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chromosomal fingerprinting or karyotyping (see Section 4.2.6.3) has shown differences in chromosome size (polymorphism) to be common. Although usually a function of chromosomal translocation, chromosomal differences may be due to more fundamental reasons. As noted in Section 4.2.2, lager strains of S. cerevisiae are a species hybrid of S. monacensis and top fermenting S. cerevisiae (Pedersen, 1995). Consequently, lager strains contain two distinct but functionally similar populations of chromosomes that resemble either S. monacensis or S. cerevisiae.

The ploidy in brewing strains is difficult to determine, particularly where it is based on measurement of cellular DNA and comparison with the DNA content of a haploid reference strain (Hammond, 1996). A comparatively crude approach to measuring DNA was used by Aigle et al. (1983) who showed two industrial yeasts to be tetra-ploid. A more recent application (Guijo et al., 1997) with enological isolates of S. cerevisiae (see below) showed strains to be diploid, triploid and tetraploid. The work of Codon et al. (1998) reported above moved ploidy analysis forward by using flow cytometry to determine DNA content. However, for most, even a crude measurement of ploidy is of little relevance! It is sufficient to accept that production-brewing yeasts are, inevitably, polyploid or aneuploid. However, polyploidy is of concern for geneticists as such strains are difficult or impossible to work with using what are now traditional genetic approaches. Such strains do not lend themselves to the classical method of'tetrad analysis' (for a review see Wickner, 1991) as they sporulate poorly and where sporulation occurs the spores are frequently inviable. This was important as tetrad analysis was very much at the core of haploid yeast genetics and, as such, was instrumental in much of the genetic mapping programme (Cox, 1995). Consequently, yeast breeding programmes and analysis of brewing strains was hampered until the arrival of new genetic approaches in the 1970s and '80s (see Section 4.3.4).

The sophisticated molecular approach reported by Hadfield et al. (1995), quantified the chromosome copy number by the use of 'ploidy probes'. Without going into detail, this approach hinges on the insertion of a DNA fragment into a specific chromosome. The fragment contains a known yeast gene that is found as a single copy in the haploid genome. The DNA probe recognises its homologous counterpart on the chromosome and replaces it. Inclusion of a 'selectable marker' (frequently resistance to an antibiotic) in the DNA probes enables the copy number of the probed chromosome to be determined.

The 'ploidy probes' approach has been used with two related top (Yl) and bottom-fermenting (Y9) ale strains (Hadfield et al., 1995). Analysis of Yl suggested this strain to be triploid with three copies of chromosomes IV, V and XV. However, in Y9 there was evidence of aneuploidy, having three copies of chromosomes IV and XV but two copies of chromosome V. It is not immediately obvious how this change can bring about changed flocculence. Of the probed chromosomes, only chromosome V has been associated with flocculation through FL08 (see Table 4.17). However, although necessarily tentative, it is tempting to conclude that the change in flocculation is related to the change in chromosomal copy number. Indeed Hadfield et al. (1995) noted that 'this may be a mechanism by which yeast can develop new characteristics in an alternative manner to gene mutation'.

An alternative, Guijo et al. (1997) reported a more traditional genetic approach to determining chromosome copy number in S. cerevisiae. Here, six enological strains of S. cerevisiae used in the fermentation and 'flor' film ageing of sherry-type wines were found to contain two, three or four copies of chromosomes. Indeed all six strains were aneuploid, with four being predominately diploid, one triploid (but disomic and tetrasomic for certain chromosomes) and one tending toward the tetraploid but tri-somic for certain chromosomes.

There is general acceptance that polyploidy is advantageous for industrial domesticated yeasts. However, specific evidence of the benefits (or disadvantages) of polyploidy in yeast is a little thin on the ground! Inevitably, the advent of the yeast genome sequence and associated analytical tools has enabled the implications of ploidy to be systematically unravelled. In a keynote paper, Galitski et al. (1999) constructed a series of isogenic or genetically identical strains that differed only in ploidy ranging from haploid («) to tetraploid (4n). The implications of increased ploidy were far-reaching and not always predictable. Although cell size increased with increasing ploidy (see Table 4.18), 3n and 4n strains grew more slowly in aerobic

Table 4.18 The impact of yeast ploidy on cell morphology (from Galitski et al., 1999).

