Genetics, Molecular and Cell Biology of Yeast

Genetics, Molecular and Cell Biology of Yeast
Roger Schneiter January 2004

Yeast Genetics, page 2

Table of Content

1. Introduction

5

1.1. What are yeasts ?

4

1.2. History of yeast and yeast in history

6

1.3. Yeast in relation to the tree of life

7

1.4. Yeast is a model eukaryote

7

1.5. Information on yeast

10

1.6. Yeast strains

11

1.7. A few basic facts about yeast

12

2. Growth and life cycles

13

3. The yeast genome

18

3.1. Sequencing project overview

20

3.2. Overview of clustered duplications in the Saccharomyces cerevisiae genome 23

3.3. Example the genetic and physical map of chromosome iii

24

4. Genetic nomenclature

25

4.1. Chromosomal genes

25

4.2. Mitochondrial genes

28

4.3. Non-mendelian determinants

30

5. Genetic analyses

31

5.1. Overviews with examples

31

5.2. Tetrad analysis

33

5.3. Complementation and its complications

36

5.3.1. Complementation groups as genes

36

5.3.2. Intragenic complementation as an indication of multiple domains

37

5.3.3. Nonallelic noncomplementation as an indication of assemblies

37

5.4. Non-mendelian inheritance

37

5.5. Suppression

38

6. Transformation

40

6.1. Yeast vector and dna fragments

41

6.2. Synthetic oligonucleotides

42

6.3 Mitochondrial transformation

42

7. Yeast vectors

43

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7.1. Yip vectors

45

7.2. Yep vectors

46

7.3. Ycp vectors

47

8. Genes important for genetic studies

48

8.1. URA3 and LYS2

48

8.2. ADE1 and ADE2

49

8.3. GAL1 promoter

50

8.4. LacZ and other reporters

51

9. Reverse genetics: manipulating the genome in vitro with plasmids

52

9.1. Coning by complementation

53

9.2. Mutagenesis in vitro

55

9.3. Two-step gene replacement

56

9.4. Gene disruption and one-step gene replacement

58

9.5. Plasmid shuffle

61

9.6. Recovering mutant alleles

64

10. Interaction of genes

65

10.1. Heterozygosity and dominant-negative mutations

65

10.2. Intragenic complementation

67

10.3. Non-allelic non-complementation

68

10.4. Suppressors

69

10.5. Synthetic enhancement and epistatic relationships

71

11. Genomic analysis

73

12. Analyses with yeast systems

76

12.1. Two-hybrid systems

76

12.2. Yeast artificial chromosomes (yacs)

79

12.3. Expression of heterologous proteins in yeast

82

13. Cell biology

84

13. Bibliography

85

1. INTRODUCTION

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1.1. What are Yeasts?
Yeast are unicellular fungi (http://www.doctorfungus.org/). The precise classification uses the characteristics of the cell, ascospore and colony. Physiological characteristics are also used to identify species. One of the more well known characteristics is the ability to ferment sugars for the production of ethanol. Budding yeasts are true fungi of the phylum Ascomycetes, class Hemiascomycetes. The true yeasts are separated into one main order Saccharomycetales.
Yeasts are characterized by a wide dispersion of natural habitats. Common on plant leaves and flowers, soil and salt water. Yeasts are also found on the skin surfaces and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites. The common "yeast infection" is typically Candidiasis is caused by the yeast-like fungus Candida albicans. In addition to being the causative agent in vaginal yeast infections Candida is also a cause of diaper rash and thrush of the mouth and throat.
Yeasts multiply as single cells that divide by budding (eg. Saccharomyces) or direct division (fission, eg. Schizosaccharomyces), or they may grow as simple irregular filaments (mycelium). In sexual reproduction most yeasts form asci, which contain up to eight haploid ascospores. These ascospores may fuse with adjoining nuclei and multiply through vegetative division or, as with certain yeasts, fuse with other ascospores.
The awesome power of yeast genetics is partially due to the ability to quickly map a phenotype-producing gene to a region of the S. cerevisiae genome. For the past two decades S. cerevisiae has been the model system for much of molecular genetic research because the basic cellular mechanics of replication, recombination, cell division and metabolism are generally conserved between yeast and larger eukaryotes, including mammals.

