What Happens When the Cohesins Do Not Work - Coursework Example

WHAT HAPPENS WHEN THE COHESINS DO NOT WORK AND HOW THAT LEADS TO CANCER Definition and Evolution of Cohesins Cohesin is a multiunit protein complex associated with chromosomes, it is usually found in eukaryotes but closely related structures are found in bacteria. The four main subunits of cohesion include “Psm1, Psm3, Rad21 and Psc3 in fission yeast, as well as a less firmly associated fifth subunit, Pds5 (orthologs of budding yeast Smc1, Smc3, Scc1, Scc3 and Pds5, respectively)” (Schmidt, Brookes and Uhlmann, 2009). Cohesin plays a vital role in cell division during segregation of chromosomes; it ensures replicated sister chromatids are aligned and stick close together. Cohesin also plays an important role in maintaining the integrity of DNA. It also performs gene regulating functions in rapidly reproducing cells- mitosis and meiosis processes-and non-reproducing cells. Cohesin discovery dates back to as early as 1971, when some scientists discovered some protein subunits-later linked to cohesion- while studying reproducing cells in Drosophilla melanogaster. Other researchers studying Saccharomyces cerevisiae in 1990s also discovered some cohesin proteins similar to those discovered in 1970s and later in 1990s by other scientists studying defective genes for DNA repair and chromosome segregation in Schizosaccharomyces pombe and Aspergillus nidulans. Some o the proteins discovered by the researchers as describes above include Smc1, Smc3, Scc1/Mcd1/Rad21, Smc1, Smc3 and Scc3/Irr1 which were later discovered to be subunits of cohesion (Schmitz, Tedeschi and Peters1, 2008) A study by Michaelis et al. (1997) showed that cohesin had an important role in segregation of chromosomes since it was observed to be present in particular phases of mitosis that involve chromosome separation and segregation- G1, interphase and metaphase- but not in anaphase where segregated chromosomes move towards opposite poles. The discovery by Michaelis and group has since been the basis of argument on the role of cohesins in segregation and many scientists and researchers alike have taken upon the discovery and have been conducting various experiments to ascertain the roles and functions of cohesins, its location, and structure among other nitty-gritty. This paper will discuss cohesins in details- their role in cell division, its structure, location, other functions and why S. pombe is commonly used in study of cohesins. Later on the various discussions will help in understanding what happens when cohesins do not perform their role appropriately especially in cell division including diseases such as cancer. Why Study Schizosaccharomyces pombe Schizosaccharomyces pombe is commonly classified as a fission yeast, it was first isolated from East African millet beer several decades ago. Various experiments have since been carried out using the organism given that it was found to have fifty genes that are responsible for various human diseases such as cystic fibrosis, cancer, diabetes among others; the largest similarities pointed to cancer (Eisen, 2002) and hence S. pombe has been studied widely in cancer researches. Over time there has been need to stretch researches to greater dimensions by intensive studies. However, there have been limitations to the approach since scientists have been arguing as to whether S.pombe is a “fission yeast”, ascomycete or a fungus given that in the Universal Translation Codes, S.pombe has been observed to have unique genes from those of man and other ascomycetes; generally S. pombe is broadly viewed as a lower fungi closely related to zygomycetes and chytridiomycetes. It is therefore not proven beyond reasonable doubt that S.pombe matches more the animal genome than other lower organisms such as yeast and Aspergillus (“Schizosaccaromyces pombe”. Though there is sufficient evidence that S. pombe has a large number of genes similar to those in humans as mentioned earlier in this section. S. pombe also presents favorable conditions for intensive genome and related studies especially due to the fact that it has the least ever number of genes coding for proteins-4940 (Wood et al. 2002)-as compared to S. cerevisiae’s 5300- hence smallest open reading frames (ORFs) for eukaryote, this makes it easier to subject the genes to sequencing among other procedures; it has 14% unique ORFs, 3% homologous to those of Caenorhabditis elegans –these genes are totally absent in S. cerevisiae. In addition, S. pombe has more most of its genes reserve; genes located on chromosomes whether essential or not are not lost but