Yeast E-Book

Horst Feldmann

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 689

Veröffentlichungsjahr: 2011

Beschreibung

Yeast is one of the oldest domesticated organisms and has both industrial and domestic applications. In addition, it is very widely used as a eukaryotic model organism in biological research and has offered valuable knowledge of genetics and basic cellular processes. In fact, studies in yeast have offered insight in mechanisms underlying ageing and diseases such as Alzheimers, Parkinsons and cancer. Yeast is also widely used in the lab as a tool for many technologies such as two-hybrid analysis, high throughput protein purification and localization and gene expression profiling. The broad range of uses and applications of this organism undoubtedly shows that it is invalubale in research, technology and industry. Written by one of the world's experts in yeast, this book offers insight in yeast biology and its use in studying cellular mechanisms.

Leseprobe

Contents

Preface

Foreword

1 Introduction

1.1 Historical Aspects

1.2 Yeast as a Eukaryotic Model System

2 The "Early Days" of Nucleic Acids

2.1 Molecular Biology of tRNA

2.2 Ribosomes, Ribosomal RNAs, and Ribosomal Proteins

2.3 Messenger RNAs

3 Genetic Engineering and Reverse Genetics

3.1 The Molecular Revolution

3.2 Transformation of Yeast Cells

3.3 More Genetic Tools from Yeast Cells

3.4 The Two-Hybrid System

4 Cell Structures and Functions

4.1 The Yeast Cell

4.2 Yeast Envelope and Cell Skeleton

4.3 Yeast Chromosome Structure and Function

5 Cellular Dynamics

5.1 Cell Propagation

5.2 Regulatory ATPases – AAA + Proteins

5.3 Protein Modification by Proteins and Programmed Protein Degradation

5.4 The Yeast Cell Cycle, Cell Division, and Chromosome Segregation

5.5 Intracellular Protein Sorting and Transport

5.6 Membrane Transporters in Yeast

6 Anatomy of the Transcription Machinery and Auxiliary Complexes: Chromatin Remodeling

6.1 Transcriptional Regulation and Transcription Factors

6.2 RNA Polymerases and Cofactors

6.3 Coupling Transcription to Pre-mRNA Processing

6.4 DNA Repair Connected to Transcription

6.5 Modification of Chromatin During Transcription

7 Molecular Signaling Cascades and Gene Regulation

7.1 MAP Kinase Pathways

7.2 General Control by Gene Repression

7.3 Gene Regulation by Nutrients

7.4 Stress Responses in Yeast

7.5 Posttranscriptional Control of Gene Expression in Yeast

7.6 Protein Splicing – Yeast Inteins

8 Yeast Organellar Biogenesis and Function

8.1 Mitochondria

8.2 Peroxisomes

9 Genome, Proteome, Transcriptome, Metabolome, and Regulatory Networks

9.1 The Yeast Genome Sequencing Project

9.2 Characteristics of the Yeast Genome

9.3 Functional Genomics

9.4 Hemiascomycetous Yeasts and Evolutionary Aspects

10 Disease Genes in Yeast

10.1 General Aspects

10.2 Trinucleotide Repeats and Neurodegenerative Diseases

10.3 Aging and Age-Related Disorders

10.4 Mitochondrial Diseases

11 Yeast in Biotechnology

11.1 Fermentation and Metabolic Engineering

11.2 Biopharmaceuticals from Health-Care Industries

11.3 Biomedical Research

11.4 Environmental Technologies: Cell-Surface Display

12 Epilogue: The Future of Yeast Research

References

Appendix: Online Resources Useful in Yeast Research

Index

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The Author

Prof. Dr. Horst Feldmann

Adolf-Butenandt-Institut

Molecular Biology

Ludwig-Maximilians-Universität Munich

Germany

Correspondence address:

Prof. Dr. Horst Feldmann

Ludwig-Thoma-Strasse 22B

85232 Bergkirchen

Germany

Cover

Budding Yeast marked with GFP

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Preface

This monography is intended to summarize the outstanding contributions the yeast Saccharomyces cerevisiae has made to molecular and cell biology over half a century. As a simple and fast growing eukaryotic organism tractable to genetic and biochemical approaches it has served as a suitable model system, in which basic cellular processes could be investigated. This is mainly due to the fact that – particularly after its complete genome had become known as the first one among eukaryotes – many components mediating such pathways were found to be evolutionary conserved from yeast to man.

Over the years, I have kept a collection of papers demonstrating the achievements in various fields. I also gained a lot of information from the weekly seminars that were arranged in the departments where I worked and from lectures and courses that I had a chance to present. I gratefully remember the many fruitful discussions with my colleagues from all over the world – at congresses or privately – which helped broaden my background.

Foremost, I wish to thank André Goffeau and Jean-Luc Souciet, who suggested and helped to compile a catalogue of research fields in molecular and cell biology to which yeast made significant contributions. I am indebted to B. Dujon, A. Goffeau, M. Ghislain, C. Marck, F. Messenguy, P. Morsomme, T. F. Outeiro, C. Rodrigues-Pousada for critical reading of the manuscript and many useful comments and suggestions. Not to forget the nice contacts I found during the Yeast Genome and the Génolevures Projects.

