Rflp

RNA (ribonucleic acid)

rRNA (ribosomal RNA)

Munich Informatioan Centre for Protein Sequences.

Semi-autonomous and self-reproducing organelles located in the cytoplasm of eukaryotic cells. These organelles are responsible for the energy conversion of most of the cellular energy metabolites into adenosine triphosphate (ATP) by oxidative phosphorylation.

The process of nuclear division in eukaryotic cells.

A diploid strain lacking one of a pair of chromosomes is monosomic in that chromosome.

A single-stranded RNA that acts as the template for the amino acid sequence of proteins.

Nonfermentable substrate.

DNA stretches that potentially encode proteins. They always have a start codon (ATG) at one end and a translation-terminating stop codon at the other end.

Genes without any recognised function.

Polymerase chain reaction.

Pulsed field gel electrophoresis.

The observable characteristics of a yeast strain.

Typically variability in chromosome size that enables differentiation of closely related strains of yeast.

Generic term that typically means that the ploidy of the yeast is greater than diploid.

Polymers composed of a linear string of amino acids.

The complete set of proteins that yeast can form.

A yeast strain that has no obvious requirements for nutritional supplements.

Random amplified polymorphic DNA.

Restriction fragment length polymorphism.

Polymer composed of a repeating backbone of phosphate and sugar subunits to which different bases are attached: adenine (A), cytosine (C), guanine (G), and uracil (U). The sugar backbone of RNA is composed of ribose subunits. RNAs can be distinguished by their different properties: mRNA, tRNA, and rRNA.

A structural and functional component of ribosomes. Ribosomes are the cellular machinery responsible for the translation of the genetic information into proteins. They are composed of rRNA and ribosomal proteins.

Abbreviation

Definition

Saccharomyces cerevisiae

SGD snRNA

spindle sporulation

TAFE telomere tetraploid transcription translation transposable element (transposon)

triploid trisomic tRNA (transfer RNA) Ty elements zygote

Taxonomic classification: Eukaryota; Plantae; Thallobionta; Eumycota; Hemiascomycetes; Endomycetales; Saccharomycetaceae. Saccharomyces is a genus of ascomycetes. They are normally diploid unicellular fungi that reproduce asexually by budding. Asci, containing four haploid ascospores, develop directly from the diploid vegetative cells by meiosis. After germination of the ascospores the haploid cells can reproduce vegetatively, or haploid cells of different mating type can fuse to form a diploid zygote. Most laboratory strains used are, in contrast to wild-type yeasts, stable haploids.

Saccharomyces Genome Database.

A set of RNAs that are typically smaller than 300 nucleotides and function in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs). The function of snRNPs is to mediate and regulate post-translational RNA processing events.

A network of fibrous microtubules and associated molecules formed during mitosis between the opposite poles (centromeres) of eukaryotic cells. It mediates the movement of the duplicated chromosomes to opposite poles.

The process of spore development. Sporulation can be induced by external signals, such as absence of nitrogen.

Transverse alternating field electrophoresis.

The terminal part of a eukaryotic chromosome, consisting of a few hundred base pairs with a defined structure. Telomeres are important for maintaining chromosomal structure and stability, as they permit replication of the ends of the linear DNA molecule.

Four copies of each chromosome (4n).

The process by which DNA is used as a template for the synthesis of an RNA molecule.

The process of protein synthesis from a mRNA template, occurring at the ribosome.

A mobile DNA sequence that can move from one site in a chromosome to another, or between different chromosomes. The transposable elements in yeast are the Ty elements.

Three copies of each chromosome (3n).

A diploid strain carrying three copies of chromosome III is trisomic for chromosome III.

A set of RNAs that act during protein synthesis as adaptor molecules, matching individual amino acids to their corresponding codon on a mRNA. For each amino acid, there is at least one tRNA.

Tys are members of a widely distributed family of eukaryotic elements called LTR-containing retrotransposons. They have the same sequence organisation as retroviruses. The complete retrotransposons are 5 to 6 kilobases long. They are bracketed by long-terminal repeats (LTR), which are 300 to 400 basepairs long.

