1 Introduction - Yeast as a Versatile Model System

1.1 Yeast History

Talking about 'yeast', which in every day language is synonomous for Saccharomyces cerevisiae
(name created for a yeast strain observed in malt, 1837), one is immediately reminded that this
species probably is the oldest domesticated organism (Figure 1-1). It lives on sugars and has been
used for beer brewing in Sumeria and Babylonia, already 6000 B.C. In parallel, S. cerevisiae strains
have been used with grape cultivation in Georgia and for dough leavening in Egypt. Levure goes back
to latin levare, and so is leaven, simultaneously used for dough and yeast as an organism able to
anaerobically release CO2 during the baking process. The English word yeast, like Dutch guist, or
even the German Hefe, are derived from a west-germanic expression haf-jon, meaning the potential to
leaven. The Greek zymi () is used simultaneously for yeast and dough and occurs as a root in
words related to beer or fermentation. Thus the modern expression 'enzymes' (en zymi = in yeast) has
been created to designate the compounds derived from yeast that are able to ferment sugar. In fact,
this observation dates back to the first studies of Louis Pasteur (tudes sur la bire) in 1857, which he
carried through during his time at Strasbourg University. The unique properties of the yeast, S.
cerevisiae, among some 700 yeast species (a subgroup from 700,000 different fungi) and its
enormous hidden potential which has been exploited for many thousands of years, made it a preferred
organism also for research. Moreover, S. cerevisiae and other yeasts yielded a vast majority of
industrial and medical applications benficial to human life (Figures 1-2 and 1-3).

Figure 1-1: Yeast history

Yeast has been introduced as an experimental organism in the mid-thirties of the 20th century
[Roman, 1981] and has since received increasing attention. The elegance of yeast genetics and the
ease of manipulation of yeast, and finally the technical breakthrough of yeast transformation to be
used in reverse genetics, have substantially contributed to the enormous growth in yeast molecular
biology [Earlier compilations: Strathern et al., 1981; Broach et al., 1991; Guthrie & Fink, 1991]. This
success is also due to the fact, which was not anticipated then a couple of years ago, that the extent

to which basic biological structures and processes have been conserved throughout eukaryotic life is
remarkable.

Figure 1-2: Biotechnologies involving yeast.

Prokaryotic products: Tetanus toxin fragment C; Streptokinase

Surface antigens of viruses: Hepatitis B; HIV; Foot and mouse disease; Influenza; Polio;
Polyoma; Epstein-Barr; Oncogenic retroviruses
Malaria antigen
Animals products: Hirudin; porcine interferon; interleukin; trypsin inhibitor
Human hormones: Insulin; parathyroid hormone; growth hormone, chorionic gonadotropin
Human growth factors: IGF1; NGF; EGF; tissue factor; CSF; GM-CSF; TNF
Human blood proteins: Hemoglobin; factors VIII and XIII; aplha-1-antitrypsin;
antithrombin III; serum albumin
Various human enzymes; CFTR; estrogen receptor; INF-alpha; INF-beta1

Figure 1-3: Therapeutic products from yeasts.

1.2 Yeast - an Experimental System for Molecular Biology

Starting around 1960, S. cerevisiae was introduced as an experimental system for molecular
biology. As early as in 1980, yeast was used to produce Hepatitis B vaccine. In 1996 yeast was the
first eukaryotic organism, of which the complete genomic sequence could be established. In the years
to follow, yeast became a useful reference against which sequences of human, animal or plant genes,
and those of a multitude of unicellular organisms under study could be compared. Moreover, the ease
of genetic manipulation in yeast opened the possibility to functionally dissect gene products from other
eukaryotes in the yeast system. In the post-genomic era, yeast was again at the forefront in functional
genomics.
It is well established that yeast is an ideal system in which cell architecture and fundamental cellular
mechanisms can be successfully investigated. Among all eukaryotic model organisms, S. cerevisiae
combines several advantages. It is a unicellular organism (Figure1-4) which, unlike more complex
eukaryotes, can be grown on defined media giving the investigator complete control over
environmental parameters. Yeast is tractable to classical genetic techniques and functions in yeast
have been studied in great detail by biochemical approaches [Earlier compilations: Strathern et al.,
1981; Broach et al., 1991; Guthrie & Fink, 1991]. In fact, a large variety of examples provide evidence
that substantial cellular functions are highly conserved from yeast to mammals and that corresponding
genes can often complement each other.
No wonder then that yeast had reached the forefront in experimental molecular biology being the first
eukaryotic organism of which the entire genome sequence has been made available [Goffeau et al.,
1996; Dujon, 1996]. The wealth of sequence information obtained in the yeast genome project was
and still is extremely useful as a reference against which sequences of human, animal or plant genes,
and those of a multitude of unicellular organisms now under study may be compared. Moreover, the
ease of genetic manipulation in yeast has opened the possibility to functionally dissect gene products
from other eukaryotes in the yeast system.