I have tried to document the developments by including more than 2000 references. Whenever possible, these references are selected such that the reader can follow achievements made over the past years. By far, this collection will not completely mirror the engagement of so many yeast laboratories. Wherever possible, I cited original papers, but in many cases I had to rely on review articles contributed during these years by competent researchers. Therefore, I apologize to all colleagues who might be disappointed that their original work has not been quoted adequately. Further, I could only briefly mention (or even had to exclude) work that aimed at deciphering biochemical pathways that otherwise would have been worth covering in more detail. It may even be that I gave preference to topics that bear a certain relationship to my own research.

With great pleasure, I wish to acknowledge the care of the team of WILEY- BLACKWELL publishers at Weinheim in editing and manufacturing this book. Dr. Evgenia Koutsouki (Publishing Editor, Life Sciences) kindly guided our first correspondence in September 2008 and invited external reviewers for the manuscript. Once the comments were positive, it was Dr. Andreas Sendtko (Publishing Manager) and his colleagues from the production department to take over. Many thanks for their excellent and accurate handling of my manuscript and the pictures, so I had little trouble with corrections.

The major headache encountered when researching this book was with Deutsche Telekom, who did not manage for a total of eight years to establish a DSL line to our new domicile in Bergkirchen. This meant that I had to use a tediously slow but expensive ISDN internet connection for all my searches.

Finally, but most importantly, I whole heartedly thank my wife Hildegard for her patience and encouragement, who for many years found me toiling over my computer at home.

München and Bergkirchen November 2009

Horst Feldmann

Foreword

Whenever somebody has developed a liking for Science . . . he will stick to it for ever.

Feldmann, H. (2008) A Life with Yeast Molecular Biology, in Stories of Success-Personal Recollections XI V.P. Skulachev and G. Semenza (Eds.), Comprehensive Biochemistry, Vol. 46, Chapter 4, pp. 275–333, Elsevier.

For most of the past 50 years, Horst Feldmann has been scrutinizing yeast, this humble servant of molecular biologists. He made breakthrough contributions to the structure of the serine-specific tRNA and a number of tRNA genes. He discovered the preferred integration of transposon elements upstream of tRNA genes. He identified and characterized the transcriptional activator Rpn4, a proteasome subunit. In 1994, he published in EMBO Journal, with 96 coauthors from 28 European laboratories, the full 807 kb sequence (410 open reading frames) of yeast chromosome II that at that time was the largest chromosome entirely sequenced.

Through his long-lasting involvement in FEBS, EMBO, and DFG boards and the organization of the famous “Spetses Summer Schools,” he acquired a thorough knowledge of the molecular biology scenery in Europe and Soviet Union. In this book, he describes the major scientific knowledge acquired by studying Sacchar-omyces cerevisiae. Nearly 50 000 scientific publications contributed by more than 1000 laboratories have been devoted to yeast. Basic scientific subfields such as tRNA structure, directed mutagenesis, protein engineering, mitochondrial biogenesis, cell cycle, DNA transcription, subcellular trafficking, intron splicing, genomics, and so on have been pioneered by scientists using yeast. Six of them were awarded Nobel Prize. The decantation of this immense pool of scientific information is a task that only someone personally involved in the birth of molecular biology and genomics could undertake. Other books have previously attempted to survey “Yeast Science,” but this one is thorough, updated, and unbiased. It reflects the precision of the chemistry training of Horst Feldmann as well as his independence and ethics in the choice of most relevant references. The illustrations bear his personal artistic stamp. A crew of specialized subeditors have checked all the information reported. Thank you, dear Horst, for this book. For many colleagues, it will remain as a legacy of our common sticky passion that is the unraveling of the molecular basis of eukaryotic life and a testimony of the uniqueness of “The Yeast Model”.

Louvain-la-Neuve, November 2009

André Goffeau

2

The "Early Days" of Nucleic Acids

2.1 Molecular Biology of tRNA

The adaptor hypothesis, formulated by Francis Crick in 1957 [13] in connection with his thoughts on the genetic code [14], proposed that during protein synthesis the single amino acids concatenated to a peptide chain on "microsomal particles" are carried by specific adaptor molecules. On the one hand, these adaptors can form a stable bond with specific amino acids and on the other hand these are capable of delivering these amino acids to the growing peptide chain by reading the information from a (microsomal) RNA template according to the same base-pairing rules as found in DNA. In the same year (1957), Hoagland, Zamecnik, and Stephenson [15] reported the discovery of what then was collectively called sRNA (soluble ribonucleic acid) and what we now call tRNA (transfer ribonucleic acid), after having shown that the activation of amino acids for the RNA-dependent synthesis of polypeptides involves the cleavage of ATP to AMP and pyrophosphate, with the intermediate formation of aminoacyl-AMP compounds.

Using sRNA from E. coli or liver cell extracts, Fritz Lipmann's group could show that the amino acids were, in fact, chemically bound via a highly reactive aminoacyl ester bond to the 3'-terminus of these molecules [16]. The aminoacylation test [15] became an excellent means to demonstrate that particular amino acids were bound to specific sRNA components. At this time, however, it was nearly impossible to fractionate sRNA from cell extracts into single species to yield individual transfer ribonucleic acids.

