. Jayaraman, A., Puranik, S., Rai, N. K., Vidapu, S., Sahu, P. P., Lata, C., et al. (2008). cDNA-AFLP analysis reveals differential gene expression in response to salt stress in foxtail millet (Setaria italica L.). Molecular Biotechnology, 40, 241–251.

    Article  CAS  Google Scholar 

  3. Xiaohu, X., Heping, L., & Chaorong, T. (2009). A silver-staining cDNA-AFLP protocol suitable for transcript profiling in the latex of Hevea brasiliensis (para rubber tree). Molecular Biotechnology, 42, 91–99.

    Article  Google Scholar 

  4. Breyne, P., Dreesen, R., Cannoot, B., et al. (2003). Quantitative cDNA-AFLP analysis for genome wide expression studies. Molecular Genetics and Genomics, 269, 173–179.

    CAS  Google Scholar 

  5. Vuylsteke, M., Peleman, J. D., & van Eijk, M. J. (2007). AFLP-based transcript profiling (cDNA-AFLP) for genome-wide expression analysis. Nature Protocols, 2, 1399–1413.

    Article  CAS  Google Scholar 

  6. Durrant, W. E., Rowland, O., Piedras, P., Hammond-Kosack, K. E., & Jones, J. D. (2000). cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. The Plant Cell, 12, 963–977.

    Article  CAS  Google Scholar 

  7. Reijans, M., Lascaris, R., Groeneger, A. O., et al. (2003). Quantitative comparison of cDNA-AFLP, microarrays, and GeneChip expression data in Saccharomyces cerevisiae. Genomics, 82, 606–618.

    Article  CAS  Google Scholar 

  8. Milioni, D., Sado, P. E., Stacey, N. J., Domingo, C., Roberts, K., & McCann, M. C. (2001). Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system. Plant Molecular Biology, 47, 221–238.

    Article  CAS  Google Scholar 

  9. He, P., Friebe, B. R., Gill, B. S., & Zhou, J. M. (2003). Allopolyploidy alters gene expression in the highly stable hexaploid wheat. Plant Molecular Biology, 52, 401–414.

    Article  CAS  Google Scholar 

  10. Bachem, C. W. B., van der Hoeven, R. S., de Bruijn, S. M., Vreugdenhil, D., Zabeau, M., & Visser, R. G. F. (1996). Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: Analysis of gene expression during potato tuber development. The Plant Journal, 5, 745–753.

    Article  Google Scholar 

  11. Chapman, N. H., Burt, C., & Nicholson, N. (2009). The identification of candidate genes associated with Pch2 eyespot resistance in wheat using cDNA-AFLP. Theoretical and Applied Genetics, 118, 1045–1105.

    Article  CAS  Google Scholar 

  12. Noel, L., Thieme, F., Nennstiel, D., & Bonas, U. (2001). cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria. Molecular Microbiology, 41, 1271–1281.

    Article  CAS  Google Scholar 

  13. Decorosi, F., Viti, C., Mengoni, A., Bazzicalupo, M., & Giovannetti, L. (2005). Improvement of the cDNA-AFLP method using fluorescent primers for transcription analysis in bacteria. Journal of Microbiological Methods, 63, 211–215.

    Article  CAS  Google Scholar 

  14. Berila, N., Borecka, S., Dzugasova, V., Bojnansky, J., & Subik, J. (2009). Mutations in the CgPDR1 and CgERG11 genes in azole-resistant Candida glabrata clinical isolates from Slovakia. International Journal of Antimicrobial Agents, 33, 574–578.

    Article  CAS  Google Scholar 

  15. Hooshdaran, M. Z., Barker, K. S., Hilliard, G. M., Kusch, H., Morschhäuser, J., & Rogers, D. P. (2004). Proteomic analysis of azole resistance in Candida albicans clinical isolates. Antimicrobial Agents and Chemotherapy, 48, 2733–2735.

    Article  CAS  Google Scholar 

  16. Weiberg, A., Pöhler, D., Morgenstern, B., & Karlovsky, P. (2008). Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA. BioMed Central Genomics, 9, 480.

    Google Scholar 

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Acknowledgment

We thank to Margaret Holmes for grammar corrections.

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Authors and Affiliations

  1. Laboratory of Molecular Microbiology, National Center of Infectious and Parasitic Diseases, Janko Sakasov 26, 1504, Sofia, Bulgaria

    Victoria Levterova, Stefan Panaiotov, Nadia Brankova & Kristin Tankova

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  1. Victoria Levterova
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  2. Stefan Panaiotov
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  3. Nadia Brankova
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  4. Kristin Tankova
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Correspondence to Stefan Panaiotov.

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Levterova, V., Panaiotov, S., Brankova, N. et al. Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique. Mol Biotechnol 45, 34–38 (2010). https://doi.org/10.1007/s12033-009-9236-y

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  • DOI: https://doi.org/10.1007/s12033-009-9236-y

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