The diploid cell resulting from the union of two haploid cells of complementary mating types.

Although undeniably a major success, the DNA sequence of S. cerevisiae is relatively small with a genome of 13.5 Mb. Indeed the second eukaryote to have its genome sequenced - the nematode worm (Caenorhabditis elegans) - has a genome of 97 Mb. This sequence published in December 1998 (C. elegans Sequencing Consortium, 1998) has enabled the comparison of two highly diverged eukaryotes, one a unicellular micro-organism and the other a multicellular animal (see Section 4.3.2.2). Of course, this is the ultimate prize although the genome of man (Homo sapiens) is -not surprisingly - much larger, being in the order of 3500 Mb (Oliver, 1996)! Inevitably there is already astonishing progress on the Human Genome Project such that the sequence of one of the smallest of the 23 pairs of chromosomes, chromosome 22, was published in December 1999 (Dunham et al., 1999).

There are two major web sites: (i) the Saccharomyces Genome Database (SGD) at http://genome-www.stanford.edu/Saccharomyces/ and (ii) the European Union funded database at MIPS (Munich Information Centre for Protein Sequences) at http://www.mips.biochem.mpg.de/mips/yeast/MIPS. As detailed elsewhere in this section, these web sites are a splendid and continually updated resource. The review by Cherry et al. (1998) breaks the SGD down into easily digested chunks: (i) (predominately) DNA sequence information, (ii) structural information of proteins (iii) underpinning literature and abstracts and (iv) maintenance of gene nomenclature. Such is the success of the SGD web site that it is 'hit' about 45 000 times a week (Cherry et al., 1998)!

The yeast genome project required the sequencing of some 12 million nucleotide bases (for an overview see Mewes et al., 1997). This encodes about 6217 potential proteins ('open reading frames' - ORFs) which account for almost 70% of the total sequence. Just how revolutionary this project is can be gleaned from our current knowledge of sequence and function. Only about half the proteins are 'known', being well-characterised biochemically or genetically. Of the rest, understanding ranges from 'some indication of their function in vivo' to 'similarities to other unchar-acterised proteins or (show) no similarities at all' (Mewes et al., 1997). The 30% or so of genes without any recognised function - so called 'orphan genes' - represent virgin territory to geneticists and protein chemists. To understand the function of these genes new ways and approaches have had to be devised that frequently challenge traditional thinking.

In principle, knowledge of the sequence of the genome should enable the theoretical synthesis of a complete set of proteins (the so-called 'proteome', Goffeau et al., 1996) that yeast can form. This fascinating opportunity arrives through computer-driven comparisons of the anticipated amino acid sequences with proteins of known function. However, only 50% or so of the predicted proteins that meet the required criteria for 'similarity score' can be functionally identified. Consequently, the distribution of proteins by functional role (Fig. 4.17) is not yet 'carved in stone'. It might be expected that, with time and new effort, the proportion of ORFs with assigned function would increase. Where known, the functions of individual ORFs are given in the 'overview' of Mewes et al. (1997). In some instances, this analysis has revealed gene products in yeast whose existence was previously in doubt.

For example, the classical 'function first' approach (Oliver, 1997) proceeds from understanding biological function to identification of a DNA sequence. Conse-

Cell rescue

Cell rescue

Protein synthesis Protein destination

Fig. 4.17 Yeast genome sorted by eleven functional categories (redrawn from Mewes et al.. 1997).

Protein synthesis Protein destination

Fig. 4.17 Yeast genome sorted by eleven functional categories (redrawn from Mewes et al.. 1997).

quently, the 'orphan' genes (without any recognised biological function) would be missed using this approach. This has led to the concept of performing 'genetic analysis in the reverse direction' (Oliver, 1997). Here, each unknown protein is selectively deleted and its biological function determined by detailed screening of the mutant's phenotype. Hampsey (1997) has described - in a useful 'hands-on' review - the screening over 70 distinct phenotypes in yeast. A more sophisticated route (Oliver, 1997; Oliver et al., 1998) couples two-dimensional gel electrophoresis with the analytical power of mass spectrometry (see Section 4.2.6.4) - to achieve a 'metabolic snapshot' of each deletant. This concept has been extended to enable direct analysis of more than 100 proteins (e.g. the yeast ribosome complex) via a combination of liquid chromatography with mass spectrometry (Link et al., 1999).