conserved-deleted- in family generations and their sub-sets hence it is easier to trace their history and follow up on certain issues along a lineage. According to Decottignies, Sanchez-Perez and Nurse (2002), S. pombe has 14 essential genes that encode for proteins commonly referred to as Protein Fate functional group which are genes involved in “protein folding, modification, and targeting”; these are the most important in relation to the human genome-as they take part in cell division and DNA repair. They include SPBC106.06, stt3, cut4, SPBC582.07c, SPBC1685.03 and sec61. Decottignies and group observed that cdc13, mob1, alp6, cut12 are essential for mitosis while cut4, SPBC106.06 and SPBC582.07c maybe necessary for the process to be successfully completed while SPBC582.11c is necessary for DNA replication initiation while SPBC1271.13 may be essential for protein synthesis to occur. Most of the essential genes discovered in S pombe are unique and have no close relative/homologs in S. cerevisiae also the genes are randomly distributed and located in relation to their functions. Cell Division Cell division is a process through which cells multiply-important for growth and reproduction; the parent cell divides into two or more daughter cells. In eukaryotes, mitosis and meiosis processes are forms in which cell division occurs. Cohesins play major role in mitosis and meiosis processes especially in ensuring cohesion of split replicated chromosomes starting at S-phase of mitosis. Chromosomes carry genetic information in form of DNA that is replicated in each cell division cycle; the replication occurs in the S-phase whereby four sets of sister chromatids are produced, to ensure that during separation the right chromatids move the right direction, cohesin ensures the sister chromatids meant for daughter cell A are strongly bound together and those meant for daughter cell B alike, move towards opposite poles in a spindle and align correctly in anaphase-stage in mitosis. After correct alignment, enzyme separase normally cleaves the cohesion away from the chromosomes-apart from that at the centromere-and is ready for another cycle of cell division. This subject has widely been studied and researchers seek to shed light on where specifically cohesins bind to chromosomes as well as differences in these locations between organisms of different species. Commonly as studied in budding yeast, cohesins are found at the RNA polymerase II transcriptional sites and not in post-transcriptional ORFs whereas in higher vertebrates such as class mammalia, cohesions are mostly found at a DNA zinc finger protein-along a chromosome- required for transcription. Although this binding site-DNA-zinc finger- is similar to that observed in Drosophilla melanogaster, but there are differences in binding patterns since in the fruit fly, binding occurs at more sites in “highly transcribed genes” across the genome (Schmidt, Brookes and Uhlmann, 2009). A protein called Mis4/Ssl3 cohesin loader which has a high affinity for cohesin is said to be responsible for identification of cohesion binding sites on a chromosome but the mechanism by which the protein-cohesin binding occurs is little studied. The structure of cohesion as documented in various studies is said to be ring-shaped-due to the nature of head to head binding and tail to tail binding of its subunits Smc1 and Smc3 with the aid of Scc1and Scc3 which stabilize the structure at the Smc head convergence points. By utilizing this shape, cohesions are said to bind DNA in an encircling manner that topologically traps it (Uhlmann, 2004); this mechanism is said to make DNA more compact. It is the Scc1 that is cleaved by separase during anaphase to ensure the cohesin ring opens to release chromatin. Alternatively, cleavages at any of the folded Smc sites will force separate the chromatin and cohesion As stated earlier, after alignment of sister chromatids at two opposite poles- in preparation for cell halving, cohesions separate from the chromatins, but amazingly, the two chromatins as joined at the centromere do not separate until a new cell cycle begins. Scc1 is cleaved by separase but the end product of the cleavage has to be a c-terminal Scc1 that will ensure no re-binding of the Smc1 and Smc3 again; the Scc1 fragment has to be removed by proteolysis before a re-binding can occur. This separation occurs in prophase but interestingly, the ring at the centre of the two chromatins-centromere-is never opened until metaphase where its cleaved by a separase to mark the beginning of anaphase; this exceptional separation has been subject to research