Article

Schekman (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21 205-215. In this article the authors use secl-1 mutant cells to improve the isolation method and obtain additional sec mutants. Characterization of the new mutants is begun by placing them into complementation groups (genes) and making phenotypic comparisons among strains carrying these different mutant genes. 1. Figure 1 shows...

Cell Fractionation

The study of subcellular components often requires purifying a large amount of a particular component. One might be interested in isolating and purifying for further analysis the components of a complex unit like a ribosome, proteasome, or spliceosome. Or one might want to measure the metabolic activity of organelles such as the mitochondrion or peroxisome. Cell fractionation methods have been developed that allow the researcher to separate relatively pure samples of subcellular components in...

Genetic Crosses And Linkage Analysis

Saccharomyces diploids undergo meiosis when placed in starvation conditions and form four haploid ascospores, or just spores for short, all contained in a single sack called an ascus. These four spores are referred to as a tetrad since each spore contains one chromatid from each of the 16 tetrads of chromatids found in prophase I of meiosis. To fully understand the crosses outlined below, it would be helpful to first review the process of meiosis including chromatid segregation patterns and...

Article 17

Tecklenburg, & R.A. Sclafani (1999) RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae. Genetics 151 965-977. The eukaryotic cell cycle is a highly regulated process requiring dozens of proteins to coordinate the series of complex events, sometimes occurring in parallel, which must proceed in an orderly manner. After years of studying the genes encoding these regulators, Hartwell and coworkers realized that a second class of...

Single gene segregation

Since genl-62 is a single alteration in the GEN1 gene, the only possible tetrad that can result from this cross is one containing two GEN1 spores and two genl-62 spores. If a thousand tetrads were dissected all would be 2 wild-type 2 mutants because only a single mutant gene is segregating in this cross. Thus, the 2 2 segregation pattern is consistent with the fact that a single genetic difference exists between the parent strains in the cross. But what if one does not know whether a mutant...

Immunoprecipitation And Related Techniques

These methods are used to physically separate a particular protein from a cell extract. They rely on a high-affinity, sequence-specific interaction between the protein and another molecule capable of specifically binding to that protein, such as an antibody. To achieve physical separation, the molecule providing the recognition specificity is bound to an inert substrate that can be separated physically from the binding reaction mix. Once purified by one of these methods, the protein can be...

Article 15

Chen, & D. Botstein (1991) A group of interacting yeast DNA replication genes. Genes Dev. 5 958-969. The authors of this article make very elegant use of genetic analysis to explore the function of a new series of essential genes involved in the initiation of DNA synthesis. Strains carrying cold-sensitive mutations at two loci, CDC45 and CDC54, arrest growth at low temperatures with a phenotype similar to cdc7 mutants that is, they arrest as large budded cells with...

Overview

Baker's brewer's yeast, Saccharomyces cerevisiae, is a molecular genetic model organism. It is a eukaryote with a nucleus and membrane-bound organelles like mitochondria, peroxisomes, endoplasmic reticulum, and a Golgi complex. As such, complex processes like chromosome replication, transcription and translation, cell division, secretion, membrane trafficking, subcellular compartment structure and function, energy metabolism, cytoskeletal structure and mechanics, and intracellular signaling...

Article 4

Fangman, & J. Rosamond (1986) Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae. Mol. Cell. Biol. 6 15901598. 1. The Introduction to this article summarizes the phenotype of strains carrying temperature-sensitive mutations of CDC7 as follows. At the nonpermissive temperature cdc7 mutants arrest prior to the initiation of DNA replication as budded cells with a single nucleus lacking an elongated spindle but having a divided...

Article 2

Manney (1974b) Mutations affecting sexual conjugation and related processes in Saccharomyces cerevisiae. II. Genetic analysis of nonmating mutants. Genetics 76 273-288. In Article 1, MacKay & Manney isolated a series of sterile mutants that they placed into 16 different phenotypic classes. Most of these mutants were not conditional, i.e. they were sterile under all growth conditions. Thus, genetic analysis of these mutants would be difficult. Mating frequency was very...

Introduction

Molecular genetics is a tool used by today's biologist interested in understanding not simply describing the underlying mechanisms of processes observed in cellular and developmental biology. It is a fusion of the biochemical and genetic approaches to problem solving developed over the past decades and the resulting synergy of these approaches has produced an extremely powerful tool for the investigation of living systems. The biochemical approach has been very productive in identifying the...