Ploidy

Cell volume (|^m3)

Cell length/width ratios

Haploid

72 + 1

1.20 + 0.01

Diploid

111 + 2

1.24 + 0.01

Triploid

152 + 3

1.29 + 0.01

Tetraploid

289 + 6

1.39 + 0.02

culture with a greater lag phase than the corresponding haploid and diploid strains. Although undeniably interesting, the real core of the work reported by Galitski et al. (1999) was the effect of ploidy on the expression of all yeast genes during aerobic exponential growth. Using DNA chip technology, they searched for genes whose expression, relative to total gene expression, increased or decreased as the ploidy changed from haploid to tetraploid. Using a stringent cut-off of a ten-fold difference in gene expression between n and An, ten genes were ploidy induced and seven genes were ploidy repressed. Of these 17 genes, 10 encode known functions. One (CLN1, which encodes a cell cycle protein) was ploidy repressed, which may explain the greater cell size associated with higher ploidy. Similarly and intriguingly FLOll, 'whose molecular function is unknown', and which determines invasive growth into agar plates was ploidy repressed. Two of the genes that were ploidy induced were of interest: CTS1, which is involved in the separation of mother and daughter cells, and CTR3, which is involved in copper transport. However, despite these fascinating insights the vast majority of yeast genes were unaffected by an increase in ploidy.

Accordingly, from the work of Galitski et al. (1999), the 'jury is still out' as to the identifiable benefits (or not) of polyploidy. A more empirical 'real world' but admittedly soft argument for polyploidy is the realisation that - as noted above -domesticated strains are at least diploid. Perhaps the real selective benefit arises from aneuploidy and chromosomal rearrangements and translocations. This suggests that there is selective advantage in maintaining a number of copies of most (if not all) chromosomes that benefit the cell in terms of gene dosage. Simplistically, increasing the copy number of important genes is of benefit and is selectively advantageous. Certainly there is evidence that increasing the dosage of genes responsible for utilisation of maltose increases the rate of maltose fermentation (Hammond, 1996). A further argument is that polyploid strains are innately more stable as genetic change will require that each copy of a gene is effected by a 'change event' (Casey, 1990; Reed & Nagodarithana, 1991; Hammond, 1996). Evidently in evolutionary terms, such genetic 'buffering' (Soltis & Soltis, 1995) is a successful strategy as the genome project suggests that chromosomal tracts have been duplicated in the haploid genome (Section 4.3.2.1). However, we may have been lulled into a false sense of security, as genetic instability in production brewing strains is increasingly being reported (see Section 4.3.2.6). Perhaps it is more appropriate to view the greater stability of polyploid strains as being relative to the more susceptible haploid isolates.

4.3.2.6 Chromosomal instability. In recent years chromosomal instability in brewing yeast has had a 'good press'. The growing use of DNA fingerprinting (Sec tions 4.2.6.1-4.2.6.3) techniques has provided evidence that the brewing yeast genome can and does change. Karyotyping has shown changes in chromosome size (Sato et al., 1994; Casey, 1996) whereas RFLP fingerprinting has succeeded in associating changes in process performance of a variant strain with genetic changes (Wightman et al., 1996). For many this is, and remains, somewhat surprising. However, the view that by nature of their polyploidy, brewing yeasts are somehow immune to genetic change is now increasingly accepted as outdated and incorrect.

Perhaps it is more surprising that there is any need to debate the concept of genetic instability or change in brewing yeast. How else can the diversity of brewing yeasts (Section 4.2.3) be explained other than through genetic change and evolution? An excellent case history is the evolution of lager strains of S. cerevisiae. As noted by Casey (1996),

'over the last one hundred and fifty years, each strain has shown tremendous ability to adapt to environmental and nutritional selection pressures unique to its propagation, maintenance and utilisation - as reflected by chromosome copy number and size differences in virtually every chromosome region.'

Without wishing to prolong the argument, it is both ironic and contradictory to accept the principle of selecting stable strains with different process characteristics and not to recognise that this must be a consequence of a genetic change. Perhaps it is a matter of semantics. Genetic evolution through rearrangement is expected, genetic instability is not!

The development of karyotyping (Section 4.2.6.3) provided the tools by which chromosomes are separated and characterised by size. With it came the 'striking discovery that most (fungal) species exhibit chromosome-length polymorphism (CLP)' (Zolan, 1995). The CLPs, are detected by their change in size, and are caused by chromosomal rearrangements, which can occur via a variety of routes. Zolan (1995) has described five scenarios where DNA can be exchanged, added or lost which leads to chromosomes increasing or decreasing in size. An even more radical rearrangement can result in the complete loss of a chromosome. The evidence that such events happen is indisputable. At a fundamental level, the genome project has shown that almost half the haploid genome is duplicated with a host of 'cluster homology regions' (see Sections 4.3.2.1). Further, the many observations of chromosomal length polymorphism and the occurrence of aneuploidy (Section 4.3.2.4), unequivocally argue that the yeast genome is labile and is prone to rearrangement.

