Section Notes The cell division cycle presents an interesting system to study because growth and division must be carefully coordinated. For many cells it is important that it reaches the correct size before divinding, otherwise cells can become too large or too small after repeated divisions. Also, it is important to ensure that chromosomes are fully replicated before segregation to prevent chromosome breakage or mis-segregation. And lastly, cells must maintain a certain growth and division rate or they can proliferate out of control as in cancer. These are just a few of the types of problems the cell cycle must solve. The basic cell cycle can be divided into distinct steps where one step must finish before the next begins. These transitions are coordianted and controlled. The figure below outlines the cell cycle steps as they are often divided. G1 is where growth after division occurs and the transition from G1 to S is one place where growth and divison can be regulated. S phase is where DNA replication occurs and then there is a G2 phase of more growth before mitosis is triggered. When trying to understand a process such as the cell cycle, it is important to decide what types of questions you are interested in answering. In lecture this was described as the level of abstraction. You could take a “big picture” approach where you try to understand the basic rules that govern a process and general requirements that must be met before steps can occur. Or you can zoom in to the molecular level and try to understand specifically which molecules trigger certain changes and how they are regulated. Each level of abstraction can help contribute to understanding a problem and it is not necessary to understand the “zoomed in” level before tackling the big picture. For the cell cycle, an early set of cell fusion experiments revealed some basic rules for the cell cycle. The experiment involved fusing cells at different stages of the cell cycle together and seeing what happens to the chromosomes. Three different fusions led to three basic ideas: 1) If one fuses a cell in mitosis with a cell in any other stage of the cell cycle, the chromosomes from the “non-mitosis” cell replicate and enter mitosis. This indicates that mitosis dominates. 2) If ones fuses a G1 cell with a cell in S phase, the G1 nucleus also enters S phase. This indicates that there is some sort of signal present that can trigger DNA replication. 3) However, fusing an S phase cell with a G2 cell does not induce the G2 nucleus to synthesize its DNA again. This suggests that the cell has a mechanism for determining that the DNA has already been synthesized and there are ways to keep the cell from re-copying it’s DNA. When addressing any scientific question, it is important to determine what type of system you want to study. Oftentimes it is helpful to study specialized cells because there aren’t as many extraneous things to obstruct what you want to study. For example, eggs are specialized to divide rapidly and the cell cycle is simplified to S and M, or replication and division. Xenopus eggs are a particularly good system because they are large and fertilized externally, thus easy to access. Fully grown oocytes are arrested until they receive a signal (progesterone) that triggers the divisions associated with meiosis I and II. After meiosis II, the cells arrest again until fertilization after which the cells begin to divide without any growth in between the divisions, resulting in small cells after each round. The cell fusion experiments suggested that there is a mitosis promoting factor (MPF) that can trigger mitosis in cells. There are pulses in the levels of MPF that correlate with each entry into mitosis and the reduction in MPF levels seems to regulate the exit from mitosis. Experiments also showed that MPF pulses do not actually depend on the DNA at all as the eggs still show pulses of MPF even after the removal of the nucleus. This suggests that the pulses are not dependent on new DNA transcription since they occur for many cycles even in the absence of the DNA. Even though the DNA is not needed for these pulses, it is known that protein synthesis is required for embryonic mitoses. This is possible in the absence if DNA because of maternal mRNAs that are deposited in the egg. An experiment involving sea urchin eggs eventually revealed the peptide responsible for the oscillations. Because maternal proteins are also deposited in an egg, it can be difficult to distinguish proteins that are newly synthesized from those that are maternal. In order to tell them apart, one must label (usually with radioactive methionine) new proteins. If you add 35S-methionine to the eggs after they have been isolated and fertilized, any newly synthesized proteins will contain 35S, which can be detected later. Then at different times points (to see what proteins are present at different points in the cell cycle), you remove samples and disrupt the egg membranes with SDS. This releases the proteins which you can then run on an SDS-PAGE gel which separates them purely based on molecular weight. The gel will show you all of the proteins present in the egg, both newly synthesized and maternal, so you then expose it to film which can detect only the radioactive proteins. This experiment revealed a protein that was synthesized and then degraded as the cells progressed through the cycle. Called cyclin, the levels of this protein peak at mitosis and subsequent degradation triggers the transition to interphase. At this point, the hypothesis was that cyclin synthesis drives cells into mitosis and its destruction pushes them out. An experiment with frog egg cytoplasm shows that cyclin is indeed the protein responsible for this transition and that it is the only factor required. This entire cycle of mitosis and interphase can be replicated in a test tube with cytoplasm removed from frog eggs. After removal, you treat the cytoplasm with RNase which destroys any existing mRNAs present. Since there is also no DNA present, there is no way for new protein synthesis. You then block the RNase and add in fresh cyclin mRNA. Since it is the mRNA present, you know that any protein synthesized will be cyclin. You can then watch and see if the cytoplasm generates cycles of interphase and mitosis, which it does. This shows that cyclin is sufficient to trigger this cycle. Once there was a basic understanding of the cell cycle at a high level of abstraction, scientists began to zoom in and look at the molecular level. They wanted to understand the genes responsible for cell cycle control and how they relate to each other. To do this, one uses genetics through the isolation of mutants that exhibit a phenotype of interest. Again, it is important to choose the organism you which to study carefully. In this case, scientists chose yeast because they have well established genetic tools, can replicate sexually or asexually, and also replicate quickly. It can be difficult to study essential genes because, by definition, the cells are dead/non-functional if you remove their function through mutation. One way to tackle this problem is to use temperature sensitive mutants, which means that the cells containing the mutation in the essential gene can grow at one temperature (permissive temp), but will express the phenotype if you switch them to a different temperature. Often the permissive temperature is cooler (such as 20°C) and the restrictive temperature is 37°C though this does not necessarily have to be the case. You find these conditional mutants through a process known as replica plating. You mutagenize the cells (induce mutations with UV radiation or a chemical) and then plate them at 20°C where all of the cells should grow even if they have a temperature sensitive mutation. You then transfer some cells from each colony using velvet to a new plate, maintaining spatial orientation. This just allows you to know which colony corresponds to which on the two plates. You then grow the new plate at the restrictive temperature of 37°C. Most of the colonies from the 20°C plate will also grow at 37°C because they are not temperature sensitive. Any colonies that contain temperature sensitive mutations though will not be present on the 37°C plate. Since these are the mutants you are interested in, you save them from the 20°C plate and discard the rest. Most of the temperature sensitive mutants isolated will have mutations in genes unrelated to the cell cycle, so how do you find the ones you are interested in? You can take populations of cells, both wild type and mutant and when you grow them at the permissive temperature and look at them under the microscope, they will be at all different stages of the cell cycle. When you move the wild type cells to the higher temperature, they are unaffected and will still contain cells at all different stages of the cell cycle. The same thing will occur for any mutants that have mutations in genes unrelated to the cell cycle. However, cell cycle mutants will arrest at a certain stage of the cell cycle when shifted to the restrictive temperature. Each mutant may arrest at a different stage depending on what the mutated gene regulates, but you can identify interesting genes by finding the mutants that have cell cycle arrest at the higher temperature. In this case, there were 148 cdc mutants identified and they affected a total of 32 different genes.