The first success of isolating tRNAs free from other cellular components seems more than fortuitous. Zamecnik and his colleagues [17] had noticed that direct extraction of yeast with aqueous phenol could be used for this purpose because under the conditions employed, little of the high molecular weight material was released from the cells. This method could be applied for large-scale preparation of total tRNA from yeast. One early application based on this approach was the large-scale preparation of yeast aminoacyl tRNA, which then was used in our experiments to establish that the amino acid was preferentially linked to the 3'-OH moiety of the terminal adenosine residue [18]. More importantly, large-scale preparation of tRNA formed the basis to obtain purified amino acid-specific tRNAs for biochemical analysis. Experiments alongthisline started in the late 1950s. One has to recollect, however, that working out appropriate fractionation procedures and applying them for mass production was a hard task. First, tons of yeast slurry had to be subjected to phenol extraction, and raw tRNA had to be precipitated from the aqueous phase with ethanol (or acetone) and further purified by column chromatography on DEAE cellulose. This scale of mass preparation of starting material was later by far surpassed when Kornberg and his colleagues "manufactured" 10 000 l of yeast cultures to obtain sufficient quantities of RNA polymerase II transcription complexes and transcription factors for X-ray studies [19, 20] (see Section 6.2).

The isolation of amino acid-specific tRNA was the most tedious task: fractionation of total tRNA by means of consecutive countercurrent distributions in various systems, column chromatography, and so on, whereby each single fraction had to be measured for amino acid acceptor activity. Of course, the subsequent analytical procedures to be applied (partial and complete digestion with more or less specific nucleases, fractionation of the fragments or components, determination of the nature of the single constituents) extended over several years. But in the end, Holley and his coworkers in the United States succeeded in determining the first sequence of a tRNA, the alanine-specific tRNA from yeast [21] (Figure 2.1a). Soon after, Hans Zachau's group from the new Institute of Genetics in Cologne reported the sequences and the structures of the modified nucleotides from two yeast serine-specific tRNAs [22, 23] (Figure 2.1b).

Figure 2.1(a) Cloverleaf structure of the alanine-specific tRNA [21]. The boxed G-C has to be removed according to Ref. [24]. (b) Cloverleaf structure of the serine-specific tRNAs [22, 23]. Substitutions in Ser tRNA I versus Ser tRNA II are marked by arrows.

The next tRNA sequences to be elucidated were those of yeast tRNATyr [25] and tRNAPhe [26]. In the years to follow, the sequences of a great variety of amino acid-specific tRNAs from yeast and some of their isoacceptors were determined in several laboratories. The groups of Guy Dirheimer at the Institute of Molecular and Cellular Biology and the Faculty of Pharmacy in Strasbourg contributed many yeast nuclear encoded tRNA structures, a subject they pursued since 1968 until the end of the 1970s [27]; sequencing in Strasbourg was continued to characterize yeast mitochondrial tRNAs, mainly in the decade to follow [27, 28]. A plethora of modified nucleosides occurring in yeast nuclear and mitochondrial tRNAs were identified and characterized by many workers as well. Characterization of the modified nucleosides was facilitated by the mass preparation of specific tRNAs, and more than 50 could be detected in yeast [29]. In most cases, it was also possible to delineate the enzymatic pathways by which the "odd" compounds are derived from their parent nucleotides. Many laboratories were involved in this research and this field is still under investigation as a few citations may illustrate [30–41]. Finally, RNA modifications could be discovered by using microarrays [42].

A rational extension of the work on yeast tRNA structure was to solve the problem of how aminoacyl tRNA synthetases specifically interact with their cognate partners in the aminoacylation reaction [43]. This afforded the purification of specificenzymes and allowed to set up appropriate methods for the isolation of tRNA/synthetase complexes to carry out X-ray studies and fast kinetic techniques to determine reaction parameters. Important contributions came from a most successful cooperation between several groups, those of Ebel, Giégé, Moras, Grosjean, and others [44–46]. This is not to deny that other organisms, mainly E. coli, have contributed a lot to this field. In all, these investigations led to the important notion that each tRNA/synthetase system has to follow intrinsic rules for recognition given by the conformational features of the partners. tRNA "identity" was also recognized as the superior criterion for the interactions between (pre)tRNAs and the nucleotide modifying enzymes.

Remarkably, the first three-dimensional structure of a tRNA molecule was also derived from a yeast tRNA, the phenylalanine-specific tRNA, by Alex Rich and coworkers in 1974–1975 [47]. Figure 2.2 shows the comparison of the conformation of yeast phenylalanine tRNA in two crystal forms. The two molecules from the orthorhombic and monoclinic unit cells have been fitted by a least squares procedure. Three group coordinates are plotted: the position of the phosphorus atom, the centroid of the five atoms in the furanose ring of ribose, and the centroid of the six atoms that make up the six-membered ring in either pyrimidines or purines.

