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Hereford, L.M. & L.H. Hartwell (1974) Sequential gene function in the initiation of

Saccharomyces cerevisiae DNA synthesis. J. Mol. Biol. 84: 445-461.

Hartwell and coworkers identified 32 Saccharomyces CDC genes required for progress through the cell division cycle. They also grouped these genes based on their execution point in the cell cycle, and to a first approximation each gene product is required at a particular time during the cell cycle. Temperature-sensitive mutations in three genes (CDC4, CDC7, and CDC28) do not initiate DNA synthesis. Cells treated with Saccharomyces «-factor, a peptide pheromone produced by cells of the a mating type, also do not initiate DNA synthesis and pheromone-treated cells arrest as unbudded cells. Thus, all act in the first portion of the cell cycle called G1 or Gap 1. The goal of this article is to determine whether or not the three gene products act independently on different processes, and if so can the process carried out by these gene products be temporally ordered in relation to one another and to the process blocked by the «-factor?

The authors use two methods to determine the temporal order of the events controlled by Cdc4p, Cdc7p, Cdc28p, and the «-factor. The first is called the reciprocal shift method. The second is referred to as the double-mutant method. Both are explained in the text of the article. The double-mutant method is also known as epistasis analysis. It is used to determine if genes with similar mutant phenotypes are in the same or different pathways, and, if in the same pathway, to place them in a linear order relative to one another based on the step in the pathway controlled by that gene.

1. Strains containing temperature-sensitive mutations in CDC4, CDC7, and CDC28 will not initiate DNA synthesis at the elevated, or nonpermissive, temperature. MATa strains treated with a-factor also are unable to initiate DNA synthesis. The morphological effects of these blocks, however, are different.

(a) Describe the morphology assumed by a mating-type cells treated with a-factor for approximately one cell cycle and by the cells of cdc4, cdc7, and cdc28 mutant strains exposed to 36°C for approximately one cell cycle.

(b) Based on these phenotypes and on your knowledge of the morphological events of the Saccharomyces cell cycle, what preliminary hypothesis(es) might you have proposed at the outset of this experiment and why? (You may just be able to group these genes into early and late G1 events.)

2. The following questions are based on Figure la.

(a) Why does the closed-circle line go smoothly up indicating a continuous DNA synthesis at a constant rate and the open-triangle curve look like steps?

(b) Approximately how long (to the quarter hour) does it take for cells of strain HI35.1.1 to complete one full division cycle? How did you determine this?

(c) How long is the S phase? How did you determine this?

(d) In the open-triangle curve, what is happening during the flat portion of the curve from about 3.75 h to about 5.0 h?

3. The following questions are based on Figure lb.

(a) Why does the open-square curve go through more than one step while the filled-square curve levels off after only one step?

(b) Cycloheximide is a powerful inhibitor of protein synthesis. Are all the proteins required for completion of the cell cycle made prior to the a-factor or Cdc4p block in G1 and on what evidence do you base this conclusion?

(c) Is it surprising that the filled-square cells arrest as unbudded cells? Explain.

4. In the cdc7 mutant strain, cell separation (cytokinesis) was delayed. How was this manifested?

5. Mutant strains carrying a recessive temperature-sensitive alteration in a (fictitious) cell division cycles gene, cdcl08, block initiation of DNA synthesis at a step that preceeds the a-factor block. Diagram the results you would expect if you were to run an experiment like the one shown in Figure 1.

6. Why do the authors put the cdc4 block before the cdc7 block? (Give two reasons.) Is CDC4 epistatic to CDC7 or vice versa?

7. It is interesting to note that in cdc4 and cdc7 mutants bud formation can be initiated in the absence of DNA synthesis while in cdc28 mutants and a-factor-treated a mating type cells, which also do not initiate DNA synthesis, cells arrest as unbudded cells.

(a) What does this suggest about the regulation of bud formation initiation and its relationship to the initiation of DNA synthesis?

(b) Is the initiation of DNA synthesis epistatic to the initiation of bud formation? If not, which model of Table 2 best fits their relationship, and why?

8. The spindle plaque (now referred to as the spindle pole body) is an elaborate multilayered platelike structure embeded in the nuclear envelope of Saccharo-myces (see Chapter 3). The Saccharomyces nuclear envelope never falls apart during cell division and the spindle pole body serves the role of the centriole.

(a) Describe the terminal morphological phenotype of the spindle pole body of a-factor-treated cells and cdc4, cdc7, and cdc28 mutant cells exposed to high temperature (work done by Beyers and coworkers).

(b) Are their results consistent with those reported here, and, if not, how do their conclusions differ?

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