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Fig. 4.16 Discriminant analysis bioplot based on FT-IR data for 22 strains of S. cerevisiae. These same strains were subject to PyMS analysis in Fig. 4.14 (redrawn from Timmins et al., 1998).

• Fatty acid profiles - FAME (fatty acid methyl ester) profiling has proved particularly successful in the typing of clinical bacterial genera and species (Gut-teridge & Priest 1996). However, in the case of yeasts, it is generally held that the profiles of saturated and unsaturated fatty acids in S. cerevisiae are essentially the same, although the profile is sufficiently distinctive and consistent to differentiate S. cerevisiae from other species of yeast encountered in the wine industry (Tredoux et al., 1987). Augustyn and Kock (1989) have proposed the exciting possibility that fatty acid profiles may be used to differentiate strains of S. cerevisiae. Central to this proposal are prescriptive protocols for the aerobic growth of each strain, defined conditions of extraction/methylation and analysis using sensitive capillary GLC columns together with mass spectroscopy. Although up to 14 different fatty acids could be identified, statistical analysis often fatty acids (as a relative percentage) enabled the differentiation of 13 strains of S. cerevisiae. However, 'as the number of strains to be differentiated increases, it is quite possible that the data generated by a study of 10 fatty acids used here will prove to be inadequate' (Augustyn & Kock, 1989).

• Protein fingerprinting - It has been estimated that there are more than 2000 different proteins in the microbial cell (Gutteridge & Priest 1996). Indeed, in yeast, the genome project has suggested almost 6000 'potential' proteins (Goffeau et al., 1996). With so much diversity it is perhaps not surprising that the extraction and electrophoretic separation of cellular proteins has provided a successful route for the identification and characterisation of, particularly, medically important bacteria. Within brewing microbiology there has only been limited application of protein fingerprinting, notably the identification of lactic acid bacteria (Gutteridge & Priest 1996). However, with S. cerevisiae, protein electrophoresis appears to offer significant discriminatory power. The work of van Vuuren and van der Meer (1987) succeeded in grouping 29 predominately wine strains of S. cerevisiae into five clusters of closely related strains. Examination of the 'electropherograms' and accompanying dendrogram of related-ness suggest that all the strains could be differentiated from each other. As with all of these methods, consistency is all (particularly growth conditions and extraction) together with sophisticated data capture and statistical treatment.

4.3 Genetics - genome, cell cycle and modification

4.3.1 Introduction

The explosion in activity and understanding of the genetics of the yeasts is ably summed up by Wheals (1995) who noted that in the first edition of The Yeasts (1969 71) 'genetics warranted a single chapter'. However, in the second edition (1987-95) of The Yeasts, coverage of 'genetics' accounted for one volume of 11 chapters, four appendices and 619 pages. With the publication of the complete sequence of the genome of S. cerevisiae in 1996 (Goffeau et al., 1996), the size of the 'genetics' element of any third edition of The Yeasts can only be speculated on.

Yeast genetics is considered to have commenced in the 1940s with the pioneering work of Winge in the Carlsberg Laboratories in Denmark and Lindegrin in Illinois, USA. Since then S. cerevisiae has become very much the organism of choice in the study of eukaryote genetics and cell biology. However, for the worldwide brewing industry, yeast genetics came of age in the 1980s with the development of gene cloning and the recognition that genetic manipulation offered the possibility to tailor yeasts to meet desired needs. In the succeeding years, a number of 'foreign' genes have been successfully introduced into brewing yeast so much so that, technically, genetic manipulation could almost be described as being 'routine'! However, as yet, labelling demands, regulatory hurdles and, more importantly, consumer negativity or concern have mitigated against the commercial use of a genetically modified yeast (see Section 4.34).

Although genetic manipulation remains 'too hot to handle', the endeavour of many committed geneticists has resulted in numerous valuable spin-offs and insights. In particular, genetic methods are now used to routinely fingerprint and differentiate brewing yeast strains (see Sections 4.2.6.1-3) and have provided hitherto unexpected insights into genetic instability and change (see Section 4.3.2.5). In the more rarefied atmosphere of applied brewing research, genetic methods increasingly feature in the routine technical armoury that is used to unravel biological function and interaction.

Although the detailed consideration of yeast genetics is beyond the scope of this book, there is room, and a need, to review the special and applied case of brewing yeast genetics. This is especially necessary as - for reasons detailed below - brewing yeast strains differ significantly from laboratory isolates of S. cerevisiae. Consequently, the important points of brewing yeast genetics are usually lost in a welter of detail and understanding that surrounds the laboratory strains. For specialist, authoritative reviews of brewing yeast genetics see Kielland-Brandt et al. (1995) and Hammond (1993, 1996). For detailed reviews of the wider vista of yeast genetics the interested reader is referred to volume 6 of The Yeasts edited by Rose et al. (1995).

4.3.1.1 Genetic nomenclature and definitions. Genetics is not alone in being impenetrable for all but the professional. The intention of this section is to provide the reader with an understanding of the 'hot' topics in brewing genetics. Inevitably, there is much jargon, which, not surprisingly can confuse and frustrate. In an attempt to minimise this pain, Table 4.14 provides a glossary of some of the terms used in this chapter.

4.3.2 The genome

4.3.2.1 Yeast genome project. Work on the sequencing of the genome of S. cerevisiae began in 1989 and finished with the publication of the sequence on 24 April 1996. The work was lauded as a milestone in biology as (i) it was the first eukaryotic genome to be sequenced and (ii) it represented the collective efforts of some 633 scientists from 96 laboratories worldwide. The project was very much of its time, using 'modern infomatics technology' (Goffeau et al., 1996) and the Internet to facilitate communications and project management. Indeed, the sequence of the genome was first 'published' on the World-Wide Web and is now easily sourced and interrogated on the Internet (for a review see Brown, 1998).

Table 4.14 Glossary of terms and abbreviations used in genetics.

Abbreviation

Definition

[rho°] allele amino acids aneuploid ascospore (spore) ascus auxotrophic base pair (bp)

bases cdc cDNA (complementary DNA)

centromere

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