The shape of its three-dimensional structure caused Francis Crick to compare it to the folded structure of protein saying that "tRNA mimics a protein." More 3D structures of tRNAs were disclosed later, for example, by the approaches of Sigler and his collaborators [49].

During the 1970s, attention was drawn to the cellular processes as to how tRNAs (the prototypes of simple and stable RNAs) are transcribed from the corresponding genes and how the resulting precursors are processed to finally result in their mature form. Necessary steps in tRNA biosynthesis included modifications of particular nucleotides and, in eukaryotes, the enzymatic addition of the universal 3' -CCA end not contained in the gene sequences to the "core" tRNA [50, 51].

Figure 2.2 Three-dimensional structure of yeast phenylalanine tRNA. Left: The solid line connecting the group coordinates represents the conformation of the molecule in the orthorhombic unit cell, while the dashed line shows its conformation in the monoclinic unit cell. Right: Secondary and tertiary hydrogen bonds between bases are shown with different shading. The numbers refer to the residues in the polynucleotide chain. (Reproduced from Ref. [48] with permission of Oxford University Press.)

From the pioneering work of Darnell on RNA precursors, it was already manifest that eukaryotic tRNA precursors must contain additional sequences at their 5'- and 3' -ends that have to be removed during maturation [52]. Blatt and Feldmann [53] using extremely short pulses of [32P] during yeast growth and subsequent fractionation of the precursors by polyacrylamide gel electrophoresis noted that some specific precursors were considerably longer than the matured molecules and that processing might occur in consecutive steps. Fractionation and characterization of a total population of specific tRNAs and tRNA precursors was later refined by a two-dimensional gel electrophoresis approach [54]. In contrast to sea urchin tRNA genes found to be arranged in clusters of tandem repeats [55], or most of the E. coli tRNA genes being arranged in a polycistronic mode [56], the yeast tRNA genes were found to occur as single transcriptional units scattered throughout the genome [57]. The only exception to this rule later turned out to be a yeast tRNAAsp–Arg pair [58]. Precursors to specific tRNAs were identified by Hopper and Kurjan [59].

A big surprise came from the sequence analysis of a yeast tRNATyr gene and the analysis of its transcript by Goodman, Olson, and Hall: the tRNATyr precursor revealed the presence of a 14-nucleotide intervening sequence located 3' to the anticodon, being removed during the maturation process [60]. So the cloning and analysis of the first tRNA genes from yeast indicated that split genes occurred not only in adenovirus [61, 62] but, as found soon after, also in protein encoding genes in mammalian cells [63–65]. The first intron in a yeast mitochondrial gene (the large rRNA coding gene) was identified in 1978 [66] though its presence was so obvious from the differences between ω+ and ω– strains as observed by Dujon in 1974 (see Section 8.1).

In the early 1980s, more yeast tRNA genes containing introns had been characterized and studies on their maturation begun [67–69]. Finally, it became evident that some 25% of the yeast tRNA genes carry introns of variable length but always at the same position, next to the 3'-side of the anticodon. It was mainly John Abelson and his collaborators who became interested in yeast tRNA splicing and after many years succeeded in unraveling the details of this maturation step and in characterizing the endonucleases involved in this process [70]. An interesting observation was that in some cases modifications of particular nucleotides depended on the presence of the intron sequences. However, the functional significance of these introns (occurring generally in eukaryotic tRNA genes) largely remains a mystery.

2.2 Ribosomes, Ribosomal RNAs, and Ribosomal Proteins

Undoubtedly, the pioneering work on ribosomes, ribosomal RNAs, and ribosomal proteins was done in the E. coli system. In the early days of molecular biology, there was no need to fall back upon eukaryotic organisms, since appropriate material was abundantly available from bacterial sources. Also, detailed work on ribosome structure and function was guided by investigations into bacterial ribosomes. Only when it became apparent by the work of Hartwell and colleagues that the constituents of eukaryotic ribosomes in several aspects differed from their bacterial counterparts, such as in type of RNA or number of ribosomal proteins [71–73], as well as in size, arrangement [74, 75], and expression of the rRNA genes, yeast became a player in this field.

Earliest research on yeast ribosomes stems from the work of J.R. Warner [76], when he investigated the assembly of ribosomes in yeast, followed by numerous publications from his laboratory on the ribosome field [77, 78]. When it became feasible to study gene regulation in yeast, the genes for ribosomal RNAs and ribosomal proteins were of particular interest. A hierarchy of elements regulating the synthesis of yeast ribosomal proteins [79–81] and later the effects of nutritional control on ribosome synthesis were described [82, 83]. REB1, a key regulator of yeast ribosome synthesis, was described in 1990 [84], and a new regulator was discovered in 2004 [85]. Description of promoter and terminator elements for rRNA synthesis as well as the trans-regulatory control proteins began in 1984 and has been continued to present [86–90]. The three-dimensional structure of the yeast ribosome has been established [91].

