Molecular Biology of the Cell - Volume 2 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1993

Cell Growth and Division
Yeast as a Model System

Yeasts are unicellular Fungi comprising a large group of rather diverse organisms. Because they reproduce almost as rapidly as Bacteria and have a Genome Size less than 1/1000th that of mammals, they have proved exceptionally useful for the genetic analysis of Introduction/5.html">Eukaryotic Cell biology. Although Xenopus eggs are an invaluable model for studying the biochemical and cytophysiological aspects of Cell Cycle regulation, they are unsuitable for genetic studies. By contrast, working with Yeast offers powerful opportunities for identifying, cloning, and characterizing the genes involved in cell cycle control. Here we will focus on two species: the budding yeast Saccharomyces cerevisiae, used by bakers and brewers, and the fission yeast Schizosaccharomyces pombe. Cells of the latter divide symmetrically into two identical daughter cells, whereas budding yeasts divide in a less common, asymmetrical manner: the mother cell produces a small bud that grows and completes the remaining Phases of the cycle before finally detaching from the parent cell (Fig. 13-16).

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Fig. 13-16. Comparison of The Cell cycles of fission and budding yeasts. In fission yeast (top), a typical eukaryotic cell cycle exhibits G₁, S, G₂, and M phases. The nuclear envelope, however, does not break down: mitotic spindle microtubules form within The Nucleus and attach to spindle pole bodies at its periphery. The cell divides in two by forming a partition (called a cell plate). In budding yeast, the cycle includes normal G₁ and S phases; however, the microtubule-based spindle begins to form very early, during S phase, and consequently a normal G₂ phase is absent. Unlike the fission yeast cycle, mitosis here does not involve visible chromosome Condensation, and the cell divides by budding. As in fission yeast (but unlike higher Eukaryotic cells), the nuclear envelope remains intact during mitosis.

It is believed that the evolutionary lineages leading to budding and fission yeasts diverged hundreds of millions of years ago. Nevertheless, their life cycles are similar. Both forms can reproduce in either a diploid or a haploid state. In addition to normal division, diploid cells can undergo Meiosis to produce haploid cells (see Chapter 15); likewise, haploid cells can undergo regular division or fuse in pairs to form diploid cells (Fig. 13-17; see also Section 10.3.2). The presence of a haploid phase facilitates genetic analysis and makes it possible to isolate loss-of-function mutants; in a diploid Organism, such Mutations would be in a recessive state (as is the case in cultured mammalian cells) and thus much harder to detect and screen. In both yeast species, Nutrition and sexual mating play a crucial role in cell cycle control; therefore, yeasts serve as an excellent model for exploring the broader question of how the division cycle is regulated by extracellular environmental factors.

Fig. 13-17. Life cycles of the budding yeast (Saccharomyces cerevisiae) and the fission yeast (Schizosaccharomyces pombe). The Proportions of the life cycle spent in the Haploid and Diploid phases vary between species and depending on environmental conditions. With an abundant food supply, normal wild-type strains of budding yeast reproduce as diploid cells with a life cycle duration of about two hours. Under starvation conditions, however, they undergo meiosis to form haploid spores, which germinate under favorable conditions to yield haploid cells. Depending on environmental conditions and genotype, these cells either divide or fuse (conjugate) in G₁ phase to re-form diploid cells. Conversely, fission yeast typically reproduces in the haploid state; under nutrient deprivation, haploid cells fuse to form diploid cells, which rapidly undergo meiosis and sporulation to restore the haploid phase. The most widely used laboratory strains of budding yeast are mutants that, much like fission yeast, reproduce primarily in the haploid phase.

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13.2.1. Each mutation affecting the yeast Cell Division cycle halts or disrupts the progression of the cycle at a specific phase [12, 13]

Identifying the genes involved in cell cycle control requires appropriate mutants and Methods for obtaining progeny from them. However, cells with a disrupted cell cycle machinery are incapable of reproduction. The way around this is to search for conditional mutants, in which the defect is manifested in the phenotype only under specific conditions. Typically, researchers look for a Gene product whose molecular Structure is slightly altered such that it loses its function in one (restrictive) Temperature range while retaining it in another (permissive) range. For such temperature-sensitive mutations, low temperatures are usually permissive, and high temperatures are restrictive. Thus, a mutant can be obtained at a low temperature and then, by raising the temperature, the altered gene can be inactivated to study the mutant phenotype.

