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GREAT EXPERIMENTS
Cell cycle genes

Lee Hartwell

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Introduction

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The cell cycle is the process by which a cell duplicates its contents and divides them into two daughter cells. It has been apparent for more than a hundred years that there is an order to the events of the cell cycle. Chromosome duplication must precede chromosome segregation, which, in turn, must precede cytokinesis. The cell cycle was initially divided into two phases, based on what could be seen through a microscope: interphase, when chromosomes could not be seen, and mitosis, when they visibly condensed and segregated. It was not until radioactive thymidine was used to study DNA replication in the early 1950s that the current four-phase cycle of G1, S, G2, and M was defined. DNA replication occurred in S phase and chromosome segregation in M. G1 and G2 were "gaps" between these events. By the middle of the 1960s, it became clear that the decision to initiate a cell cycle was controlled in G1, suggesting that the control of cell division was distinct from the events of cell division. What was missing were ways of finding out how the events within the cell cycle were coordinated, and of identifying the molecular components that ordered these events. I decided to take a genetic approach, with the ultimate goal of identifying genes that functioned at discrete steps of the cell cycle.

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Background

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I became interested in cell division when I was a postdoctoral fellow with Renato Dulbecco. I was fascinated by the differences between normal and cancerous cells that were evident even in cells removed from the body and grown in culture. My decision to use genetics to study the problem in yeast grew out of frustration. I had spent over a year in Dulbecco's laboratory trying to develop a project on cell division utilizing mammalian cells, but each of the approaches that I tried failed.

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My desire to apply genetics to the problem was strongly influenced by my experience as an undergraduate at Cal Tech. There I had the wonderful experience of working with Bob Edgar when he began using temperature-sensitive (ts) and nonsense mutants to define all of the genes of the virus T4. Over a few years, he and Bill Wood used these mutants to work out the complicated process of building the T4 virus structure. There was no way this could have been done without genetics. Mutants revealed the incomplete intermediates of morphogenesis, and cell extracts from mutant-infected bacteria provided the starting materials for in vitro morphogenesis experiments. I wanted to be able to do something equally definitive with cell division. I thought that genetics would be ideally suited to studying cell division because, in several fundamental respects, cell division was like phage reproduction. In both cases, DNA replication was followed by a series of macromolecular assemblies that built chromosomes. In phage, this was followed by phage particle assembly, and in cells, by formation of the mitotic spindle and the cytokinetic apparatus. It seemed reasonable to expect that mutants could be used to reveal the pathway of morphogenetic events leading to cell division, much as they had been for phage reproduction.

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Figure 1  
A scanning electron micrograph of a number of Saccharomyces cerevisiae cells. Many of the cells have buds, which differ in size from cell to cell. Photo courtesy of Ira Herskowitz.

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Figure 2  
The bud grows continuously as the sequence of events that replicates and separates the chromosomes takes place. Major cell cycle events that can be assayed are indicated.

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Genetic studies with mammalian cells were impossible, so Dan Wulff, a colleague of mine, suggested finding a model eukaryotic cell amenable to genetic analysis. A casual perusal of the journal Genetics revealed many elegant studies with the yeast Saccharomyces cerevisiae, which is shown in Figure 1. At that time, yeast geneticists were preoccupied with genetic recombination, and most of the published work was about that phenomenon. However, Howard Douglas and Dan Hawthorne had also done some nice work on the genetics of gene regulation with the galactose pathway. Overall, S. cerevisiae looked ideal for my purposes. It grew mitotically as either a haploid or a diploid, which meant that one could isolate recessive mutants in the haploid phase and use the diploid phase to place them in complementation groups. It also grew as single cells, unlike most other fungi. A third advantage I did not appreciate until much later: S. cerevisiae is a budding yeast, and each new cell grows from a bud on the parent cell. Because the bud grows continuously over the course of the cell cycle, the size of the bud reflects the position of the cell within the cycle. This is shown in Figure 2. Bud size proved to be a key morphological marker that made the recognition and analysis of cell cycle mutants very easy. The cell cycle in yeast was also known to be similar to those in larger eukaryotes. C. F. Robinow had shown that yeast has a mitotic spindle and well-defined spindle poles (2989), and Don Williamson had demonstrated that S. cerevisiae had a cell cycle with the classic G1, S, G2, and M phases (2990). Before I could begin working with yeast, however, I needed some instruction, so I made a short visit to two of the leading yeast genetics labs, those of Bob Mortimer and Herschel Roman, after which I enthusiastically decided to work with yeast.