Nuclear congression

KAR2 KAR5 KAR7 SEC71 KAR8 SEC63 SEC72 Figure 3.7 Mating and nuclear fusion in Saccharomyces. The pathway of events followed by the nuclei of mating cells and the zygote is depicted. The spindle pole bodies with their attached cytoplasmic microtubules orient toward each other and toward the schmoo projection, which has formed at this position in response to the higher concentration of pheromone. Fusion of the cytoplasms allows the cytoplasmic microtubules to make contact and to draw the two...

Flow Cytometry

Cells can be sorted into different classes using a fluorescence activated cell sorter (FACS). For this, cells are selectively labeled with a fluorescent dye and sorted into classes based on the extent of labeling. As described above, fluorescence labeling can be done using an antibody-conjugated fluorescent dye or a fluorescent dye such as DAPI that binds a specific cellular component. For example, an antibody specific to a particular cell surface protein is conjugated to a fluorescent dye....

Article 5

Hollingsworth, R.R., R.M Ostroff, M.B. Klein, L.A. Niswander, & R.A. Sclafani (1992) Molecular genetic studies of the Ccd7 protein kinase and induced mutagenesis in yeast. This article is an example of mutation analysis, the detailed characterization of several mutant alleles of a gene. It is an important first sep in determining the specific function or functions of the gene product of a gene and the molecular mechanism by which it carries out those functions. Article 4 reported that Cdc7p...

Corinne A Michels

Department of Biology, Queen's College of the City University of New York, New York, USA Copyright 2002 by John Wiley & Sons, Ltd Baffins Lane, Chichester, West Sussex P019 1UD, England e-mail (for orders and customer service enquiries) cs-books wiley.co.uk Visit our Home Page on http www.wiley.co.uk or http www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical,...

Article 13

Rowley, & C.A. Kaiser (1997) Control of amino acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7 and LST8. Genetics 147 1569-1584. The initial goal of this study was to identify new genes encoding proteins involved in ER to Golgi vesicle formation. Mutations in SEC13 were found to produce reduced numbers of 50 nm vesicles (ER to Golgi intermediates) and to exhibit synthetic lethal interactions with mutations in...

Case Studies from the Saccharomyces Genetic Literature

The case studies in Section III are an integral part of the learning experience to be gained from this book. The role of the case study is to reinforce the theory presented in each chapter of Section II by providing the reader with actual bench experiments that put the theory into practice. Unlike much of biological research that tends to be quite descriptive, genetic analysis techniques are abstract and in many ways similar to mathematics. In molecular genetics, one studies genes and proteins...

Article 16

Huang, I. Herskowitz, & M. Peter (1998) The role of Farlp in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating. Science 282 1511-1516. In addition to identifying farl-s mutants, Chenevert et al. (1994) isolated the mutant alleles of several other genes involved in polarized morphogenesis during mating (schmoo formation) as well as in vegetative cell division. These included alterations in CDC24, a GDP-GTP exchange...

Article 7

Kurhara, J. Rothblatt, J. Way, & P. Silver (1989) A yeast gene important for protein assembly into the endoplasmic reticulum and the nucleus has homology to DnaJ, an Escherichia coli heat shock protein. J. Cell Biol. 109 2665-2675. This article describes the cloning of yeast SEC63. It also illustrates how the same gene can be identified in different selection screens that use different phenotypes as the basis of the selection screen. The authors had developed a...

Article 11

Thorner, & D.D. Jenness (1994) Mutational activation of the STE5 gene product bypasses the requirement for G protein (3 and 7 subunits in the yeast pheromone response pathway. Mol. Cell. Biol. 14 1054-1065. This article describes the isolation of constitutive STE5 mutations. Such mutations generate constitutive signaling via the mating-type response pathway and cause cell cycle arrest in haploids. Therefore, investigators isolating such mutations must do so in...

Article 8

Schekman (1992) Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. Mol. Cell. Biol. 12 3288-3296. Sequence analysis of Sec63 protein suggested that this is an integral membrane protein and, given its function in the early stages of ER translocation, Sec63p is probably localized to the ER. This must be experimentally determined. In addition the functional importance of the DnaJ-like...

Article 9

Richardson, & C. Wittenberg (1989) Analysis of the Cdc28 protein kinase complex by dosage suppression. J. Cell Sci. Suppl. 12 29-37. This article describes the isolation of multicopy suppressors of two mutant alleles of CDC28. We will only concern ourselves with the suppressors of the temperature-sensitive allele cdc28-4. 1. Describe the strategy behind the authors' decision to search for multicopy suppressors of cdc28-4. (a) What multicopy suppressor genes...

