Practical on Life Cell Cycle of Fission Yeast - Lab Report Example

Practical Report on Life Cell Cycle of S.pombe Practical Report on Life Cell Cycle of S.pombe The cell cycle, which is also known as the cell division cycle, refers to a series of events taking place inside a cell. These events lead to division and replication of cells producing two daughter cells. Prokaryotic cells that lack a nucleus undergo cell division through the process of binary fusion. This is the splitting of the cell into two to produce new products. The eukaryotic cells that have a nucleus undergo cell cycle in three different phases: Interphase, mitotic and cytokinesis phase. The method involved colorimetric estimation of DNA through the spectrophotometry to determine the absorbance at various levels. Calibration standard curve was obtained to draw a cell cycle curve of S.pombe to compare different cell phases with that of the human being. In conclusion interphase period was evident from 0 to 80 minute time interval with G1 phase at 0 to 20 minutes, G2 at 20 to 80 minute and mitotic phase at 80 to 100 minute time. This was in correlation with the process of cell cycle of larger eukaryotes and this phenomenon can lead to studies of different diseases infections in human cells. Introduction The study of cell cycle involves a favorite organism like the fission yeast S. pombe. This is because of its faster growth as compared to other species from eukaryotic cells (Knutsel et al. 2011). Its ability to divide through medial fission and its rod shape enables its cycle be easily estimated even through visualization. Further exemplification by BATTOGTOKH, 2007 & Csikasz, 2009, suggests that the yeast is also the most preferred organism in cytological study of chromosomal dynamics since it has three chromosomes. The yeast has been widely used experimentally to investigate the timing and also control of cell cycle. Pure DNA extracts may be easily estimated by the method of spectrophotometry. This is because DNA at 260nm can absorb maximally. On the other hand, crude extracts containing extracts of DNA requires a chemical process in their estimation. This is because the homogenates contain other substances that will impede with spectrophotometry as they are majorly based on double bonds that are conjugated to a molecule. As a result, it is not regarded as a specific DNA assay (Piel& Tran, 2009). The colorimetric methods for the detection of DNA as clarified by Zhao et al., 2013, are majorly based on the reaction of pentose groups of nucleic acids. Presence of strong acids like sulphuric and glacial acids, the main links between the purines and the deoxyribose are completely hydrolyzed. The resulting residues are later converted to react with chemical diphenylamine producing a blue pigment as the end inference. This pigment can absorb at 600nm. The hypothesis of the experiment is to determine the concentration of the unknown DNA when a standard curve of DNA is constructed. The curve is to range from 0-400 absorbance (ug.ml-1). The Schizosaccharomyces pombe yeast cells have been grown in culture that is synchronous after the size of cell selection procedure that selected cells of similar volume from the given culture. Therefore at the start, all the cells are approximately the same in size thus assumed they are in the same stage of cell cycle. Aim To determine the timing of the different stages of cell cycle of Schizosaccharomyces pombe, this is fission yeast. Objectives 1. To determine the amount of DNA present in the samples. 