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Based on the phage T4 paradigm, I isolated over 400 temperature-sensitive mutants and studied the patterns of macromolecular synthesis and cell division following a shift from permissive to restrictive temperature. A subset had patterns suggesting specific defects in protein synthesis, RNA synthesis, DNA synthesis, or cell division (2992). The ultimate goal, of course, was to identify proteins that function in cell physiology, but the current approach to this problem, gene cloning, had not even been conceived of. Showing that one could go from mutant to protein was essential to validating the project. Fortunately, by this time Calvin McLaughlin, an expert in protein synthesis, had arrived at UC Irvine, where I was a faculty member at the time. Together we studied the protein synthesis mutants and were able to identify two genes as the structural genes for aminoacyl-tRNA synthetases (2993).

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The experiments

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Our survey of macromolecular synthesis and cell division in the temperature-sensitive mutants had identified some with the properties we expected for cell cycle mutants. That is, after a shift from permissive to restrictive temperature, cell division stopped while DNA, RNA, and protein synthesis continued. However, this criterion was not very precise and did not discriminate between blocks at different phases of the cell cycle. The key breakthrough that opened up the cell cycle to genetic analysis was the realization that cell cycle progression could be studied very conveniently by time-lapse photomicroscopy. This insight came quite serendipitously. Brian Reid, an undergraduate in the laboratory, was studying some mutants that formed elongated shapes and it made sense to analyze them by photomicroscopy. As soon as we started looking at the photographs, we realized how informative they were for studying cell division. This was because S. cerevisiae grows by budding; since the bud grows continuously throughout the cell cycle, simply looking at the size of the bud revealed where a cell was in the cycle.

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Figure 3  
Cell cycle mutants can be identified visually by following a population of cells over time after the temperature is raised. A population of normal cells or cells that are mutant in non?cell cycle functions will always contain cells at all stages of the cycle. The cells in a population of a cell cycle mutant, however, will accumulate at one stage of the cycle. Such a population is easily identified because all the cells will have the same shape.

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We screened through hundreds of our ts mutants and discovered that about ten percent of them appeared to have a specific block in the cell cycle. That is, when a population of cells was shifted from the permissive temperature at which they could reproduce to the restrictive temperature where they could not, all of the cells stopped at one stage of the cycle for example, with a small bud. The process is shown in Figure 3. We were insecure about whether the external morphology of the cells alone was telling us the whole story, so Joe Culotti, a graduate student, learned how to stain nuclei, and analyzed the nuclear morphologies as well. His findings were consistent with cell cycle defects in that the nuclei were also uniformly arrested in each mutant population.

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Figure 4  
Normal cells and three cdc mutants after several hours at the restrictive temperature.

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Our initial screen identified thirty-five cell cycle genes (2038). The phenotypes of mutants in several of them are shown in Figure 4. To study them, we developed methods for rapidly synchronizing cultures of mutant cells so that we could follow events after a shift to the restrictive temperature. In order to determine the specific defect in each mutant, we needed to be able to monitor as many of the events of the cell cycle as possible. We developed assays for budding, DNA replication, nuclear division, cytokinesis, and growth in the overall size of the cell. Breck Byers became interested in the mutants and added several parameters that at the time could only be monitored by electron microscopy: spindle pole duplication, separation, and segregation, as well as spindle elongation (2994). Within the initial set of 35 genes, we found blocks specific to every one of these events.

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Figure 5  
Analysis of many cdc mutants produced this branched pathway describing the interdependencies among events in the cell cycle of yeast. Any event downstream of another is dependent on it; events in the different branches are independent of one another. Cytokinesis depends on all the other events, but reinitiation of the cell cycle is independent of cytokinesis. The numbers indicate the point at which the corresponding cdc mutation blocks a branch.