Article 14

Holzmacher, P. Teung, & C.A. Kaiser (1995) Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p. J. Cell Biol. 131 311-324. SEC12, SEC13, SEC16, and SEC23 are required for vesicle formation for ER to Golgi transport. This article begins to explore the role of the encoded proteins, in particular Secl6p. 1. Review the biochemical functions of Sarlp, Secl2p, and Sec23p. What evidence suggests that S EC 16 genetically interacts...

Article 10

Sugino, & A. Sugino (1992) Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G S cell cycle transition. Genetics 131 21-29. Temperature-sensitive mutations in DBF4 had been isolated in an independent screen for cdc mutants (Chapman & Johnston, 1989). These dbf4 ts alleles do not initiate DNA synthesis at the nonpermissive temperature and arrest with a dumbbell-shaped morphology similar...

Article 1

Schekman (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccahromyces cerevisiae. Proc. Natl Acad. Sci. USA 76 1858-1862. Secretion is an essential process. It provides a mechanism for releasing proteins to the extracellular space and for the insertion of integral membrane proteins into the lipid bilayer of cellular membranes. The process of secretion directs proteins to compartments such as the plasma membrane and vacuole, and...

Article 3

Hartwell, L.H. (1980) Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J. Cell Biol. 85 811-822. In this article Hartwell uses the G1 arrest response to isolate mutants in the pheromone response pathway. Since a-factor is easily purified from the culture medium of mating type a cells, Hartwell was able to add the pheromone directly to the culture medium. Hartwell avoided the problems in genetic analysis encountered by MacKay & Manney as...

Article 6

Kozutsumi, M-J. Gething, & J. Sambrook (1989) S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell 57 1223-1236. BiP is an ER localized member of the HSP70 (heat shock protein 70) family. Studies of BiP function in mammalian cells showed that it bound transiently to a number of nascent secreted proteins and strongly to improperly folded proteins blocking their exit from the ER. As such, it was hypothesized that BiP...

Expression Vectors

Expression vectors are vectors that allow one to construct gene fusions that replace the native promoter of a gene with another promoter for any of a variety of reasons. For example, the native promoter might initiate transcription at a very low rate, too low to allow for purification or detection of the protein product of the gene, or only under very special conditions. Placing the ORF of the gene of interest under the control of a high-level constitutive promoter in a YEp vector would...

Nucleus

The Saccharomyces nucleus is an ovoid structure localized off to one side of the vacuole in the unbudded cell. It contains the chromosomes and in the electron microscope one can observe a nucleolus. For an in-depth review of nuclear structure and function the reader is referred to Wente et al. (1997). The Saccharomyces nuclear envelope that separates the contents of the nucleus from the cytoplasm consists of a double membrane, each having a lipid bilayer. One unique feature of Saccharomyces is...

Protein Extraction And Purification

Standard analytical methods for protein purification and characterization are routinely used in the study of Saccharomyces proteins. It is important to have a Figure 2.6 Immuno-gold localization. a-Factor receptor Ste2p is localized to the plasma membrane of a mating type cells. In response to the binding of a-factor, Ste2p is internalized in the form of vesicles by a process called endocytosis. Immunogold labeling methods are used here to visualize the subcellular location of Ste2p during this...

Reading List

Mutant Hunts To Select or to Screen (Perhaps Even by Brute Force) Novick, P. & R. Schekman (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccahromyces cerevisiae. Proc. Natl Acad. Sci. USA 76 1858-1862. Complementation Analysis How Many Genes are Involved Novick, P., C. Field, & R. Schekman (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21 205-215. Mutant Hunts and...

References And Further Reading

Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, & K. Struhl, editors (2001) Current Protocols in Molecular Biology. John Wiley & Sons, Ltd, New York. Brown, A.J.P. & M. Tuite (1998) Methods in Microbiology. Vol. 26 Yeast Gene Analysis. Academic Press, New York. Chalfie, M Y. Tu, G. Euskirchen, W.W. Ward, & D.C. Prasher (1994) Green fluorescent protein as a marker for gene expression. Science 263 802- 805. De Vit, M.J., J.A. Waddle, & M. Johnston...