overtime. Shugoshins (Sgo1) which are proteins, are said to be responsible of shielding meiotic cohesions from cleavage in mitosis and meiosis 1 (McGuiness et al, 2005) and hence ensuring chromatids stay together. The proteins are located on a cohesion specific site which is at the centromere and a 50 kb region surrounding it. Shugoshins require 12 base pairs, Bub1 kinetochore component and Spo13 meiosis factor to bind to the centromere (Kiburz et al. 2005) Chromosomes are made up of five major proteins known as histones, these are arginine or lysines based and are therefore subject to phosphorylation, methylation or acetylation hence varied functions such as readiness for replication, transcription or packaging. Histones are specifically found at the center of chromatin as small particles, around which DNA winds in a helical form. The chromatin is then arranged in the nucleosome which ct as a packaging container; five histone units make up a chromatin-four in the interior and one on the external. The four internal histone molecules include H2A, H2B, H3 and H4 and the external histone molecule is H1. Structural changes in the amino acid basic format-arginine and lysines-determines whether DNA packaging will occur in cellsor in the nucleosome. During replications, structural changes of histones enable formation of new DNA strands while the former histones-in their unchanged structure form the template strand which then undergo transcription and translation with help of DNA polymerases and ribonucleic consitituents (mRNA, rRNA, tRNA) leading toformation of new proteins (“Chapter 37: Eucaryotic Chromosomes and Gene Expression”). Application After the exciting stories of how cells divide and multiply, there are the unfortunate stories of what may happen when cell division does not occur as programmed. In case errors occur during cell division in processes such as mitosis and meiosis or in transcription, translation and replication, there are resultant consequences such as malformations and abnormalities, rare disorders and diseases such as cancer. Cancer is among the leading killer diseases in the world according to 2009 statistics, 13% of total deaths worldwide are attributed to cancer. Previously known as a disease of the rich but in the present; it has highest prevalence in low –income/developing nations. Normally cancer is thought to be more of a lifestyle disease associated with oxidative foods, inheritance and harmful rays and radiations, but in the actual sense most people do not understand how cancer emerges for instance from food. The mechanisms involved have something to do with disruption of cells in particular the cell division processes that are responsible for growth and development. The most common cancers include colon, prostate, breast and lung cancers. Cancers are named based on the site of origin and there are four broad categories; sarcoma, leukemia, carcinoma and lymphoma. Sarcomas are those that originate from the connective and supportive tissues such as the bone, blood vessels, muscles and cartilage. Carcinomas originate from the skin and related line of tissues. Leukemia on the other hand has roots in blood forming tissues such as bone marrow whereby anomalies in blood cells lead to the cancer. Lymphomas have their origin in the cells of the immune system. Another “silent” category is that of the central nervous system (CNS) whereby cancers originate from the CNS such as the brain and spinal cord. Cancers can also be categorized broadly based on the group of the population affected-males and females. For instance men are prone to prostate, colon and lung cancers. Women are susceptible to breast and cervical cancers. In children, leukemia, lymphoma and brain tumors are common. m Cell division is a proliferation process and cancer is a disease due to proliferation gone wrong. Therefore in short something goes wrong somewhere in the cell cycle for cells to be termed cancerous. The cell-division cycle does not occur continuously but is a periodical event separated by resting phases and it is also under tight control whereby is there is a problem with the regulatory mechanisms and the proliferation processes take place continuously, the resultant feature will be a piled up mass of cell which are known as tumors. It is quite interesting to note that cancer doesn’t develop overnight but a series of three or more changes/mutations have to occur for cells to turn cancerous. In nature, cells have ways of sending messages to others inform of chemical signals; a cell can inform neighboring cells to divide by releasing a growth-promoting signal, these communication systems