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The most well-known and commercially significant yeasts are the related species and strains of Saccharomyces cerevisiae. These organisms have long been utilized to ferment the sugars of rice, wheat, barley, and corn to produce alcoholic beverages and in the baking industry to expand, or raise, dough. Saccharomyces cerevisiae is commonly used as baker's yeast and for some types of fermentation. Yeast is often taken as a vitamin supplement because it is 50 percent protein and is a rich source of B vitamins, niacin, and folic acid.
In brewing, Saccharomyces carlsbergensis, named after the Carlsberg Brewery in Copenhagen, where it was first isolated in pure culture by Dr. Emile Christian Hansen around 1888, is used in the production of several types of beers including lagers. S. carlsbergensis is used for bottom fermentation. S. cerevisiae used for the production of ales and conducts top fermentation, in which the yeast rise to the surface of the brewing vessel. In modern brewing many of the original top fermentation strains have been modified to be bottom fermenters. Currently the S. carlsbergensis designation is not used, the S. cerevisiae classification is used instead.
The yeast's function in baking is to ferment sugars present in the flour or added to the dough. This fermentation gives off carbon dioxide and ethanol. The carbon dioxide is trapped within tiny bubbles and results in the dough expanding, or rising. Sourdough bread, is not produced with baker's yeast, rather a combination of wild yeast (often Candida milleri) and an acid-generating bacteria (Lactobacillus sanfrancisco sp. nov). It has been reported that the ratio of wild yeast to bacteria in San Francisco sourdough cultures is about 1:100. The C. milleri strengthens the gluten and the L. sanfrancisco ferments the maltose. For more information about sourdough see rec.food.sourdough FAQ.
The fermentation of wine is initiated by naturally occurring yeasts present in the vineyards. Many wineries still use nature strains, however many use modern methods of strain maintenance and isolation. The bubbles in sparkling wines are trapped carbon dioxide, the result of yeast fermenting sugars in the grape juice. One yeast cell can ferment approximately its own weight of glucose per hour. Under optimal conditions S. cerevisiae can produce up to 18 percent, by volume, ethanol

Yeast Genetics, page 6 with 15 to 16 percent being the norm. The sulfur dioxide present in commercially produced wine is actually added just after the grapes are crushed to kill the naturally present bacteria, molds, and yeasts.
The yeast like fungus, Candida albicans, is commonly found in the mouth, vagina, and intestinal tract. Candida is a normal inhabitant of humans and normally causes no ill effects. However, among infants and individuals with other illness a variety of conditions can occur. Candidiasis of the mucous membranes of the mouth is known as thrush. Candidiasis of the vagina is called vaginitis. Candida also causes severe disease in persons with AIDS and chemotherapy patients.
1.2. History of Yeast and Yeast in History
Most anthropologist consider humans to established agrarian, wheat-growing civilization in 10,000 B.C.E. in the Fertile Crescent of Sumaria, present day Iraq. Beer making began in the same area, some six thousand years ago, i.e. before the birth of Abraham. In the upper, left hand corner of this ancient seal you can see a representation of Sumarians drinking beer through straws.
Fermentation of mashed grains was probably considered a magical property of a properly cared-for vessel. We have been carrying around these vessels ever since and thus the cultivation of yeast has always been closely linked with human culture. It was not until Louis Pasteur's time that yeast was colony-purified. Saccharomyces cerevisiae was purified from European beers. Schizosaccharomyces pombe was purified from an African millet beer.

1.3. Yeast in Relation to the Tree of Life

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The five kingdom classification scheme (plants, animals, fungi, protista and monera) was developed prior to existence of nucleic acid comparisons between living things. Plants, animals and fungi are all closely related eukarya. This tree of life is a figure we have revised from Norm Pace.

1.4. Yeast is a Model Eukaryote
The yeast Saccharomyces cerevisiae is clearly the most ideal eukaryotic microorganism for biological studies. The "awesome power of yeast genetics" has become legendary and is the envy of those who work with higher eukaryotes. The complete sequence of its genome has proved to be extremely useful as a reference towards the sequences of human and other higher eukaryotic genes. Furthermore, the ease of genetic manipulation of yeast allows its use for conveniently analyzing and functionally dissecting gene products from other eukaryotes.