2. To estimate the timing of G1, S and G2 phases of the cell cycle for this yeast organism under the growth. MATERIALS AND METHODOLOGY Materials 11 test tubes and reagents of diphenylamine at 10g/1 in glacial acid and 25ml of sulphuric acid. Samples labelled A-F. The samples were formed through sampling selection procedure done at intervals of every 20 minutes. These samples were from the growing culture that were synchronous and treated firstly through, homogenization of lysed cells with buffer solution containing Triton X-100. Secondly, through removal of RNA and protein through incubation with an enzyme proteinase K. This is at a concentration of 0.1 mg/ml, and ribonuclease concentration at 0.1 ug.ml-1 for one hour. Lastly, through the process of addition of ethanol to the sample in ratio of 2:1 to cell lysate. This precipitates the DNA, which dissolves at pH of 7.5. Procedure The test tubes number, DNA standard, water volume and samples were tabulated as follows: Tube number DNA standard Water Sample 1 0 ml 2.0 ml 2 0.5 ml 1.5 ml 3 1.0 ml 1.0 ml 4 1.5 ml 0.5 ml 5 2.0 ml 0 ml 6 2 ml A 7 2 ml B 8 2 ml C 9 2 ml D 10 2 ml E 11 2 ml F (Where test tube 1-5 are the standards and test tube 6-11 are the samples) 4.0 ml of the diphenylamine reagent at 10g/1 and 25 ml of H2SO4 was added to the 11 test tubes. Then the screw tops on the tubes were loosened, and the mixing done gently. The tubes were then placed in the water bath for 10 minutes and boiled then allowed to cool. Absorbance reading of tubes 1- 11 was made where tube 1 was used as a reagent blank at 600nm mark and recorded in Table 1. A calibration curve of absorbance and DNA concentration of the standard was then constructed (See Figure 2 in the appendix). The curve was then used to calculate the concentration of the DNA samples in the unknown samples. Results Table 1: Showing DNA volume, concentration, and absorbance. Test tube DNA volume DNA Concentration Absorbance 1 0 0 0 2 0.5 100 0.073 3 1 200 0.186 4 1.5 300 0.254 5 2 400 0.276 6 0.551159618 115.4285714 0.092 7 0.61255116 128.2857143 0.101 8 0.58526603 122.5714286 0.097 9 1.246930423 261.1428571 0.194 10 1.369713506 286.8571429 0.212 11 1.383356071 289.7142857 0.214 The table above shows DNA volume, DNA concentration, and Absorbance. The concentration of the samples was obtained from the equation of the standard curve that had a line equation of y= 0.0007x + 0.0112. Therefore using the equation the unknown concentrations were calculated as follows: For example sample, 6 had an absorbance of 0.09. Therefore the concentration was calculated as follows: y= 0.0007x + 0.0112 where y is the absorbance and x is the DNA concentration. Therefore, x= = = 115.4286 µg/ml. The rest of the sample concentrations were also calculated in the same way. After finding the DNA concentration the DNA concentration per cell was calculated using the following formula: DNA concentration per cell = For example, the DNA concentration per cell for sample 6 was calculated as follows: DNA concentration per cell = = 1.44 * 10-6 The rest of the DNA concentration was also calculated using the same formula and the result recorded in Table 2. Table 2: showing DNA concentration per cell at different times. Test tube DNA volume DNA Concentration cell/ml time DNA concentration/ cell 1 0 0 2 0.5 100 3 1 200 4 1.5 300 5 2 400 6 0.551159618 115.4285714 8.00E+07 0 1.44286E-06 7 0.61255116 128.2857143 1.50E+08 20 8.55238E-07 8 0.58526603 122.5714286 1.50E+08 40 8.17143E-07 9 1.246930423 261.1428571 1.50E+08 60 1.74095E-06 10 1.369713506 286.8571429 1.50E+08 80 1.91238E-06 11 1.383356071 289.7142857 3.00E+08 100 9.65714E-07 In order to study the cell cycle, a graph of DNA concentration per cell and time was then drawn as shown in Figure 1 below. The curve showed the different phases in the cell cycle of the fission yeast. Figure 1: Cell cycle of S.pombe From the graph, it is evident that the concentration increased with the increase in absorbance from sample 6 to sample 11. This clearly explains the difference timing of the stages in the cell cycle. DISCUSSION The concentration of DNA increased with the increase in absorbance. This illustrated the timing of every stage in the cell cycle. The yeast divides through asexual means because it is haploid in the life cycle. Most of the time in the cycle is spent in the stage of G2 since most of the cell cycle is controlled here. From the graph, therefore, it is evident from time intervals of between 20 to 80 minutes. This means more cells are growing and, therefore, high concentration of DNA. Between 0 to 40 minutes the cell is in G1 phase as it goes through biosynthetic activities and the supply of proteins increases. S