Article 17

Malczynski, & M. Snyder (1998) Pheromone-regulated genes required for yeast mating differentiation. J. Cell Biol. 140 461-483. This article uses the genome-wide transposon mutagenesis approach to identify additional genes involved in cellular processes involved in mating such as agglutination, polarized growth for the formation of the mating projection, cell fusion, and nuclear fusion. Searches for mutants exhibiting defects in mating have identified a number of genes...

References And Further Reading

Botstein, D. (1993) From phage to yeast. In The Early Days of Yeast Genetics, M.N. Hall & Linder, eds. Cold Spring Harbor Laboratory Press, New York. Botstein, D. & G.R. Fink (1998) Yeast an experimental organism for modern biology. Science 240 1439-1443. Botstein, D., S.A. Chervitz, & J.M. Cherry (1997) Yeast as a model organism. Science 277 1259-1260. Hall, M.N. & P. Linder, editors (1993) The Early Days of Yeast Genetics. Cold Spring Harbor Laboratory Press, New York. Lander,...

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...

Reading List

Mutant Hunts To Select or to Screen (Perhaps Even by Brute Force) Hartwell, L.H. (1967) Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93 1662-1670. Complementation Analysis How Many Genes are Involved Hartwell, L.H., R.K. Mortimer, J. Culotti, & M. Culotti (1973) Genetic control of the cell division cycle in yeast V. Genetic analysis of cdc mutants. Genetics 74 267-286. Hereford, L.M. & L.H. Hartwell (1974). Sequential gene function in the initiation of...

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...

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

Saccharomyces Cell Wall Glucan

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...