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Application of PCR-TTGE and PCR-RFLP for Intraspecific and Interspecific Characterization of the Genus Saccharomyces Using Actin Gene (ACT1) Primers

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Abstract

In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism (RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.

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References

  1. Barnett JA, Payne RW, Yarrow D (2000) Yeasts: characteristics and identification, 3rd edn. Cambridge University Press, Cambridge

    Google Scholar 

  2. Belmont LD, Drubin DG (2001) Actin structure function relationships revealed by yeast molecular genetics. Results Probl Cell Differ 32:103–121

    PubMed  CAS  Google Scholar 

  3. Briones A, Úbeda J, Grando S (1996) Differentiation of Saccharomyces cerevisiae strains isolated from fermenting must according to their karyotype patterns. Int J Food Microbiol 28:369–377

    Article  PubMed  CAS  Google Scholar 

  4. Fernández-Espinar MT, Esteve-Zarzoso B, Querol A, Barrio E (2000) RFLP analysis of the ribosomal internal transcribed spacers and the 5.8S rRNA gene region of the genus Saccharomyces: a fast method for species identification and the differentiation of flor yeasts. Ant van Leeuwen 78:87–97

    Article  Google Scholar 

  5. Fernández-González M, Espinosa JC, Úbeda JF, Briones AI (2001) Yeasts present during wine fermentation:comparative analysis of conventional plating and PCR-TTGE. Syst Appl Microbiol 24:634–638

    Article  PubMed  Google Scholar 

  6. Guillamón JM, Sabaté J, Barrio E, Cano J, Querol A (1998) Rapid identification of wine yeast species based on RFLP analysis of the ribosomal ITS region. Arch Microbiol 169:387–392

    Article  PubMed  Google Scholar 

  7. Hernán-Gómez S, Espinosa JC, Úbeda JF (2000) Characterization of wine yeast by temperature gradient gel electrophoresis (TGGE). FEMS Microbiol Lett 193:45–50

    Article  PubMed  Google Scholar 

  8. James SA, Cai J, Roberts N, Collins MO (1997) A phylogenetic analysis of the genus Saccharomyces based on 18S rRNA gene secuences: descripcion of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. Int J Syst Bacteriol 47:453–460

    Article  PubMed  CAS  Google Scholar 

  9. Josepa S, Guillamón JM, Cano J (2000) PCR differentiation of Saccharomyces cerevisiae from Saccharomyces bayanus/Saccharomyces pastorianus using specific primers. FEMS Microbiol Lett 193:255–259

    Article  PubMed  CAS  Google Scholar 

  10. Manzano M, Cocolin L, Lacumin L, Cantoni C, Comi G (2005) A PCR-TGGE technique to assess differentiation among enological Saccharomyces cerevisiae strains. Int J Food Microbiol 101:333–339

    Article  PubMed  CAS  Google Scholar 

  11. Manzano M, Medrala D, Giusto C, Bartolomeoli I, Urso R, Comi G (2006) Classical and molecular analyses to characterize commercial dry yeasts used in wine fermentations. J Appl Microbiol 100:599–607

    Article  PubMed  CAS  Google Scholar 

  12. Muyzer G, Somalla K (1998) Application of denaturing gradient electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Ant van Leeuwen 73:127–141

    Article  CAS  Google Scholar 

  13. Nügel R, Abelson J (1980) Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 77:3912–3916

    Article  Google Scholar 

  14. Querol A, Barrio E, Ramón D (1992) A comparative study of different methods of yeast strain characterization. Syst Appl Microbiol 15:439–446

    Google Scholar 

  15. Rose MD, Winston F, Ph Hieter (1990) Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 131–132

    Google Scholar 

  16. Smole-Mozina S, Dlauchy D, Deak T, Raspor P (1997) Identification of Saccharomyces sensu stricto and Torulaspora yeast by PCR ribotyping. Lett Appl Microbiol 24:311–315

    Article  PubMed  CAS  Google Scholar 

  17. Tominaga T (2004) Rapid identification of pickle yeasts by fluorescent PCR and microtemperature-gradient gel electrophoresis. FEMS Microbiol Lett 238:43–48

    Article  PubMed  CAS  Google Scholar 

  18. Torriani S, Zapparoli G, Suzzi G (1999) Genetic and phenotypic diversity of Saccharomyces sensu stricto strains isolates from Amarone wine. Diversity of Saccharomyces strains from Amarone wine. Ant van Leeuwen 75:207–215

    Article  CAS  Google Scholar 

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Acknowledgment

The authors are grateful to the Regional Government (JJCCM) for the funds assigned to Project PCI 08-0095-8912.

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Correspondence to J. F. Úbeda.

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Úbeda, J.F., Fernández-González, M. & Briones, A.I. Application of PCR-TTGE and PCR-RFLP for Intraspecific and Interspecific Characterization of the Genus Saccharomyces Using Actin Gene (ACT1) Primers. Curr Microbiol 58, 58–63 (2009). https://doi.org/10.1007/s00284-008-9283-9

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  • DOI: https://doi.org/10.1007/s00284-008-9283-9

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