are controlled by proto­oncogenes and tumor suppressor genes such as p53; change in any of the genes leads to cancer as a result of uncontrolled cell proliferation. Similarly cancerous cells have the potential to instruct other cells to proliferate since they possess characteristics of normal cells and further they possess unique counteracting mechanisms that alter normal cell check point activities. For instance, cancerous cells have unlimited longevity because they have a mutating effect on cell life-span controlling genes; they produce a protein called telomerase which ensure telomeres do not shorten as designed under natural death program but instead remain longer and capable of several cell divisions hence making cancerous cells immortal. Further still cancerous cells can be able to survive outside the original space of the original normal cells; normally have their own space and unique features such as PH and exist on their own but are connected by an extracellular matrix. Cancerous cells on the other hand can exist freely on their own without support of other cells; they also have the potential of shifting through a process called metastasis whereby for instance a cancerous cell with origin in the liver can enter and survive in the bone and divide there hence becoming malignant tumors (Skebber, 2008-2009; KupChella). Cell cycle Cell cycle refers to the processes through which a cell undergoes cell division and replication. There are four major phases- G1, S, G2 and M phases and two checkpoints at G1/S phase and G2/ M phases. G1, S, G2 are commonly referred to as interphase while M phase is the mitotic phase. The mitotic phase consists of four phases prophase, metaphase, anaphase and telophase. At G1 cells pick up after the last cell division cycle and prepare most for the next phase-S-phase; this is a restriction point and in cultures, it is estimated and not less than 24 hours (Lodish, Berk and Zipursky et al., 2000). The check point at G2/M phase occurs when cell growth stops giving room to division of the cell into daughter cells. The transition between the cell cycle phases is controlled by cyclin-dependent kinases (Cdk); four Cdks-1, 2, 4 and 6-in S. cerevisiae, there homologs in mammals are Cdk A, B, D, E; A and B regulate the S and G2 phases while D and E are expressed at G1. Once a signal is relayed to G1, the Cdks that are specific to GI-D and E- are activated and prepare the cell to enter into the S-phase by increasing expression of transcription factors (TF) which then activate S cyclins and other components including enzymes required for DNA replication. On the other hand cyclins at G2-commonly referred to as mitotic cyclins, promote the start and continuity of the mitosis process by enabling necessary proteins for chromosome and spindle formation are availed. Other regulatory factors include early response and delayed response genes; these regulate the speed at which cells in the resting phase (G0) enter into the cell cycle. Mutations especially due to viral infections due to retroviruses- c-Fos and c-Jun, in these groups of genes can lead to cancer. For a cell to bypass the regulatory sites a protein called retinoblastoma must be present; for instance Cdk D-the first cyclin produced in a cell cycle when signaled by maybe a growth factor binds to existing Cdk4 forming a complex-D-Cdk4. D-Cdk4 phosphorylates the retinoblastoma protein which exists as an inactive E2F/DP1/Rb making E2F genes more active; E2F leads to formation of cyclins important for the next cell cycle stages, including cyclin A and E. Cyclin E for instance binds to Cdk2 forming E-Cdk2 complex which ensure the cell moves through the G1/S restriction point. Cyclin B on the other hand binds Cdk1 and the resultant complex leads to initiation of the mitotic phase. The transition processes in the cell cycle are controlled by inhibitors broadly categorized as cip/kip family and INK4a/ARF family; members of cip/kip such as p21, p27 and p57 inactivate the D-Cdk4 complex hence stopping the cell cycle at G1 phase. In case of DNA damage, regulatory genes p53 lead to activation of p21 which in turn slows down progression of the cell cycle hence ensuring the damage is not replicated in the S-phase (Collins, Jacks and Pavletich, 1997). p16INK4a which is a member of INK4a/ARF family binds onto Cdk4 hence stopping the cell cycle at G1. p14arf another member of the INK4a/ARF family prevents degeneration of p53 hence ensuring regulation of the cell cycle at points controlled by p53 as described above; it is important in arresting cancerous developments at earlier stages by ensuring mutant cells