Measured in terms of sequence determination, rRNA genes and ribosomal protein genes from yeast were somewhat behind what had been established in E. coli. Sequences of the small ribosomal RNAs from E. coli were established in 1967 [92], and those of the large rRNA genes in 1978 and 1980, respectively [93, 94]. In comparison, the sequences of the yeast ribosomal RNA genes were solved a few years later: 5S [95], 5.8S [96], 18S [97], and 28S [98]. The first DNA sequences containing yeast ribosomal protein genes were isolated in 1979 [99], and later on these genes were characterized [100].

2.3 Messenger RNAs

2.3.1 Structure of Yeast mRNAs

Studies on yeast messenger RNA started around 1969–1970 in the laboratory of Lee Hartwell with the participation of Warner and McLaughlin [101]. As soon as it became known that mammalian messenger RNA is polyadenylated [102, 103], McLaughlin et al. [104] were able to show that messenger RNAs from yeast contain polyadenylic acid sequences of ~50 nucleotides in length at their 3'-ends, and a few years later it was established that the 5'-termini of mRNA from yeast are blocked by methylated nucleotides [105]. Interestingly, even yeast histone mRNA was found to contain 3'-poly(A) sequences [106].

Yeast messenger RNAs for specific proteins were isolated and characterized only late [107]. For example, mRNAs for glycolytic enzymes were identified in 1978 [108] and ribosomal protein genes in 1980 [109]. A fact mentioned in Michael Smith's Nobel lecture [110] is that his first cooperation for applying his newly developed approaches of using synthetic deoxyribo-oligonucleotides for monitoring gene isolation involved the people working on the yeast iso-1-cytochrome c gene, the laboratories of Fred Sherman and Benjamin D. Hall. Stewart and Sherman [111] had identified frameshift mutations by sequence changes in iso-1-cytochrome c. This led to the enzymatic synthesis of oligonucleotides of defined sequence for identifying this gene [112], its isolation, and sequence determination [113,114]. The sequence of the iso-1-cytochrome c (CYC1) mRNA was also determined [115], as well as its 5'-end positioned by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs [116]. Later, Guarente and collaborators studied the regulation of CYC1 [117, 118] and CYT1 (cytochrome c1) by heme via the HAP complex [119].

Both the aforementioned techniques were applied to the SUP4 tRNATyr locus [120,121]. Thus, these initial approaches made clear that "synthetic DNA" became an invaluable tool for many applications: as a probe for gene isolation, in direct sequencing of double-stranded DNA by the enzymatic method of Sanger's laboratory [122] using synthetic oligonucleotide primers, for the precise identification of point mutations produced by classical genetic techniques at a given locus, or in the development of oligonucleotide-directed mutagenesis.

The interest in isolating specific mRNAs from yeast probably faded away as soon as the cloning of specific yeast genes became feasible. The lack of large introns in yeast genes and an average size of yeast genes of some kilobases meant a huge advantage in the cloning strategies over genes from higher eukaryotes, where introns could be manifold and of considerable length.

2.3.2 Introns and Processing of pre-mRNA

It was in 1977 that the occurrence of introns in mammalian genes was pinned down and that splicing was detected as the decisive step in maturation of pre-mRNA to mature m RNA by the Nobel Prizewinners of1993, R. Roberts [123] and P. Sharp [124], not to forget the merits of others [61–65]. Only 3 years later, the actin gene from yeast was shown by Gallwitz and Sures to possess an intron sequence near its 5'-end [125].

Though it became clear much later that only 4–5% of the yeast genes possess introns, the sophisticated splicing machinery of eukaryotic organisms has been fully retained in yeast. Finally, more than a hundred different genes encode products important for RNA splicing, comprising about 2% of the total yeast genome. In fact, yeast has served as a model system that has substantially contributed to fully disentangle the "splice cycle" genetically and biochemically, mainly initiated by the work of C. Guthrie, J. Abelson, J. Beggs, and their collaborators [126,127]. Nonetheless, details of the splicing mechanism are still under study to date [128–130].

In 1983, Langford and Gallwitz [131] described a (unique) intron-contained sequence in yeast required for splicing, the so-called branch point, which was also observed in polyadenylated RNA from other sources [132]. In yeast, this site was identified as a particular A residue within the (unique) intron sequence TACTAAC [133]. In the same year, several groups were able to show that lariat structures are the in vivo intermediates of the splicing process, similarly occurring in yeast and in mammalian systems [134–139]. Biochemically, the branch site could be defined as a 2'/3'-ester bond [140]. Shortly before, in 1981, it had been recognized by Breathnach and Chambon [141] that there was a limited set of conserved sequences (preferably 5'-GU. . .AG-3') at each intron boundary, and these consensus sequences were found to be common for vertebrate, plant, and yeast cells [142].