A more direct approach to understanding the function of yeast genes is to systematically create deletion mutants of all 6000 ORFs found in the yeast genome. As with the original genome sequencing project, this phase of work is being addressed by a consortium of laboratories in the USA, Canada, Japan and in Europe (European Functional Analysis Network or EUROFAN).

This project is also enabling gene functions to be probed in other ways. Work is ongoing to construct a 'minimalist' genome where every gene is an essential one

(Oliver, 1997). This approach was triggered by the realisation that parts of the genome are gene poor and apparently redundant. Further examination of the sequence shows that almost half the genome is duplicated. In all, 55 large 'blocks' or 'cluster homology regions' (CHRs) together with many other smaller regions (Seoighe & Wolfe, 1998) have been identified through computer analysis where clusters of genes on one chromosome have homologues somewhere else in the genome (Wolfe & Shields, 1997). These workers argue that the entire yeast genome underwent duplication about 108 years ago. They suggest that two ancestral diploid yeast cells with eight chromosomes fused to form a tetraploid which then was reduced to a diploid (with 16 chromosomes) through deletion of about 85% of the duplicate copies. Of interest to the brewer is CHR 'Block 1' (Wolfe & Shields, 1997) where a flocculation gene is one of seven duplicated regions found on chromosome I and VIII. The location of these regions of homology is easily visualised via the 'genome browser' search facility of the splendid MIPS web site.

Subsequent evolutionary events have ensured that gene function is not necessarily duplicated in these blocks. A CHR found in chromosomes XIV and III contains genes that encode the enzyme citrate synthase. However, the cellular location of these enzymes differs in that one (chromosome III) is found in the peroxisome and the other (chromosome XIV) in the mitochondria (Goffeau, 1996). In evolutionary terms, such duplication is a successful strategy (Mewes et al., 1997) as modifications can be made to one copy of a gene without affecting the function - which may be critical - of the other. Indeed, Wolf and Shields (1997) argue that genome duplication may have been critical in the evolutionary adaptation to anaerobic growth. Alternatively, gene duplication may simply have evolved to produce higher levels of a gene product.

4.3.2.2 Yeast genome project, human disease and pathogenicity. One of the more effecting themes from the genome project is the view that an understanding of the yeast proteome is 'a prerequisite for understanding the more complex human pro-teome' (Goffeau, 1996). One of the 'big wins' is that 'nearly half of the proteins known to be defective in human heritable diseases show some amino acid sequence similarity to yeast proteins' (Goffeau, 1996). Although perhaps oversold - 'about 30% of human disease associated genes significantly match yeast genes' (Foury, 1997) - the ease with which yeast genes can be manipulated and modified can be anticipated to provide considerable insight into understanding the molecular basis of human disease. By way of example, some of the yeast genes with a high sequence similarity to human disease genes are detailed in Table 4.15. Such 'comparative genomics' are clearly of major academic and commercial significance, which inevitably will extend beyond S. cerevisiae to include other sequenced model organisms. For example Chervitz et al. (1998) have compared the yeast genome with that of the multicellular nematode worm, C. elegans. From this it is clear that most of the core biological functions (intermediary metabolism, DNA and RNA metabolism etc.) are carried out by orthologous proteins in both S. cerevisiae and C. elegans. These 'orthologous proteins' are proteins of different species that can be traced back to a common hypothetical ancestor. However, those genes (e.g. signalling) involved in multi-cellularity have no yeast orthologs. Taken together these are powerful insights into eukaryote biology and provide strong support for the basic assumption that model

Table 4.15 Yeast homologues (with high sequence similarity) of human disease-associated genes (from Bassett et al., 1996; Foury, 1997).

Yeast gene Function in yeast Human gene Human disease

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