are not replicated. Further the cell cycle is regulated by transcription components that work hand in hand with the Cdk-cyclins. Structure of Cohesin Cohesins are made up of our main subunits namely scc1, smc1, scc3 and smc3. Cohesin Smc3 and Smc1 are members of the Structural Maintenance of Chromosome (SMC) family (Arumugam et al., 2006). The SMC proteins have two functional units-an ATPase and hinge region, the ATpase and hinge region connect in a highly coiled form and hence forming dimmers with ATpases at the end and hinge region at the center. The coiling and opening of the ring-like dimmers is made possible by ATP hydrolysis. Scc1 and Scc3 further make the coiled structure stable by binding Smc1 and Smc3 at the ATpase sites. Scc3 also binds the Scc1 at their C terminals whenever the Scc1 attach to the SMC proteins. The opening and closure of the coiled-ring like structure of cohesion is also controlled by Scc1 whose selective binding to both smc1 and smc3 leads to a closed ring and an open ring when it binds to one of the SMC proteins. Studies have also reveled that cohesion proteins arrange themselves as dimers held together by Scc3 in a handcuff shape (Zhang et al., 2008) The functions of Cohesin The core function of cohesin that has widely been studied is its cohesive role on the sister chromatids in cell division especially at the end of metaphase. In anaphase the cohesive forces have to weaken to allow the sister chromatids to separate to the poles. If cohesion is not established sister chromatids will separate way before mitosis begins this is normally referred to as condition called chromosome missegregation, therefore there has to be a tight regulatory mechanism to ensure cohesion when necessary and to give room for separation as required. But surprisingly no checkpoints are known with this regard and anomalies in chromosome separation do not stop the cell cycle; however such anomalies are very rare. Cohesins are also said to facilitate the binding of spindles to chromosomes, this occurs in a non-uniform manner with high binding affinity observed at the kinetochores, adenosine triphosphate rich sites and convergent intergenic regions. Evidence that cohesions play these important roles is the existence of enzyme sepatase that is responsible for separation of chromosomes by cleaving the Scc1/Mcd1 subunit. Cohesins also accelerate the rate of DNA restoration after damage. This is said to be due to its cohesive role; it manages to keep the broken chromatid closer to its sister chromatid hence accelerating the repair process. In addition, cohesion is required at the G1, S, G2/M checkpoints to check for damages as the cell cycle progresses through the stages. How cohesins work at the checkpoints has not been very clear although some studies contemplate that cohesins are activators and also mobilize proteins required in the DNA repair (Jessberger, 2009) Location of cohesion Cohesins are generally located along chromosome arms with concentration at pericentric/centromere regions; at the chromosome arm the cohesions have been observed to bind to AT-rich-DNA sequences while at the centromere, cohesions bind to kinetochores. In mechanisms not very clear, cohesion also tends to have a high affinity for intergenic regions located at transcription convergent zones. Cohesin tend to be conservative about their locations but research has show that there position can be changed through transcription by use of RNA polymerase or rather other researchers suggest that cohesion locate themselves based on the transcriptional status (Bausch et al., 2007). The direction of transcription is determined by gene orientation and there are three major orientations that genes take, namely head to head, head to tail and tail to tail. For instance the tail to tail orientation is thought to cause transcription convergence and a change in these orientations probably causes different occurrences related to transcription hence need for cohesions to change their locations. Mechanisms of mitosis & meiosis Mitosis is a form of cell division through which cells multiply hence in eukaryotes, it is a core growth and development process. It is divided into 6 phases namely interphase, prophase, prometaphase, metaphase, anaphase and telophase. Interphase is not a formal part of mitosis but rather more of a preparation phase into mitosis. It comprises of the G1, S, G2 stages of cell division (discussed in sections above) and cells engage in metabolic activity in readiness for the division process. Cells are not visible in the nucleus. At Prophase each cell has a complete set of genes; most eukaryotes