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This chapter deals only with the yeast S. cerevisiae, and related interbreeding species. The fission yeast Schizosaccharomyces pombe, which is only distantly related to S. cerevisiae, has equally important features, but is not as well characterized. The general principles of the numerous classical and modern approaches for investigating S. cerevisiae are described, and the explanation of terms and nomenclature used in current yeast studies are emphasized. This article should be particularly useful to the uninitiated who are exposed for the first time to experimental studies of yeast. Detailed protocols are described in the primary literature and in a number of reviews in the books listed in the Bibliography. The original citations for the material covered in this chapter also can be found in these comprehensive reviews.
Although yeasts have greater genetic complexity than bacteria, containing 3.5 times more DNA than Escherichia coli cells, they share many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Unlike many other microorganisms, S. cerevisiae is viable with numerous markers. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal bakers’ yeast are commercially available and can provide a cheap source for biochemical studies. Can be grown on chemically defined media.
Unlike most other microorganisms, strains of S. cerevisiae have both a stable haploid and diploid state. Thus, recessive mutations can be conveniently isolated and manifested in haploid strains, and complementation tests can be carried out in diploid strains. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Exogenous DNA with at least partial

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homologous segments can therefore be directed at will to specific locations in the genome. Also, homologous recombination, coupled with yeasts’ high levels of gene conversion, has led to the development of techniques for the direct replacement of genetically engineered DNA sequences into their normal chromosome locations. Thus, normal wild-type genes, even those having no previously known mutations, can be conveniently replaced with altered and disrupted alleles. The phenotypes arising after disruption of yeast genes has contributed significantly toward understanding of the function of certain proteins in vivo. Many investigators have been shocked to find viable mutants with little of no detrimental phenotypes after disrupting genes that were previously assumed to be essential. Also unique to yeast, transformation can be carried out directly with synthetic oligonucleotides, permitting the convenient productions of numerous altered forms of proteins. These techniques have been extensively exploited in the analysis of gene regulation, structure-function relationships of proteins, chromosome structure, and other general questions in cell biology. The overriding virtues of yeast are illustrated by the fact that mammalian genes are being introduced into yeast for systematic analyses of the functions of the corresponding gene products.
In addition, yeast has proved to be valuable for studies of other organisms, including the use of the two-hybrid screening system for the general detection of protein-protein interactions, the use of YACs for cloning large fragments of DNA, and expression systems for the laboratory and commercial preparation of heterologous proteins. Many of these techniques are described herein.
During the last two decades, an ever-increasing number of molecular biologists have taken up yeast as their primary research system, resulting in a virtually autocatalytic stimulus for continuing investigations of all aspects of molecular and cell biology. Most significantly, knowledge of the DNA sequence of the complete genome, which was completed in 1996, has altered the way molecular and cell biologist approach and carry out their studies (see Dujon, 1996; Goffeau et al., 1996). In addition, plans are under way to systematically investigate the possible functions of all yeast genes by examining the phenotypes of strains having disrupted genes.

1.5. Information on Yeast

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A general introduction to a few selected topics on yeast can be found in the book chapters "Yeast as the E. coli of Eucaryotic Cells" and "Recombinant DNA at Work" (Watson et al., 1987). Comprehensive and excellent reviews of the genetics and molecular biology of S. cerevisiae are contained in three volumes entitled "Molecular Biology of the Yeast Saccharomyces" (Broach et al., 1991; Jones et al., 1992; Pringle et al., 1997). An important source for methods used in genetics and molecular biology of yeast is contained in the book edited by Guthrie and Fink (1991). Overviews of numerous subjects are also covered in other sources (Broach et al., 1991; Brown & Tuite, 1998; Jones et al., 1992; Pringle et al.,1997; Wheals et al., 1995), including protocols applicable to yeasts (Fields & Johnson, 1993) and introductory material (Walker, 1998). A more comprehensive listing of earlier reviews can be found in Sherman (1991). Interesting and amusing accounts of developments in the field are covered in The Early Days of Yeast Genetics (Hall & Linder, 1992). The journal Yeast publishes original research articles, reviews, short communications, sequencing reports, and selective lists of current articles on all aspects of Saccharomyces and other yeast genera.

Current and frequently-updated information and databases on yeast can be conveniently retrieved on the Internet through World Wide Web, including the "Saccharomyces Genomic Information Resource" (http://genomewww.stanford.edu/Saccharomyces/) and linked files containing DNA sequences, lists of genes, home pages of yeast workers, and other useful information concerning yeast. From the MIPS page (http://www.mips.biochem.mpg.de/) you can access the annotated sequence information of the genome of Saccharomyces cerevisiae and view the chromosomes graphically or as text, and more. The Incyte / YPD page (http://www.incyte.com/sequence/proteome/index.shtml) contains a protein database with emphasis on the physical and functional properties of the yeast proteins (subscription only).

This script is based on: “An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae” by Fred Sherman (http://dbb.urmc.rochester.edu /labs/Sherman_f/yeast/Index.html)