From then on, several groups were engaged in characterizing the cellular components involved in the splicing process and in elaborating the detailed mechanism of this process. In the end, it turned out that mRNA processing followed similar routes in yeast and in higher eukaryotes. The first functionally important components surmised to be involved in splicing were the small nuclear RNAs (snRNAs) [143], which are ubiquitous and had been found in all organisms from bacteria to humans, andinmany viruses. While the snRNAs in higher eukaryotes are encoded by up to 100 gene copies each, the laboratory of C. Guthrie detected that yeast contains five small nuclear RNAs (U1, U2, U4, U5, and U6 snRNAs), each encoded by a single copy of an essential gene [144]. The mutual interactions of snRNAs and their interaction with pre-mRNA, as well as the interdependence of particular splicing steps with particular snRNAs, were studied in detail. The fact that the intermediate state, consisting of two RNAs, was efficiently converted to the final products strongly suggested that these RNAs remain bound in a complex and, given the importance of the snRNAs in splicing, suggested the existence of a "splice cycle" and finally led to the eminently important discovery of the "spliceosome" [145, 146]. Moreover, the spliceosome was recognized as a particle (much like a ribosome) in which the RNA components were associated with a number of proteins forming stable cellular RNA–protein complexes (PRPs) [147].

The single steps in the spliceosome cycle, where particular PRP proteins are required, are consistent with the cycle as defined by kinetic and biochemical methods. Most transitions between specific forms of the spliceosome require one or more specific proteins. Furthermore, a number of PRP mutants were shown to be defective in splicing because of their inability to reassemble snRNPs for further splicing. Thus, both genetic and biochemical results proved that the spliceosome cycle is the process responsible for excision of introns from split genes (Figure 2.3)

The nearly 100 different proteins shown to cooperate in splicing belong to various types, such as zinc-finger proteins, small G proteins, and ATP-dependent RNA helicases of the DEAD-box or DEXH-box families [148–153]. Although the basic mechanisms of pre-mRNA splicing had been resolved in about 15 years from the discovery of spliced genes [126,127,154–156], in sort of a competition between yeast and higher eukaryotes, the aspects of alternative splicing and trans-splicing had to wait their resolution with the aid of organisms other than yeast, since these routes scarcely exist in yeast. Furthermore, yeast could contribute only little to solve questions about the evolution of introns and exons.

Figure 2.3 The splice cycle in yeast. (After Ref. [126].)

Several scientists had speculated that genes originally evolved as exons and that the progenote organism from which current prokaryotic and eukaryotic organisms evolved may have had a split gene structure [157–160]. These primordial exons are pictured as encoding sequences for stable protein folding domains. Assembly of a number of exon sequences by RNA splicing would be expected to produce a protein composed of stable folding domains that have a high probability of being functional either structurally or catalytically. If genes originally evolved in this fashion, the arrangement of introns in relation to protein secondary structure might not be random. Evidence to support this hypothesis has been sought in the exon–intron structure of evolutionarily old proteins critical for energy metabolism.

Phylogenetic comparison of the sequences of homologous genes from a variety of organisms revealed that intron sequences had drifted much more rapidly than exon sequences. This suggested that intron sequences might generally not be functional, at least in the context of requiring long tracts of specific sequences. Furthermore, the length of introns in homologous genes significantly varied during evolution, suggesting little constraint. Finally, it became clear that specific introns could be lost during evolution. The mechanism responsible for the exact deletion of introns is probably related to gene conversion using a cDNA copy of the mRNA or a partially spliced intermediate RNA. This process has been documented for the removal of introns from yeast genes by G. Fink [161] and raises the question of why introns persisted during evolution [162].

However, as will be discussed in a separate chapter below (Section 8.1), yeast mitochondria revealed introns in several of its genes completely differing in structure from nuclear genes. One outstanding finding was that introns (at least in part) coded for particular functions, a fact that later also became apparent for many nuclear genes from higher organisms, and another surprise was that mitochondrial introns behaved like mobile genetic elements [163].

Summary

In mid-1960s, efforts to work out the genetic code raised an interest in determining the primary structures of tRNAs, those molecules that had been postulated by Francis Crick to function as adaptors in protein synthesis. Biochemical analysis afforded (i) the large-scale preparation of tRNA free from other cellular components and (ii) the fractionation of bulk tRNA into amino acid-specific species. In 1960, Monier, Stephenson, and Zamecnik had devised an approach to isolate low molecular weight RNA from yeast by simple phenol extraction and precipitation of the soluble RNA from the aqueous phase with ethanol, which made this organism a most useful source for successive work. Fractionation and subsequent analysis of purified tRNA species was much more tedious and took several years. In the end, the first sequence of a tRNA to be determined in 1965 was that of an alanine-specific tRNA from yeast followed by the sequences of yeast serine, tyrosine, and phenylalanine tRNAs. In all cases, these sequences could be arranged in a "cloverleaf" structure, with the anticodon triplet exposed in the anticodon loop. Some 10 years later, the three-dimensional structure of yeast phenylalanine tRNA was resolved. This model formed a basis to investigate the interactions of tRNA with its cognate partners, the amino acid tRNA synthetases and nucleotide modifying enzymes. The newly developed molecular techniques, such as cloning and sequencing DNA, were successfully applied to study the genomic arrangement of yeast tRNA genes and to follow the biogenesis of mature tRNA from their precursors. Research in 1977 was highlighted by the detection of introns in mammalian messenger RNAs, but it came as a similar surprise the same year that yeast tRNA genes do also contain "intervening sequences" that have to be processed out from the transcripts during maturation (a procedure that later was confirmed for many eukaryotic tRNA genes in general). Although only some 20% of the nuclear yeast tRNA genes were later recognized to possess intervening sequences, tRNA precursors could successfully be used to characterize the enzymes involved in the cleavage and ligation reactions.