Fig. 13-18. These diagrams illustrate how a temperature-sensitive cell division cycle (cdc) mutant can be distinguished from other temperature-sensitive mutants. When the temperature is raised to the restrictive level, where the mutant gene product cannot function normally, the mutant will continue its cell cycle until it reaches a step it cannot pass (in this case, the initiation of S phase). Because the cell continues to grow despite the cell cycle block, cdc mutants become abnormally large (not shown). Meanwhile, with other mutations that disrupt processes required for growth throughout the entire cycle (such as ATP synthesis), the cell will arrest at any stage of the cycle as soon as its biochemical reserves are depleted.

Mutations that specifically affect individual Components of the cell cycle machinery cannot be identified solely by the loss of the mutant cells' ability to divide, since any lethal defect will lead to this outcome. Cell division cycle (cdc) mutations are more reliably identified by how they block or arrest a specific phase of the cell cycle at the permissive temperature (Fig. 13-18). In budding yeast, the presence and size of the bud serve as a simple visual indicator showing which stage of the cell cycle is blocked in a given cdc mutant; in the case of fission yeast, more sophisticated approaches using the cell cycle analysis methods discussed above are required.

Between 40 and 50 cdc genes have been identified in each of the two aforementioned yeast species. In A number of cases, biochemical analysis has made it possible to precisely determine the function of the gene product. For example, certain cdc mutants arrested in S phase proved to be defective in genes encoding DNA ligase or Enzymes required for the synthesis of DNA precursors. As will be described below, Recombinant DNA technology can serve as a general approach for characterizing all Proteins encoded by cdc genes. However, some important insights can be gleaned even without this information.

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13.2.2. Yeast cdc mutants can be used to analyze the coupling between cell cycle events

When the temperature is raised to the restrictive level, the cell cycle arrests in most cdc mutants at the stage where the cdc gene product acts. As a rule, the cell loses The ability to progress to the next stage of the cycle, which means that the initiation of each process depends on the completion of the preceding process. Thus, in yeasts, as in mammals, most steps of the cell cycle appear to be linked together like links in a single chain. This relationship has been more thoroughly analyzed in experiments with cells carrying various combinations of different cdc mutations. The results have shown that the events of the chromosome cycle form a series of interdependent steps that are not rigidly coupled to the events of the cytoplasmic cycle (Fig. 13-19). For instance, although cytokinesis will not occur if nuclear division is prevented, cdc mutants unable to undergo cytokinesis due to defects in bud formation machinery nevertheless carry out repeated cycles of DNA Synthesis AND nuclear division. It appears to be a general rule not only for yeast but also for mammalian, insect, and many other organism cells that the chromosome cycle can continue even if cytokinesis is prevented. Indeed, multinucleated cells frequently arise in this manner during normal development as well (Section 16.5.2).

Fig. 13-19. Causal relationships among certain cell cycle events and their relation to cdc genes in budding yeast. The spindle pole body (SPB) in yeast is the equivalent of the centrosome. "Start" marks the point of irreversible commitment of the cell to the division cycle and the loss of the ability to undergo sexual conjugation (conjugation can take place only in G₁ phase). A. General scheme of the cycle; an arrow leading from event a (or from events a and b) to event c indicates that event c cannot occur prior to event a (or both a and b together). Numbers indicate specific cdc mutations that lead to cell arrest at that point in the cycle at the restrictive temperature. For example, cells carrying the cdc8 mutation arrest during DNA synthesis. Note that the chromosome cycle (DNA cycle), cytoplasmic cycle (budding cycle), and centrosomal cycle (SPB cycle) are partially independent. B. Blocked states of the four cdc mutants indicated in scheme A at high temperature. [Based on L. H. Hartwell, J. Cell Biol. 77: 627-637, 1978; J. R. Pringle, L. H. Hartwell. In: The Molecular Biology of the Yeast Saccharomyces (J. N. Strathern et al., eds.), pp. 97-142. Cold Spring Harbor Laboratory, 1981.]