have diploid cells; two complete sets of genome. Chromatin condenses and cells can be visible under a microscope, centrioles- homologous of microtubules in plants-move towards the opposite poles of the cell and the spindle begins to form. At prometaphase, the nuclear membrane disappears, proteins attach to the chromosome at the centromere and form kinetochores which help the chromosomes to move. In metaphase, chromosomes move along the spindle fibres and align themselves at the center of the cell nucleus; this prepares the cell for division in that when the cell will finally divide each daughter cell will receive a copy of each chromosome. At anaphase, the pair of chromosomes separate and move along the spindle towards opposite poles of the cell; this is aided by the motion created by kinetochores and polar attractions. In Telophase, the separated chromatids are located at extremes of each other-at the poles- and a nuclear envelope begins to form around each pair of chromatid, the chromosomes are not visible by light microscope, the spindle fibers disappear and the cell mainly redirects its energy to the division of the daughter cells. In animals, the daughter cells separate through a process called cytokinesis, whereby the cell pinches at the center leading to separation of the daughter cells. In plants, because of the cell wall, pinching by the actin protein is not possible and a cell membrane is synthesized for each of the daughter cells. This is the process by which eukaryotes proliferate, grow and develop.. Meiosis is a special form of cell division that is necessary for sexual reproduction; it involves splitting of gamete cells. For instance in humans, there are 23 pairs of chromosomes, 22 of which are normal cells and a pair of sex chromosomes; the pair of gametes is what undergoes meiosis. Unlike in mitosis, sex chromosomes do not replicate and instead half the set(s) of chromosomes. Meiosis occurs in two phases-meiosis 1 and meiosis II. Various processes such as gametogenesis and oogenesis depend on the meiotic process. Meiosis 1, is the reduction phase while Meiosis II is the dividing phase. Meiosis 1 is divided into four stages namely prophase 1, metaphase 1, anaphase 1 and telophase 1. In prophase 1, there are four chromatids or two pairs of chromosomes, one from each parent. Chromosomes condense, and start being visible by microscope, homologous chromosomes pair up and there is exchange genetic information via recombination or through formation of chiasmata. Homologous chromosomes separate, chromosomes condense further and are now visible in the nucleus, nuclear membrane disappears and spindle fibers begin to form. In metaphase 1, homologous pairs move to the center of the nucleus-at the metaphase plate, centrioles attach to chromosomes forming kinetochores, homologous chromosomes align further along the spindle fibers in an equatorial plane. At anaphase 1, homologous chromosomes move towards opposite poles by aid of kinetochores and polar attractions, the cell elongates and at begins to prepare for division. At telophase 1, chromosomes are at the poles, the spindle fiber disappears and a nuclear membrane forms around each of the haploid set of cells, chromosomes uncoil back to chromatin, cell undergoes cytokinesis and two daughter cells with half the number of chromosomes of the initial cell is formed. The cell may rest at this point in interphase II or may proceed to meiosis II and undergo our stages-prophase II, metaphase II, anaphase II and telophase II. The stages are similar to those in mitosis such that the two haploid cells produced after meiosis I undergo division hence at the end of meiosis II, there are four haploid cells (Farabee, 2010) The selective mitosis and meiosis processes ensure that eukaryotes maintain their original number of chromosomes throughout their entire generations. Further meiotic processes ensure variations and diversity in organisms due to the exchange of genetic information. The highly conservative processes also ensure abnormalities are in check, but in rare cases mutations or failure at checkpoints may lead to rare disorders and abnormalities. To better understand meiosis, the oogenesis process will be described in details in this section. Oocytes generally reproduce by mitosis but once they undergo differentiation, primary oocytes are formed which then enter the meiotic cycle. The oocytes do not go beyond prophase1 but instead remain dormant until ovulation; interestingly these processes take place in females before birth but activation of the oocytes-out of the dormant phase –occurs in puberty. Just before ovulation, the