Reporter And Other Types Of Fusion Gene

A reporter gene is used to follow gene expression in vivo. It is a fusion between all or part of a gene of interest with another gene whose product is easy to detect or measure qualitatively and or quantitatively. Most often, the researcher will choose to use a reporter gene if the product of the gene of interest is difficult to assay or detect. Thus, the reporter gene product acts as a surrogate. Figure 1.6 Reporter gene fusion constructions A fusion gene between the gene of interest and the...

Advanced Concepts in Molecular Genetic Analysis

With the advent of recombinant DNA technology and the development of methods for the purification and sequencing of proteins, it became possible to clone the gene encoding any purified protein. With the cloned gene in hand, one can use any one of a number of techniques to introduce random mutations into a cloned sequence, or one can induce mutations in regions of interest, such as sites of putative functional motifs, using a variety of in vitro techniques. The mutant alleles are then tested in...

Saccharomyces Cell Structure

Saccharomyces cerevisiae is a eukaryote and as such contains the subcellular organelles commonly found in eukaryotes. The structure and function of these organelles is fundamentally the same as it is in other eukaryotes with less versatile systems for genetic analysis, and for this reason Saccharomyces is the organism of choice for many cell biologists. For a truly in-depth review of Saccharomyces cell structure and function, the reader is referred to The Molecular and Cellular Biology of the...

Two Hybrid Analysis

The two-hybrid method was conceived and developed by Stan Fields and coworkers as a technique to detect binding physical interaction between two proteins under in vivo conditions Fields amp Song, 1989 . The concept underlying the two-hybrid method is based on the modular structure of some transcription activators and on the use of reporter genes. The reader should be sure to carefully review these subjects before attempting to understand the two-hybrid method. Stan Fields conceived of the idea...

Mutant Hunts To Select or to Screen Perhaps Even by Brute Force

The first step in a genetic analysis of a process it to isolate mutant individuals that are unable to carry out that process or carry it out in an aberrant way. The researcher must hypothesize what characteristics, changes in growth capabilities, morphology, etc. will be exhibited by this individual and to develop a means of identifying such individuals from among a larger group of normal individuals. A mutation is a heritable change in the genetic material, and mutagenesis is the process of...

Gene Disruptiondeletion In Saccharomyces Onestep Gene Replacement

Gene disruption is a method by which a DNA fragment is used to replace a genome sequence with a selectable marker gene, such as HIS3 or kanavanine resistance. In so doing, a deletion is created. The process occurs by homologous recombination and uses the enzymes of the homologous recombination pathway, such as Rad52p. The ends of the exchange fragment must be long enough and have sufficient homology to the chromosomal site so that homologous recombination can occur. Moreover, the size of the...

Glucose Grown Remove Glucose

Figure 2.4 Green fluorescent protein fusions for visualization of living cells. The photo shows a time-course of Migl repressor exit from the nucleus. These cells are expressing a fully functional Miglp GFP fusion protein. Following growth on glucose, the cells are harvested and placed in medium lacking glucose. The first panel shows the cells 30 seconds after glucose removal. Miglp-GFP is seen localized to a discrete subcellular site that, by DAPI staining, is shown to be the nucleus data not...

Genomic Analysis

With the completion of the sequence of the S. cerevisiae genome in April 1996 came a new challenge, namely functional analysis of the genome. Methods needed to be developed on a genome-wide scale to provide the tools for understanding the roles of the approximately 6000 gene products, their expression patterns, and how they interact to create a eukaryotic organism capable of complex processes like growth, cell division, and the response to extracellular signals. Several groups from throughout...

Enhancement and Synthetic Phenotypes

Enhancement is the opposite of suppression. In suppression the two mutations act together to produce a phenotype that is similar to the wild-type. In enhancement the two mutations act together to produce a mutant phenotype that is more severe than that exhibited by either mutation alone. Examples of enhancement are often discovered by chance. An investigator carries out a selection procedure for a particular mutant phenotype, but, when the mutant strains obtained are analyzed, it is discovered...

Microscopy Techniques

In standard light bright-field microscropy, a beam of light from a source usually placed below the specimen is focused onto a specimen, passes through the specimen, is focused by a second series of lenses, and is then observed by eye or photographed. Samples are usually fixed to denature the proteins in the specimen, sectioned into thin slices if needed , attached to a solid substrate the slide , and stained using any of a series of chemicals that specifically react with cellular components...