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The phenotypes of the mutants revealed a pathway of cell cycle events in which downstream events were dependent on completion of some but not all upstream events. For example, cells that did not bud still replicated their DNA and completed nuclear division, showing that the nuclear cycle was independent of the cycle of budding, cytokinesis, and cell division. However, mutants that failed to replicate DNA did not complete nuclear division or cell division, demonstrating a dependence of these events on one another. We used two additional methods to confirm and extend this pathway. First, we made double mutants between all mutants with different phenotypes. For example, two mutants failed to initiate DNA replication but they differed in budding — one (cdc7) arrested with a large bud and the other (cdc4) continued budding for multiple cell cycles. The double mutant (cdc7, cdc4) budded for multiple cell cycles, suggesting that the cdc4 step preceded the cdc7 step. We also used a series of shifts between temperature and inhibitors to order steps. As shown in Figure 5, the result was a picture of the cell cycle as a relatively simple pathway of dependent events leading to cell division. The pathway was composed of many sequential, gene-controlled steps, with genes assigned to every event that we had been able to assay. These included spindle pole body enlargement, duplication, and separation; initiation of DNA replication; DNA elongation; three stages of nuclear division; cytokinesis; and cell separation (824).

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One gene in the cell cycle drew special attention, CDC28. It was the first gene in the pathway, and neither budding nor DNA replication could occur if it did not function. Second, it functioned at the stage at which growth was integrated with division. That is, if growth was abruptly prevented by withdrawal of nitrogen (or other nutrients), cells that had started the cycle by producing a bud continued until they completed the cycle, even though there was no growth (2995). Small cells produced during nutrient starvation were arrested before the step controlled by CDC28, and when nutrients were replaced, the tiny cells grew in size and did not complete the CDC28 step until they reached normal size. Moreover, cell division was controlled during mating at the very same step (2996; 2997; 2998; 2999). Haploid yeast cells come in two different types (called mating types) and mate by fusing with a cell of the opposite type. During this process, the cell cycles of the two cells must be synchronized. To achieve this, each cell type secretes a pheromone that arrests the cell cycle of cells of the opposite type. We mimicked the process experimentally by adding purified pheromone to cells of the responsive mating type, and found that this arrest also occurs at the CDC28-controlled step. Stimulated by the central role of the CDC28 gene, Steve Reed, a postdoc in my lab, and Kim Nasmyth, a postdoc in Ben Hall's lab, collaborated to clone the gene, the first cell cycle gene cloned by functional complementation (3001). When Steve later sequenced it in his own laboratory, he inferred that it encoded a protein kinase, which ultimately proved to be the cyclin-dependent kinase (3003).

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The legacy

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Understanding the role of the cyclin-dependent kinase as the major controlling element of the cell cycle came from a convergence of several lines of research in different laboratories. Most notable was the elegant and seminal work of Paul Nurse working on the cdc2 gene of Schizosaccharomyces pombe (see The discovery of as the key regulator of the cell cycle). A long history of studies on the maturation-promoting factor (MPF) from the frog Xenopus laevis, and Tim Hunt's discovery of the periodic synthesis and decay of cyclin also provided key insights. The CDC28 gene from S. cerevisiae was shown to be the budding yeast homologue of the S. pombe cdc2 gene and to encode one of the two proteins that make up MPF. The historical development of these insights has been reviewed in detail (3004).

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For me, what is most instructive about the cell cycle field for understanding how science progresses comes from analyzing the assumptions and motivations driving each of these three areas of work. As I noted above, the genetic analysis of the S. cerevisiae cycle was driven by the paradigm of phage morphogenesis and yielded a view of the cell cycle entirely consistent with that paradigm. CDC28 only achieved prominence because it was the first gene in the pathway and functioned at the stage where division was controlled by cell growth and mating pheromone. CDC2 of S. pombe was identified as important for an entirely different reason. Paul Nurse assumed that rate-limiting steps were the most important and focused on CDC2 because some alleles created cells that divided slower than normal, while others created cells that divided faster than normal (3005). Marc Kirschner focused on MPF because it was a clock that oscillated independently of the cell cycle and appeared to provide the timing mechanism (1570). Even though all three views are partly right and partly incomplete, science as a process led all three quests to the same answer, one that was more encompassing than any of us had expected.

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The author

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Lee Hartwell is president and director of the Fred Hutchinson Cancer Research Center in Seattle, as well as professor of genetics at the University of Washington. He has been on the faculty there since 1968. He has worked with yeast for more than 35 years and sees it as a model organism for human biology. His current research focuses on using yeast to understand how individual organisms of the same species can tolerate the large amount of genetic variation found in populations, and how genetic variation affects the susceptibility of humans to disease.