Although research on yeast ribosomes and ribosome synthesis started in the early 1970s, the fundamental knowledge to this field was mainly contributed from the studies of prokaryotes, preferably the bacterium E. coli, or mammalian cells. Nonetheless, the yeast system provided useful details on eukaryotic ribosomal components and on the regulation of ribosome biosynthesis.

A field to which yeast made significant contributions was to unravel the mechanism of splicing of eukaryotic pre-mRNAs. Although only 4–5% of the protein encoding genes from yeast do possess introns, a comparison of the "splice cycle" in yeast and mammals revealed great similarity. This finding again underlined the notion that basic cellular mechanisms and components have been conserved throughout evolution. It took about 15 years (1984–1998) to work out a detailed picture. The spliceosome was recognized as a particle in which the RNA components (pre-mRNA as the substrate and auxiliary snRNAs) were associated with particular proteins (PRPs) forming stable subcellular RNA–protein complexes during the splicing process. In all, over hundred of such proteins were characterized. Of invaluable help in defining the single steps within the cycle were a multitude of PRP mutants from yeast, which were defective in splicing because of their inability to assemble specific subcomplexes.

3

Genetic Engineering and Reverse Genetics

3.1 The Molecular Revolution

In the early 1970s, three events generally revolutionized molecular biology: (i) the discovery of restriction mechanisms in bacterial cells by Werner Arber [164,165] on which basis the first specific restriction endonucleases could be isolated and applied by Smith and Wilcox [166] and by Adler and Nathans [167]; (ii) the possibility of creating recombinant DNA in vitro and to transfer it into host cells where it was capable of exerting particular functions, first demonstrated by Paul Berg and collaborators [168,169]; and (iii) the development of methods allowing the determination of DNA sequences by Walter Gilbert and coworkers [170, 171] and Frederick Sanger and collaborators [122,172], which had to follow principles different from the ones applied to RNA sequencing. Not surprisingly, yeast molecular biology was soon caught by these new potentials.

Until 1976 or 1977, any nucleic acid material from yeast had to be isolated from cell preparations. Information on particular genes, and their regulation or interactions was largely derived from genetic experiments, a privilege that was also offered by other organisms that had been used as genetic model systems, such as bacteria and their phages, Neurospora, or Drosophila.

The beginning of genetic engineering undoubtedly was marked by the successful approach of Paul Berg and his collaborators to show that recombinant DNA could be maintained in a host cell [168]. I vividly remember a long night session with full moon in the courtyard of a monastery at a Summer School 1971 held in Erice, where Berg, Sanger, and Tomkins chaired a discussion on the above three paradigm shifts. Restriction enzymes from a variety of sources became available soon and were applied to generate recombinant DNA. Methods allowing the determination of DNA sequences became reality in the years to follow and were used in yeast.

Although since 1972 several methods had been developed for cloning and characterizing recombinant DNA molecules [173], it was only after the Asilomar Conference on Recombinant DNA [174] that safe and simple procedures and vehicles could be propagated for extensive use of cloning recombinant DNA molecules. Refined cloning systems along these lines were developed in the years to follow. Clearly, the ease of cloning was manifested by the use of plasmid vectors [175–179], but the big advantage of cloning vehicles based on phage lambda was the larger size of DNA sequences that could be accommodated [180, 181]. These properties were shared by the cosmids, plasmid gene-cloning vectors packageable in phage lambda heads [182]. A technical innovation, the colony hybridization, became extremely useful in isolating specific genes from yeast, too [173].

Already before the safer cloning vehicles were available, plasmids and phage lambda had been used to clone gene containing DNA fragments from a variety of organisms [183–185] including yeast [186]. For the first time, in vitro synthesized gene sequences were cloned, for example, the β-globin gene [187]. As soon as the new cloning systems appeared on the market, they were widely used to clone DNA fragments and particular genes from various sources including yeast [188].

3.2 Transformation of Yeast Cells

The transformation of yeast cells by replicating hybrid plasmids was independently developed by two laboratories as early as in 1978, those of Jean Beggs [9] and Albert Hinnen [10]. This first successful transformation of a eukaryotic cell marked a breakthrough in (yeast) molecular biology. Several types of episomal (designated YEp), highly replicating (YRp), or chromosomally integrating shuttle vectors (YIp) were designed, carrying various selectable markers and/or various elements allowing the expression of particular yeast – or even foreign – genes [189–191]. They became thus widely applicable for studying single genetic entities or for reverse genetic approaches. Of particular interest was the notion that a chimeric plasmid endowed with a segment of centromeric DNA (YCp) and transformed in yeast cells stabilizes this plasmid. During mitotic cell division, this plasmid will be normally replicated once and the two copies are segregated between mother and daughter cells in a 1: 1 ratio. In meiotic division, the four copies will be segregated at a ratio 2:2. Hundreds of different vectors of these types have been devised till date and have been made commercially available.

