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Journal of Clinical Microbiology, April 1998, p. 1035-1038, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Intergenic Transcribed Spacer PCR Ribotyping for Differentiation of Saccharomyces Species and Interspecific Hybrids

Michael J. McCullough,1,2,3 Karl V. Clemons,1,2,3 John H. McCusker,4 and David A. Stevens1,2,3,*

Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California 943051; California Institute for Medical Research2 and Department of Medicine, Division of Infectious Diseases,3 Santa Clara Valley Medical Center, San Jose, California 95128; and Department of Microbiology, Duke University Medical Center, Durham, North Carolina 277104

Received 23 July 1997/Returned for modification 18 November 1997/Accepted 14 January 1998

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.

* Corresponding author. Mailing address: Division of Infectious Diseases, Santa Clara Valley Medical Center, 751 South Bascom Ave., San Jose, CA 95128. Phone: (408) 885-4313. Fax: (408) 885-4306. E-mail: oflahren{at}wpgate.hhs.co.santa-clara.ca.us.

Journal of Clinical Microbiology, April 1998, p. 1035-1038, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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