Hartwell was born in Los Angeles and was the first member of his family to attend college, graduating in 1961 from the California Institute of Technology. He earned a Ph.D. in 1964 with Boris Magasanik at MIT, and engaged in postdoctoral work at the Salk Institute with Renato Dulbecco. He was a member of the faculty of the University of California at Irvine before moving to the University of Washington.

Hartwell has received many awards, including the 2001 Nobel Prize in Physiology or Medicine.


Lee Hartwell
President & Director
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N., D1-060
Seattle, WA 98109-1024
E-mail: lhartwel@fhcrc.org

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Last Revised on October 22, 2004

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reviews
  • 3004 Nasmyth, K. (2001).  A prize for proliferation.  Cell 107, 689-701.  PubMed   Journal
reviews
  • 824 Hartwell, L., Culotti, J., Pringle, J. R., and Reid, B. J. (1974).  Genetic control of the cell division cycle in yeast.  Science 183, 46-51.  PubMed  
  • 1570 Gerhart, J., Wu, M., and Kirschner, M. (1984).  Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.  J. Cell Biol. 98, 1247-1255.  PubMed  
  • 2038 Hartwell, L. H., Culotti, J., and Reid, B. (1970).  Genetic control of the cell-division cycle in yeast. I. Detection of mutants.  Proc. Natl. Acad. Sci. USA 66, 352-359.  PubMed  
  • 2989 Robinow, C. F. and Marak, J. (1966).  A fiber apparatus in the nucleus of the yeast cell.  J. Cell Biol. 29, 129-151.  PubMed  
  • 2990 Williamson, D. H. (1965).  The timing of deoxyribonucleic acid synthesis in the cell cycle of S. cerevisiae.  J. Cell Biol. 25, 517-528.  PubMed  
  • 2992 Hartwell, L. H. (1967).  Macromolecule synthesis in temperature-sensitive mutants of yeast.  J. Bacteriol. 93, 1662-1670.  PubMed  
  • 2993 Hartwell, L. H. and McLaughlin, C. S. (1968).  Mutants of yeast with temperature-sensitive isoleucyl-tRNA synthetases.  Proc. Natl. Acad. Sci. USA 59, 422-428.  PubMed  
  • 2994 Byers, B. and Goetsch, L. (1975).  Behavior of spindles and spindle plaques in the cell cycle and conjugation of S. cerevisiae.  J. Bacteriol. 124, 511-523.  PubMed  
  • 2995 Johnston, G. C., Pringle, J. R., and Hartwell, L. H. (1977).  Coordination of growth with cell division in the yeast S. cerevisiae.  Exp. Cell Res. 105, 79-98.  PubMed  
  • 2996 Hereford, L. M. and Hartwell, L. H. (1974).  Sequential gene function in the initiation of S. cerevisiae DNA synthesis.  J. Mol. Biol. 84, 445-461.  PubMed  
  • 2997 Backing-Throm, E., Duntze, W., Hartwell, L. H., Manney, T.R. (1973).  Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor.  Exp. Cell Res. 76, 99-110.  PubMed  
  • 2998 Hartwell, L. H. (1973).  Synchronization of haploid yeast cell cycles, a prelude to conjugation.  Exp. Cell Res. 76, 111-117.  PubMed  
  • 2999 Wilkinson, L. E. and Pringle, J. R. (1974).  Transient G1 arrest of S. cerevisiae cells of mating type alpha by a factor produced by cells of mating type a.  Exp. Cell Res. 89, 175-187.  PubMed  
  • 3001 Nasmyth, K. A. and Reed, S. I. (1980).  Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene.  Proc. Natl. Acad. Sci. USA 77, 2119-2123.  PubMed  
  • 3003 Reed, S. I., Hadwiger, J. A., and Lörincz, A. T. (1985).  Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28.  Proc. Natl. Acad. Sci. USA 82, 4055-4059.  PubMed  
  • 3005 Nurse, P. and Thuriaux, P. (1980).  Regulatory genes controlling mitosis in the fission yeast S. pombe.  Genetics 96, 627-637.  PubMed  

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