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 are clones containing billions of genetically identical individual cells. Dividing cells are said to be in the logarithmic phase of growth because the number of cells is doubling at a rate that is dependent on the nutritional quality of the medium. When one or more essential nutrients become limiting, growth and division will slow or even stop and the cells are said to be in stationary phase and the culture is referred to as a saturated culture. This terminology is most often used to describe a liquid culture, but cells in colonies also go through similar phases.
Both rich and synthetic minimal media are used to culture Saccharomyces. Rich medium, called YEP or YP, is made from commercially available yeast extract and peptone (a complex protein digestion product). It contains all essential nutrients including ammonia (a rich nitrogen source), phosphate, sulfate, sodium, magnesium, calcium, copper, iron, etc. and certain other compounds that all Saccharomyces strains are unable to synthesize. In addition, rich medium provides many macromolecular precursors such as amino acids and nucleotides that wildtype Saccharomyces strains are able to synthesize if necessary. A sugar or other carbon energy source must be added, such as glucose (dextrose), sucrose, lactic acid, or others depending on the genotype of the strain and its ability to utilize various carbon sources. Glucose is the richest and most readily available carbon source and a rich medium containing glucose is referred to as YEPD or YPD. Because of the abundant nutrient supply, cells divide rapidly on a rich medium with a division time of about 90 minutes and easily visible colonies are formed in about 2 days.
Synthetic minimal medium, referred to as SM, is made from commercially available yeast nitrogen base plus a carbon source, usually glucose unless specified. It provides the essential nutrients listed above but lacks the amino acids, nucleotides, and other precursors that are in a rich medium. Thus, a strain must be able to synthesize these in order to grow and divide on SM medium. Growth is significantly slower on SM medium, with a doubling time of about 4 hours. Saccharomyces can be grown on a completely chemically defined medium made from about two dozen organic and inorganic compounds, but for most research this is not necessary. A strain capable of growing in a defined minimal medium is called a prototrope. Ideally this minimal medium contains only a carbon source plus inorganic salts, but it is usual for wild-type microorganisms to require supplements, such as a vitamin, to this ideal minimal medium. Despite this, the wild-type genotype is generally considered to be a prototrope. Mutant strains unable to synthesize an essential nutrient are an auxotrope for that particular nutrient.
The following points are very important for the geneticist to note and understand. If a strain is unable to synthesize a particular essential nutrient, then that nutrient will have to be added to the synthetic minimal media to allow the strain to grow on an SM medium. For example, a strain containing a mutation in an ADE gene encoding an enzyme for the biosynthesis of adenine is unable to synthesize adenine and must have adenine added to the synthetic minimal medium to allow it to grow. This mutant strain is an adenine auxotrope. Thus, an ade2 mutant strain requires adenine in the growth medium. In contrast, if a strain is unable to utilize a particular carbon source, for example sucrose, then the strain will not be able to grow on media that provide that carbon source as the sole carbon source. A strain that contains a mutation in a SUC gene is unable to utilize sucrose because it does not synthesize functional invertase, the enzyme required to hydrolyze sucrose to glucose and fructose. Thus, a suc2 mutant strain will not grow if sucrose is the only carbon source provided and some other carbon source, such as glucose, must be available. In summary, strains carrying mutations in anabolic pathways require the product of the pathway for growth while strains carrying mutations in catabolic pathways cannot grow if the substrate of the pathway is provided.
SACCHAROMYCES CEREVIS1AE AS A GENETIC MODEL ORGANISM THE MITOTIC LIFE CYCLE
Saccharomyces is a budding yeast, that is, the ovoid (or egg-shaped) mother cell produces a small protrusion or bud on its surface that grows in size during the course of interphase of the cell cycle into what will become the daughter cell. After the S phase is complete and the DNA has been replicated, the nucleus localizes to the neck region between the mother and the bud, divides into two nuclei, and one nucleus enters the bud while the other remains in the mother (karyokinesis). Following karyokinesis the cytoplasms of the mother and daughter cells divide with the formation of separate plasma membranes and cell walls (cytokinesis), and eventually the daughter cell grows to the size of the mother. Both cells are then capable of dividing again. This is outlined in Figure 1.1. A more in-depth description of the cytological changes that occur during mitosis is presented in Chapter 3.
Both haploid and diploid Saccharomyces cell types can divide by mitotic division. Many eukaryotic organisms favor either the haploid (lower plants, slime molds, many fungi) or diploid (animals, higher plants) portion of the life cycle and proceed through the alternate stage very rapidly. For Saccharomyces the existence of stable haploid and diploid cell types means that the researcher can culture large numbers of genetically identical individuals (clones) and use them for analysis of the phenotype via cytological or biochemical analysis. Other than dealing with different numbers of chromosomes, mitosis of diploid and haploid strains is essentially the same at the level of the chromosome. There are some cytological differences between haploid and diploid cells during mitosis, particularly in bud-site selection, that are discussed in Chapter 3. These do not affect the genetic analysis of other traits.
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