13.2.3. Regulation of Cell size depends on cell cycle control factors acting at Start

The growth rate of simple free-living organisms such as yeast depends primarily on nutrient availability. Under starvation conditions, daughter cells would become extremely small during rapid cell division cycles; therefore, cells require a mechanism that regulates The rate of progression through the cell cycle, and particularly the chromosome cycle, in accordance with the Cell Growth Rate (Fig. 13-20). How is this regulation achieved?

Both DNA synthesis and mitosis are complex dynamic processes that are difficult to slow down or interrupt under nutrient-deprivation conditions. In yeast, as in most other eukaryotic organisms, the duration of these cycle phases remains more or less constant despite large variations in external conditions. Instead, starvation typically prolongs the G₁ phase, although fission yeast also possesses an important regulatory mechanism known as mitotic control, which acts in the G₂ phase.

If the duration of the G₁ phase can vary under the Influence of External factors while the S phase remains unchanged, then there must be a critical point within G₁ where The sequence of S phase events is triggered, after which external factors no longer affect the subsequent progression of the cell cycle. This critical point is called Start. For most eukaryotic cells, Start (or the equivalent restriction point in mammalian cells) marks the transition to the unyielding completion of the cell division cycle.

13.2.4. Cells pass through Start only after reaching a critical size [14]

For a budding yeast cell in a nutrient-poor medium, the G₁ phase is a period of slow growth during which the chromosome cycle appears to be suspended; exit from G₁—that is, passing through Start—occurs only after the cell has attained a certain standard size (Fig. 13-20, B). In a richer medium, G₁ is shorter, but the cell size at passage through Start is practically the same; and if growth conditions are adjusted such that daughter cells are abnormally large or abnormally small following division, they will accordingly shorten or prolong their time spent in G₁ so as to pass through Start at a standard size.

Fig. 13-20. The relationship between growth rate, cell size, and the cell division cycle in a free-living organism such as yeast. A. If cells continue to divide at the same rate despite a shortage of nutrients, the daughter cells will become progressively smaller after each division until the mass of each daughter cell equals the small amount of material synthesized during a single cycle. B. Normally, when nutrients are scarce, yeast cells slow down their rate of division: because a cell cannot pass a specific point in the cycle without reaching certain "standard" dimensions, division is delayed and cell size remains more or less constant. (The observed duration of the cycle under nutrient-rich conditions is chosen as the unit of time.)

Little is known about how cells "sense" their own size, although abundant evidence suggests that some mechanism for this exists. For example, if the Cytoplasm of a growing giant amoeba, Amoeba proteus, is repeatedly excised to prevent the cell from reaching normal size, it will fail to divide even for several weeks despite vigorous growth, whereas a control cell divides roughly once a day. A possible clue to how a cell "perceives" its dimensions lies in the fact that eukaryotic cell size is generally proportional to its ploidy: a diploid cell is twice as large as a haploid one, and a tetraploid cell is twice as large as a diploid one (see Figs. 13-40 and 13-41). It can be hypothesized that the crucial factor is The ratio of cell volume to the copy number of a specific gene (or set of genes) or to the total amount of DNA (rather than the ratio of cell volume to surface area, for instance). For example, a hypothetical soluble molecule M (say, a specific RNA) might be synthesized continuously in a DNA-dependent manner; if M is unstable with a constant half-life, the total amount of M in each cell will remain constant and maintain a definite proportion to The amount of DNA. As the cell volume increases, the concentration of M will decrease; a drop in concentration below a certain critical level could serve as a signal to trigger the start point.