primary oocytes undergo the first meiotic division to half the cells-diploid to haploid forms as described above. At meiosis 1, during cytokinesis, two unequal cells are formed- a secondary oocyte and a polar body. The secondary oocytes then enter into meiosis II, the polar body also does. Two cells are the results of the second meiotic process with one bigger than the other; the bigger cell formed after meiosis II is the ovum. In general, the entire meiotic process results in an ovum and three polar bodies. Polar bodies die but the ovum is viable for fertilization. Fertilization leads to formation of a zygote which has complete set of chromosomes-22 pairs of somatic cells and 1 pair of sex chromosomes/gametes. The zygote undergoes mitosis and meiotic process as part of the growth and development processes until adulthood. These two processes mitosis and meiosis are very crucial in ensuring cell viability and failure at various security points may result into serious anomalies such as non disjunction, some of which are inheritable hence leading to rare disorders such as down syndrome-results because of trisomy at chromosome 21 and turner syndrome which come about as a result of lack of one female chromosome. Females are normally represented as XX, in turners syndrome the female cells have one X. For instance in meiosis, the halving of cells ensures the exact number of chromosomes in the parent cell is maintained in new formed cells otherwise daughter cells would have had exponentially high numbers of chromosomes. In mitosis, anomalies occur most during transcription as a result of deletions, additions, substitutions, translocations, in the chromosomal structures hence the genetic makeup. Function of PSC3&PSM3 These are cohesion subunits as identified in S.pombe, together with Psm1 and Rad21, they play major cohesion function especially in anaphase where homologous sister chromatids move towards opposite poles. They are homologs of the scc1, smc1, scc3 and smc3 identified in yeast and eukaryotes (Tomonago et al., 2000). Were it not for cohesions, chromosome segregation would be too chaotic and unspecific and abnormalities and disorders would be too many to handle. In a bid to understand the role of PSC3 and PSM3, we focus on a case study- a research conducted by Tomonaga et al (2000) on “characterization of fission yeast cohesion”. In the research, genes were extracted from S. pombe and their disruption phenotypes. When the disrupted genes were cultured in DAP1 and antitubulin antibody at 330C for 10 hours and the segregation patterns of chromosomes observed; the segregations were aberrant and followed a similar pattern in all cells. The control experiment constituted mitotic cells which were also cultured in same media but at 260C. After staining by DAP1 mutant cells expressing a mis12-GFP (GFP is a kinetochore protein) were observed and it was discovered that sister kinetochores separated early than is usual in the cell division processes. Further the mutant genes now characterized as Psm1, Psm3 and rad21 were subjected to a ura4 gene marker from S. pombe to ascertain their role in the cell cycle, later under genomic Southern hybridization, all the haploid cells marked with ura4 were found to be negative of the marker hence the conclusion that all the subunits of the genes were essential for normal cell activities. In a different experiment but within the context of the research by Tomonaga et al, the mutant genes were cultured in absence of uracil, then stained usond DAP1 and antitubulin antibody observed under a fluorescent microscope. Results showed that the mutant genes were incapable of mitosis given their short spindles; hence the role of Psm3 in mitosis was clearly established. References Arumugam et al. (2006). Cohesin's ATPase Activity Is Stimulated by the C-Terminal Winged-Helix Domain of Its Kleisin Subunit. Retrieved from www.ncbi.nlm.nih.gov/pubmed/17055978 20 April, 2011 Bausch, C. et al. (2007). Transcription Alters chromosomal locations of cohesions in S. cerevisiae. Retrieved from www.ncbi.nlm.nih.gov 19 April, 2011 Collins, K. Jacks, T and Pavletich, P.N. (1997). The cell cycle and cancer. Retrieved from www.pnas.org/content/94/7/2776.full 19 April, 2011 Decottignies, A., Sanchez-Perez, I. and Nurse, P. (2002). Schizosaccharomyces pombe Essential Genes: A Pilot Study. Retrieved from genome.cshlp.org/content/13/3/399.full 19 April, 2011 Eisen, J.A. (2002). Genome sequencing: Brouhaha over the other yeast. Nature 415, 845- 848 Farabee, J.M. (2010). CELL DIVISION: MEIOSIS AND SEXUAL REPRODUCTION. 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