The growth and division of cells (mitosis) and the formation of sperm and eggs (meiosis) are important for almost all organism

Using Saccharomyces cerevisiae to study cell cycle genes in meiosis

Anne Galbraith, Dept of Biology, University of Wisconsin-La Crosse.

The growth and division of cells (mitosis) and the formation of sperm and eggs (meiosis) are important for almost all organisms, including humans. We all began as a single cell that resulted from our father’s sperm fertilizing our mother’s egg. This single cell then divided by mitosis into two cells (daughter cells) which then grew and divided into four cells (Figure 1A). This process of mitosis continued until we were born. It then continued again until we were “grown up”. It is still happening right now as some of our cells use mitosis to replace old dead cells, such as skin cells. Meiosis, on the other hand, is a special form of mitosis that occurs only in a special subset of our cells to form eggs and sperm. In meiosis, one cell divides twice in a row to form four daughter cells from one cell (Figure 1B). Those cells are then modified to become eggs or sperm. Mitosis and meiosis, then, are similar processes, but result in very different types of cells.

Figure 1. A) In mitosis, a single cell (circle on the left) divides to form two daughter cells. These cells grow, and then divide to form a total of four cells. Those four cells grow and divide to form eight cells, etc. B) In meiosis, a single cell divides twice, resulting in four daughter cells that do not grow and divide again. Instead, these cells are modified to become eggs or sperm in humans.

One of the most important requirements of a successful mitosis or meiosis is that the blueprint of life, the DNA, is provided in equal amounts to each of the daughter cells. The DNA in all human cells except eggs and sperm is housed in 46 chromosomes. Eggs and sperm consist of only half that number, 23 chromosomes per cell. Every person inherits one set of chromosomes from their mother (in the egg) and the other set of chromosomes from their father (in the sperm). It is imperative that when mitosis and meiosis are complete, the appropriate number of chromosomes exists in each cell. If there are extra or missing chromosomes, the cells usually do not live. To ensure that there are enough chromosomes to be divided evenly between the two daughter cells during mitosis, an essential step precedes cell division that allows the chromosomes to be copied, a step called DNA replication. In mitosis, (1) the DNA is replicated, (2) the chromosomes are separated into two equal sets, and (3) the cell divides. These steps must occur in this exact order or the resulting cells will have an incorrect number of chromosomes and may not live.

A similar set of steps occurs in meiosis: (1) the DNA is replicated, (2) the chromosomes are separated twice into four equal sets, and (3) the cell divides into four cells. Each of these daughter cells has to contain 23 chromosomes, half of the original 46. Again these steps must occur in this exact order or the resulting cells will have an incorrect number of chromosomes.

It has probably become obvious at this point that mitosis and meiosis are not simple processes. In fact, there are literally hundreds of genes that are needed to control the many steps that are involved and to be sure that those steps occur in the correct order. Any mistakes can be deadly. For example, it is necessary to the cell’s survival that the DNA gets replicated and the chromosomes are separated equally so that all daughter cells get the correct number of chromosomes. It is also necessary to control the frequency at which cells divide; if cells divide more often than they should, then cancerous tumors are often the result. The death rate due to cancer in the United States dropped between 1991 and 1995 for the first time since the 1930s, translating into the saving of 10,000-15,000 lives per year (National Cancer Institute, 1999). Despite this decline in the cancer death rate, however, the prognosis is grim. Over 550,000 Americans are expected to die of cancer this year and just under half of all men and just over one-third of all women will contract cancer sometime during their lives (American Cancer Society, 2002). Cancer is currently the second leading cause of death, and, by 2004, cancer will be the number one cause of death in the United States (American Cancer Society, 2002).

Although cancer is a very complex disease, it has recently become clear that many of our very own genes that are supposed to be working in our cells to control cell division (mitosis and meiosis) are responsible for the cancer epidemic. These normal genes are altered or mutated, possibly due to our exposure to such things as tobacco smoke (which contains at least 50 known cancer-causing agents), UV light from the sun (or from the tanning booth), certain preservatives in our foods, and a host of other environmental assaults (American Cancer Society, 2002). When these normal genes are mutated, they are no longer able to do their jobs effectively, a problem that can lead to cancer.

It is very difficult to study such a basic problem as cell division in humans, although some researchers do. It has become increasingly common, however, to use model organisms, organisms that are similar to humans in terms of how their genes work. One organism that is used worldwide by thousands of researchers in the study of cell division is Saccharomyces cerevisiae. This organism is a single-celled yeast, a fungus, and is the same one that is used to brew beer and make wine and bread. Yeast grows quickly, producing a new generation of cells in about two hours. (Human cells in culture take about 24 hours to produce a new generation). It is easy to manipulate, cheap to maintain, and is nonpathogenic. (Human cells die easily, require expensive equipment and media, and special safety precautions must sometimes be taken to work with them). Yeast have been studied by researchers for nearly 100 years, resulting in innumerable experimental techniques that have been designed for use with this organism. (There are many experiments that simply cannot be done with human cells because the techniques have not been developed or they just don't work). Finally, although yeast does not look anything like a human, it still grows and divides by the processes of mitosis and meiosis and uses most of the same genes to control those processes.