The potential frequency of adaptive mutations in yeast was first reported by Paquin and Adams (1983). Using a glucose limited chemostat, the frequency of adaptation over time for haploid and diploid strains was scored against parameters such as resistance to cycloheximide, canavanine and 5-fluorouracil. The authors concluded that 'genetic changes in both haploid and diploid populations occur surprisingly frequently'.

Subsequent work by Adams and co-workers (Adams et al., 1992) extended this approach to demonstrate the sheer scale of chromosomal rearrangement that can occur during continuous cultivation of yeast. After about 50 generations in a phosphate-limited chemostat, both haploid and diploid strains of S. cerevisiae exhibited occasional physiological changes. These were interpreted as being 'popu-

lation replacements' where 'the predominate clone in the population is replaced by one with a greater adaptation to the environment' (Adams et al., 1992). Analysis of these populations by electrophoretic karyotyping together with chromosome-specific DNA probes, showed these changes to be genetic-either translocations, deletions or additions. The scale of these genetic rearrangements was such that nine of the sixteen chromosomes showed changes. Perhaps the most dramatic change was the appearance, in one population, of a new large (1100 kb) chromosome made up of sequences homologous to chromosomes II and XVI. Another population gained an extra copy of chromosome V containing a 50 kb deletion. Numerically, the most changes were seen in chromosome XII which, subsequently, has been shown to be subject to size changes in the ribosomal DNA cluster region (Chindamporn et al., 1993). Although chromosomal deletions were limited to no more than 50 kb, the more common additions ranged from 10 to a substantial 390 kb. The magnitude of these additions is put into perspective by the realisation that chromosome I is only 230 kb!

The approach of Adams (Adams et al., 1992; Paquin & Adams, 1983) has been extended to good effect by Ferea et al. (1999). Here, a related diploid strain of S. cerevisiae was cultured continuously in three glucose-limited aerobic chemostats for between 250 and 500 generations. All three populations changed similarly by becoming more efficient in the use of the limiting carbon source, glucose. Compared to the parental strain, the evolved strains produced at steady state c. four-fold more cells, c. three-fold more biomass and at least an order of magnitude less ethanol. Analysis of gene expression showed that at least 3% of the yeast genome - 184 genes -exhibited a two-fold difference in expression between parental and evolved strains. Of these genes, 88 could be identified via the Saccharomyces Genome database. Two groups could be identified as exhibiting decreased expression (glycolysis) or increased oxidative phosphorylation, TCA cycle and mitochondrial structural proteins. This clearly suggests that the phenotype is not carved in stone but, in this case, adapts presumably via 'a small set of mutations' so that the metabolism of glucose is energetically more effective. This is perhaps not the complete picture, as Ferea et al. (1999) note that a similar number of uncharacterised genes also changed patterns of expression in evolved strains.

Since the work of Adams et al. (1992) there have been numerous observations of chromosomal changes in both laboratory and brewing strains of S. cerevisiae. Table 4.19 summarises the various reported changes across the yeast genome. Currently only three chromosomes (VII, IX and XV) in S. cerevisiae remain unscathed as to reports of chromosome polymorphism. What triggers such changes remains a matter of conjecture and debate. Globally polymorphisms are reported to arise through major events such meiosis and sporulation (Yoda et al., 1993) and vegetative growth (mitosis) (Longo & Vezinhet, 1993). Whether such changes can be bracketed under the umbrella of being 'selectively advantageous' (Adams et al., 1992) remains to be seen. Whatever, it is noteworthy that in these 'academic' studies, chromosomal changes are anything but rapid, requiring 275 (Longo & Vezinhet, 1993) to upwards of 1000 generations.

Prior to the introduction of genetic fingerprinting methods, evidence for what was assumed to be genetic change was inferred from observations of altered behaviour. As remains the case today, such observations frequently revolve around changes in

Table 4.19 Reports in the literature of genetic change in S. cerevisiae (sorted by chromosome).

Chromosome

Size (kb)

Ploidy

Change

Reference

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