Whatever the underlying mechanism, passing through the start point must correspond to a switch-like transition in the state of some molecular switch. Four cdc genes in budding yeast and two in fission yeast operate at or near the start point, and they likely encode components of such a regulatory mechanism. Cells bearing temperature-sensitive mutations in these genes fail to enter the chromosome cycle and grow abnormally large if the temperature is raised to a restrictive level before they have reached the critical size required to pass the start point. Below, we discuss in detail one of the start-control genes (cdc28) in budding yeast and its counterpart (cdc2) in fission yeast. These two genes are notable for an additional function, particularly pronounced in fission yeast: their products are required not only for passing the start point, but also for a second checkpoint in the cycle—the onset of mitosis.

As we will see later, higher eukaryotic cells possess a checkpoint in the G1 phase analogous to the start point, although the rules for passing through it are more complex than in yeast. Violations of these rules lead to Cancer. For this reason alone, the genes involved in the start-point mechanism are of exceptional interest.

13.2.5. Passing through the Start Point Depends on a Protein Kinase Related to M-Phase Promoting Factor (MPF) [15]

Yeast, with its rapid reproduction and simple unicellular Organization, is an attractive subject for Genetic Engineering and can easily be used to incorporate DNA added to the nutrient medium. In principle, this makes it possible to clone the normal ("wild-type") form of any cdc gene. As shown in Fig. 13-21, the clone of interest can be readily isolated owing to its ability to rescue the corresponding cdc mutant from its abnormalities.

This approach was used to clone the budding yeast cdc28 gene and the fission yeast cdc2 gene, revealing several surprising similarities between these Two Types of yeast, as well as between yeast and vertebrates, regarding the control of the start point and mitosis. The cdc2 and cdc28 genes in yeast are homologous in both nucleotide sequence and function: in fission yeast carrying a cdc2 mutation, the functional defect can be rescued by introducing the cdc28 gene from budding yeast. Furthermore, a similar result can be achieved in these same cdc2 mutants by introducing a cloned fragment of human DNA, which evidently also contains a sequence homologous to cdc2/28. Apparently, this component of the cell-cycle control machinery is shared by yeast, mammals, and likely all other eukaryotes.

Having a cloned cdc2/28 gene, it is relatively straightforward to isolate the protein encoded by this gene. It is a protein kinase and, based on the full range of Structural and functional criteria, appears to be the yeast homolog of the kinase subunit of vertebrate MPF. Moreover, Gene cloning has revealed that another fission yeast cdc gene, cdc13—whose product interacts with the product of the cdc2 gene—is highly homologous to the cyclin gene (Section 1.1.11). These findings indicate that MPF and cyclin probably have a universal significance in The Eukaryotic Cell cycle; and the dual role of cdc2/28 in yeast (at the onset of M phase and at the start point) suggests that in vertebrates, the corresponding MPF subunit or a closely related molecule may similarly participate in controlling the initiation of the division cycle during the G₁ phase.

Fig. 13-21. Method for isolating cdc genes from a DNA library. A rare DNA clone containing a normal wild-type copy of a specific cdc gene is easily selected because the plasmid carrying it renders the corresponding mutant cell capable of growing at high temperatures. Both the fission yeast cdc2 gene and the budding yeast cdc28 gene were originally isolated in this manner. By repeating this Procedure with cDNA clones from a human DNA library in a suitable plasmid, researchers succeeded in isolating human genes capable of substituting for certain yeast cdc genes.

Indeed, studies in fission yeast indicate that Changes in the phosphorylation state of this regulatory molecule may be the mechanism by which cells coordinate their readiness for the division cycle with environmental conditions.

Conclusion

Yeast is a unicellular eukaryotic organism exceptionally well suited for genetic analysis. In both budding and fission yeasts, numerous mutations affecting the cell division cycle (cdc) have been identified, and their corresponding wild-type genes cloned. In yeast and many other eukaryotic cells, despite fluctuating nutritional conditions, standard cell size is maintained by a mechanism that prevents cells from passing a critical transition point (termed the start point) and triggers the division cycle once they attain a threshold size. In yeast, several key cdc genes involved in this control have been identified and their nucleotide sequences determined. One of them (designated cdc2 in fission yeast and cdc28 in budding yeast) encodes a protein kinase homologous to MPF; another gene (cdc13) encodes the yeast homolog of cyclin.