Two of the many genes that yeast and higher organisms have in common are CDC7 and DBF4. The CDC7 gene was identified in yeast about 30 years ago (Hartwell, 1976) and was shown to be needed for the important DNA replication step in mitosis. Since then, many research labs have discovered that this CDC7 gene actually switches DNA replication "on" in mitosis in yeast. Yeast cells that have a mutation in the CDC7 gene cannot even start the DNA replication step and therefore become stuck or “arrested” in mitosis as single cells that eventually die (Jackson, et al., 1993). Many research labs have become interested in studying CDC7 and its role in mitosis, especially since a human version of this gene was recently isolated and found to be altered in tumor cells (Hess, et al., 1998), associating this gene with cancer formation.

The DBF4 gene was implicated as a partner for CDC7 in the process of DNA replication when Kitada and colleagues obtained genetic evidence that the two gene’s protein products interact (Kitada, et al., 1992). More recent biochemical experiments have supported this hypothesis by showing directly that the Cdc7 and Dbf4 proteins really do interact, and they do so only at the initiation of DNA replication during mitotic growth (Oshiro, et al., 1999). It is clear from these experiments that the Cdc7 and Dbf4 proteins work together to initiate DNA replication during mitosis.

Although a fair amount is known about the roles of CDC7 and DBF4 in mitosis, research on meiosis has been far less intense. Nearly 25 years ago, a paper was published that presented the results of experiments designed to determine the role of CDC7 in meiosis in yeast (Schild and Byers, 1978). These scientists concluded that although CDC7 was needed for the chromosomes to replicate during mitosis, it was not needed for the chromosomes to replicate during meiosis. How strange! Why would DNA replication be controlled differently in mitosis and meiosis when the two types of cell division are so similar otherwise? Although scientists who studied DNA replication were confused by these results, the work done by Schild and Byers seemed clear enough and few scientists tried to explain the paradox. Because of this, papers about CDC7’s role in meiosis have been scarce since 1978. Unbelievably, even less work has been done on the role of the DBF4 gene in meiosis. Kitada, et al. (1992) showed that dbf4 mutants are unable to complete meiosis and form spores, but that is the extent of published work on the meiotic phenotype of dbf4.

My students and I have been working for the past few years to begin to elucidate the roles of these two genes in meiosis. We have made important advances and our work has been received with increasing interest at national scientific meetings (e.g., Wheeler, et al., 2002). It is clear at these meetings that more and more researchers are interested in the regulation of meiosis and the genes that are involved in this regulation.

Figure 2. Colonies of yeast growing on specific media types in the lab. Each single colony (for example, at the tip of the arrow) contains millions of individual cells.

Figure 3. Yeast cells treated with DAPI, a dye that stains DNA and causes nuclei to fluoresce when observed using a certain wavelength of light. These are wild-type sporulating yeast cells sampled from different times during meiosis. The bright spots in each cell are the DNA-containing nuclei. (A) All cells have only one bright spot of DNA, indicating that the first division of the chromosomes has not yet occurred in these cells. (B) One hour later, there are three cells out of those in the photo that have two bright spots of DNA, indicating that these cells have gone through the first of the two chromosome divisions. (C) Six hours later, there is a mix of cells whose chromosomes have not divided (one spot of DNA), divided once (two spots of DNA), or divided both times (four spots of DNA). As expected for a wild-type strain, most have divided twice and therefore have four spots of DNA. Photos taken from Galbraith (1995).

References

· American Cancer Society (2002) [http://www.cancer.org/eprise/main/docroot/stt/stt_0]

· Galbraith, A. M. (1995). A genetic and molecular characterization of the REC104 gene and its involvement in meiotic recombination in the yeast Saccharomyces cerevisiae, UMI Dissertation Services.

· Hartwell, L. (1976). Sequential function of gene products relative to DNA synthesis in the yeast cell cycle. Journal of Molecular Biology 104:803.

· Hess, G.F., R. F. Drong, K. L. Weiland, J. L. Sligthom, R. A. Sclafani, and R. E. Hollingsworth, Jr. (1998). A human homolog of the yeast CDC7 gene is overexpressed in various tumors and transformed cell lines. Gene 211:133.

· Jackson, A.. L., P. M. B. Pahl, K. Harrison, J. Rosamond, and R. A. Sclafani (1993). Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein. Molecular and Cellular Biology 13:2899.

· Kitada, K., L. H. Johnston, T. Sugino, and A. Sugino (1992). Temperature-sensitive cdc7 mutations of Sacchmaromyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition. Genetics 131:21.

· National Cancer Institute (1999) [http://wwwosp.nci.nih.gov/bypass99/director/index.html]

· Oshiro, G., J. C. Owens, Y. Shellman, R. A. Sclafani, and J. Li (1999). Cell cycle control of Cdc7p kinase activity through regulation of Dbf4p stability. Molecular and Cellular Biology 19:4888.

· Schild, D. and B. Byers (1978). Meiotic effects of DNA-defective cell division cycle mutations of Saccharomyces cerevisiae. Chromosoma 70:109.

· Wheeler, J., S. Hanson, N. Krause, C. Zabel, R. Rohrer, and A. Galbraith (2002). Determining the roles of the CDC7 and DBF4 genes in meiosis in Saccharomyces cerevisiae, poster presentation, Yeast Genetics and Molecular Biology Meeting, Madison, WI, August 2002.