Mating Type Mating And The Sexual Life Cycle

Haploid Life Cycle Saccharomyces

In nature most strains of Saccharomyces are diploid and carry the functional allele of the HO gene, homothalic diploids. Laboratory research strains carry mutant ho and can be grown as stable haploid cells. Haploids occur in two mating types, the a mating type and the a mating type, and these differ from one another at a single locus called the MAT locus. The two alleles of this locus are referred to as MA 7a and MATa. Stable a or a strains divide mitotically to produce genetically identical...

Classes Of Saccharomyces Cloning Plasmid Vectors

Saccharomyces plasmids were developed from Escherichia coli plasmid vectors. The basic E. coli vector is small 2-4 kilobasepairs kbp of DNA and includes genes needed for plasmid replication, an origin of replication ORI derived from an E. coli plasmid, and a selectable marker gene such as AMPr for ampicillin resistance to be used to identify E. coli transformants containing the plasmid. The E. coli ORI allows the plasmid to replicate independent of the E. coli chromosome as an extrachromosomal...

Saccharomyces Genome And Nomenclature

Saccharomyces cerevisiae has a haploid chromosome number of 16. The entire Saccharomyces genome of strain S288C is sequenced and available on the Saccharomyces Genome Database called SGD at http genome-www.stanford.edu Saccharomyces. The site has a variety of tools for sequence analysis that are particularly useful for the Saccharomyces researcher, including gene and restriction maps of the chromosomes. The site is interconnected with genome databases for other genetic model organisms and sites...

Cell Shape And Growth Patterns

Under usual culture conditions, Saccharomyces is ellipsoidal ovoid in shape and approximately 5-10 j,m long by 3-7 m wide. This is referred to as the yeast form. Figure 3.1 shows a scanning electron micrograph SEM of a cell in the yeast form. Cell division is by budding that is, a smaller ovoid daughter cell forms as a projection from the surface of the mother cell. Haploid cells are generally about one-half the volume of diploid cells. The characteristic shape is maintained by a rigid cell...

Glutamate Urea Mg2

Figure 2.1 Equilibrium density gradient analysis. Shown are the results of an equilibrium density gradient analysis carried out to determine the subcellular localization of Gaplp, the general amino acid permease, under different culture conditions glutamate or glutamate transferred to urea . Taken from Roberg et al., 1997. The density gradient is 20 -60 sucrose with and without Mg2 . The upper row of panels shows, for each fraction, the sucrose concentration open circles and the relative levels...

Gap Repair

This is a method frequently used to recover a specific sequence from the chromosome onto an episomal plasmid. Gap repair utilizes the host cell's recombination repair and DNA replication machinery to fill an artificially created deletion in a homologous sequence carried on the plasmid. Its most common use is for cloning different alleles of a cloned gene. One starts with the DNA fragment of interest cloned into a plasmid vector that is maintained as an extrachromosomal element, such as YRp or...

Complementation Analysis How Many Genes are Involved

Complementation analysis is used to determine whether two independent mutations are alterations in the same gene that is, they are alleles, or are alterations in different genes. In essence, a complementation analysis is a functional test used to define a gene. If a researcher has isolated a number of mutants with a similar phenotype, the next question asked is 'How many genes have I identified '. If there are 10 mutant strains, are they each in different genes, does each mutant carry a...

Suppression Analysis

Suppression analysis is an elegant and highly favored molecular genetic tool used to identify genes that are functionally related to the gene of interest. It dates back to the very earliest days of genetics and the work of Sturtevant 1920 and Beadle amp Ephrussi 1936 but it was not until the 1960s that the variety of suppression mechanisms and the capabilities of suppressor analysis were fully appreciated. Increased use of suppressor analysis, particularly in model genetic organisms like...

Cell Wall Cell Surface Morphology And Morphological Variation

Cerevisiae Bud Diploid

The Saccharomyces cell wall is about 200 nm thick and completely surrounds the cell. Its function is to preserve the osmotic integrity of the cell and define morphology but it has several other roles. Proteins involved in cell-cell recognition and adhesion, such as occurs during mating, are found in the cell wall. Other proteins are immobilized or retained in the periplasm, the space between the plasma membrane and the cell wall, by the cell wall. These are secreted proteins and localize...

Culture Conditions

Saccharomyces can be grown in defined media, either liquid or solid, that provide the energy and nutrients required for growth and proliferation. In a liquid medium, in which the components are dissolved in water, the individual cells are in suspension. Agar is added to a liquid medium to make solid media. Individual cells placed on the surface of a solid medium grow and divide many times using the nutrients that diffuse to them from the surrounding medium. They form well-defined colonies that...