3.3 More Genetic Tools from Yeast Cells

The construction of yeast cosmids, on the one hand, and the development of yeast cells carrying artificial chromosomes, on the other hand, can be viewed as extensions of the possibilities of transforming yeast cells into useful tools. The first yeast cosmid construct was described by Hohn and Collins [192], more sophisticated vectors were later employed to build ordered, genomewide yeast cosmid libraries [193, 194] that could be used in the yeast genome sequencing project (see Chapter 9). The cloning of large segments of exogenous DNA into yeast by means of yeast artificial chromosome vectors (YACs) was developed by Olson and his collaborators [195]. A prerequisite in this approach was the electrophoretic karyotyping of yeast [196]. In the beginning, when applied to human DNA, the YACs were of considerable advantage for mapping human chromosomes. Unfortunately, the high preposition of yeast to recombination via short (~70 bp) homology regions resulted in too many mapping failures.

3.4 The Two-Hybrid System

A novel technique revolutionizing the detection of protein–protein interactions of any kind was established in 1989by Fields and Song [197]. The yeast two-hybrid system has been developed as a potent tool to identify cDNAs, carried on one plasmid, which codes for proteins that interact with a target protein specified by a DNA sequence carried on another plasmid. This simple approach was based on the unique properties of the yeast Gal4p transcriptional activator regulating the expression of GAL4 and hence other galactose genes in yeast (see Section 9.2): the Gal4p transcriptional activator is composed of two physically separable, functionally independent activation and binding domains (Gal4-AD and Gal4-BD, respectively). The cloning vectors, which are endowed with different markers, are used to create fusions of the GAL4 domains with genes for proteins that potentially interact. After introduction of these entities into yeast strains that carry an appropriate reporter gene (HIS3 or lacZ) with a GAL4 upstream activating sequence (UAS) element in its promoter, only upon interaction of the two domains the DNA-BD will be tethered to the AD and will reconstitute the Gal4p transcriptional activator, which then results in the activation of the reporter gene. A selection of positive clones can be achieved by screening them for His + or lacZ + positives, and the GAL4-AD/library fusion plasmid can efficiently be retrieved from such colonies. The method has been improved since its invention [198], particularly to minimize the appearance of false positives, which, however, still seems to be a problem not completely overcome. In addition, a yeast three-hybrid system for detecting small ligand–protein receptor interactions was developed in the late 1990s [199, 200]. Bacterial two- and n-hybrid systems later came into use as well [201, 202].

Summary

The successful transformation of yeast cells by hybrid plasmids in 1978 marked a milestone in molecular biology. Depending on their molecular shaping, these plasmids would autonomously replicate in yeast (as single or multicopy entities) or integrate as singular copies into defined loci within the yeast genome. A plasmid endowed with a segment of centromeric DNA and transformed in yeast cells is stabilized and during mitotic cell division this plasmid will be normally replicated once and the two copies segregated according to the rules known for the yeast chromosomes. Shuttle vectors, capable of propagating both in yeast and in bacterial cells, allowed reciprocal transfer of genetic material from one host to the other. Selection of transformed cells was facilitated by inclusion of appropriate genetic markers into the plasmid sequences. Replica plating of yeast cells grown on solid agar or colony hybridization was as easy as for bacterial cells. Expression plasmids carrying yeast-specific promoter (and terminator) sequences could be used to express foreign genes in the yeast system and even to design them for export. Remarkably, this approach also proved that in a multitude of cases human genes were capable of functionally complementing their homologous counterparts in appropriate yeast mutants.

A suitable extension of yeast transformation by plasmids was offered by the finding that appropriate cosmids of considerable length (up to 40 kb) could serve as shuttle vectors as well. This technique was later applied for the construction of ordered yeast genomic libraries, which turned out to be much more advantageous in the sequencing project than plasmid or phage libraries. A similar line was followed in the construction of “yeast artificial chromosomes.” Thus, human DNA fragments up to 1 Mb could be accommodated and propagated. The big hope of using this tool in mapping the human genome, however, finally turned into a disappointment because the YACs suffered rearrangements in the yeast due to its preposition of frequent recombination via shorthomology regions. This preposition has been employed in “one-step gene replacement” of yeast genes as only 20 bp at each border were sufficient to effect disruption and subsequent substitution of a genomic sequence by another.

A most successful technique, developed in 1989 and still applied, is the yeast two-hybrid system for the detection of protein–protein interactions.

4

Cell Structures and Functions

4.1 The Yeast Cell

For a better understanding of what I will discuss in this chapter, Figure 4.1 presents a micrograph of a dividing yeast cell pointing to some of its major components and organelles. In the next Section 4.2, we will deal with the yeast envelope and the cell skeleton. Section 4.3 is devoted to the major genetic material distributed throughout the 16 chromosomes residing within the nucleus. Other genetic elements such as the retrotransposons and some extrachromosomal elements will be considered in Sections 4.3.4 and 4.3.5. More chapters are devoted to aspects of cellular dynamics and the structures of the cell organelles involved in these processes (Chapter 5), the transcription machinery (Chapter 6), gene regulation and signaling pathways (Chapter 7), and function of organelles (Chapter 8).