1.3 Experimental Approaches in Yeast Molecular Biology

It may be useful to briefly outline some of the characteristics of the yeast system. With its 12.8 Mb, the
yeast genome is about 200 times smaller than the human genome but less than four times bigger
than that of E.coli. At the onset of the sequencing project (Figure 1-5), knowledge about some 1200
genes encoding either RNA or protein products had accumulated [Mortimer et al., 1992]. The complete
genome sequence now defines some 6000 open reading frames (ORFs) most of which are likely to
encode specific proteins. A protein-encoding gene is found every two kb in the yeast genome, with
nearly 70% of the total sequence being covered [Dujon, 1996]. In addition to the protein-encoding
genes, the yeast genome contains some 120 ribosomal RNA genes in a large tandem array on
chromosome XII, 40 genes encoding small nuclear RNAs (sRNAs), 274 tRNA genes (belonging to 42
families) which are scattered throughout the genome, and some 50 copies of the yeast
retrotransposons (Ty elements). Finally, the sequences of non-chromosomal elements, such as the 6
kb of the 2 plasmid DNA, the killer plasmids present in some strains, and the yeast mitochondrial
genome (ca.75 kb) have to be considered.
The genome of S.cerevisiae is divided up into 16 chromosomes ranging in size between 250 kb and
>2500 kb. Choosing appropriate conditions, it is feasible to separate all 16 chromosomes in pulsed
field gel electrophoresis (PFGE). This provides definition of 'electrophoretic karyotypes' of strains by

sizing the chromosomes [Carle & Olson, 1985]. The gels can be utilized for Southern blotting followed
by hybridization, or to isolate chromosome-specific DNA.

Figure 1-4: Life cycle of yeast.

The first genetic map of S. cerevisiae was published by Lindegren in 1949 [Lindegren, 19949]; many
revisions and refinements have appeared since. Both meiotic and mitotic approaches have been
developed to map yeast genes. The life cycle of S. cerevisiae normally alternates between
diplophase and haplophase. Both ploidies can exist as stable cultures. In heterothallic strains, haploid
cells are of two mating types, a and . Mating of a and cells results in a/ diploids that are unable to
mate but can undergo meiosis. The four haploid products resulting from meiosis of a diploid cell are
contained within the wall of the mother cell (the ascus). Digestion of the ascus and separation of the
spores by micromanipulation yield the four haploid meiotic products. Analysis of the segregation
patterns of different heterozygous markers among the four spores constitutes tetrad analysis and
reveals the linkage between two genes (or between a gene and its centromere) [Mortimer & Schild,
1991]. On the whole, genetic distance in yeast appears to be remarkably proportional to physical
distance, with a global average of 3 kb/cM.
A large variety of protocols for genetic manipulation in yeast are available [Guthrie & Fink,1991;
Johnston, 1994]. Yeast has a generation time of ca 80 min and mass production of cells is easy.
Simple procedures for the isolation of high molecular weight DNA, rDNA, mRNA, and tRNA are at
hand. It is possible to isolate intact nuclei or cell organelles such as intact mitochondria (maintaining
respiratory competence). High efficiency transformation of yeast cells is achieved, for example, by the

lithium acetate procedure [Ito et al., 1983] or by electroporation. A large variety of vectors have been
designed to introduce and to maintain or express recombinant DNA in yeast cells [e.g. Guthrie &
Fink,1991; Johnston, 1994].
Furthermore, a large number of yeast strains carrying auxotrophic markers, drug resistance markers
or defined mutations are available. Culture collections are maintained, for example, at the Yeast
Genetic Stock Center (YGSC) and the American Type Culture Collection (ATCC). Mutant strains with
defined gene deletions together with clones carrying the corresponding gene cassettes have emerged
from the EUROFAN and TRANSATLANTIC projects (see chapter 6). Ordered cosmid libraries using
different vectors were constructed during the yeast sequencing project [e.g. Thierry et al., 1993; Riles
et al., 1993; Stucka & Feldmann, 1994].

Figure 1-5: Yeast before the genome project.

The ease of gene disruptions and single step gene replacements is unique in S.cerevisiae and offers
an outstanding advantage for experimentation (Figure 1-5). Yeast genes can functionally be
expressed when fused to the green fluorescent protein (GFP) thus allowing to localize gene products
in the living cell by fluorescence microscopy [Niedenthal et al., 1996]. The yeast system has also
proven an invaluable tool to clone and to maintain large segments of foreign DNA in yeast artificial
chromosomes (YACs) being extremely useful for other genome projects [Burke et al., 1987] and to
search for protein-protein interactions using the two-hybrid approach [Fields & Song, 1989].

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