phase is at time 80 minutes point as the replication commences, and DNA synthesis is quickly completed before the cell enters the mitotic phase where there is a reduction in DNA concentration. This is because chromosome pairs condense leading to the reduction. This is shown with the time interval of 80 to 100 minutes. The other stages from G2, therefore, as illustrated by Yurkovsky & Nachman, 2013; Howarth, 2003; Boe et al. 2008, have equal shares of the remaining time. In the fission yeast, the cycles of cytokinesis and nuclear division have synchronically timing (Bernan et al., 2012& Navaro, Weston& Nurse, 2012). Diphenylamine assay did the estimation of the absolute DNA mass and the absolute DNA content differences between the populations of cells. The assumption was based on all cells in G0 or G1 phases were in synthetic cycle. On the other hand, if the cells are in the exponential growth stage and also synthesize DNA, population portions are in S or G2 phases and, therefore, the assay will overestimate total DNA mass per cell (Egel et al., 1980). These assumptions are the limitations of this method used. Another method that can be used instead of the method above is the mutagen analysis method. It involves determining changes in genome organization throughout the cell cycles. Different mutagens are used to identify the effects of every phase, and other eukaryotic cells can correlate this. CONCLUSION Meiosis in larger eukaryotic cells can be easily understood on how they operate by understanding the phenomenon of cycle of S.pombe. Transcription mainly takes place in the S phase, and this explains how DNA replication timing can be determined in the phases. Interphase or the preparatory phase; in this stage, the cell grows as it acquires all the nutrients available for it to enter the cell division. It majorly takes place in cells and nucleus that are newly formed. The total average time the phase lasts is 90% of the time required for cell cycle. The period continues in three different stages that are G1, S, and G2. In the G1also called the growth phase involves biosynthetic activities that during M phase were slowed down considerably are resumed at very high rates. The supply of proteins increases within the cell as well as the number of organelles increases in size .Mitotic phase; in this phase the eukaryotic cell separates chromosomes in the cell nucleus to produce two nuclei that are identical. The chromosome pair condenses, and fibres attach pulling sister chromatids in the opposite sides (Fedyanina, 2012). Cytokinesis phase: the process of cytokinesis results in division of the cytoplasm, cell membrane, nuclei and organelles into two new cells that contains the same cellular components (Pollard& Jian-Olu, 2010). The application of this biochemical analysis is used in the detection of various diseases infecting organelles in the eukaryotic cells. For instance, the study is shown by Schmidt et al., 2007 on the mitochondria and endoplasmic reticulum infections, is easily comprehended through the process of cell cycle of the fission yeast. This, therefore, explains the correlation between the process of cell cycle of fission yeast and that of the human being. Additionally, through observation of dynamics of yeast chromosome and also levels of protein expression and regulation, various systems of organelles in human cells like Golgi apparatus can be studied (Schmidt et al., 2007). References Abenza, J, Chessel, A, Raynaud, W, & Carazo-Salas, R 2014, Dynamics of Cell Shape Inheritance in Fission Yeast, Plos ONE, 9, 9, pp. 1-15 BATTOGTOKH, D 2007, FORCED SYNCHRONIZATION OF EUKARYOTIC CELLS, Modern Physics Letters B, 21, 30, pp. 2033-2053 Bernal, M, Sanchez-Romero, M, Salas-Pino, S, & Daga, R 2012, Regulation of Fission Yeast Morphogenesis by PP2A Activator pta2, Plos ONE, 7, 3, pp. 1-11 Bøe, C, Garcia, I, Chen-Chun, P, Sharom, J, Skjølberg, H, Boye, E, Kearsey, S, MacNeill, S, Tyers, M, & Grallert, B 2008, Rapid regulation of protein activity in fission yeast, BMC Cell Biology, 9, pp. 1-11 Csikász-Nagy, A 2009, Computational systems biology of the cell cycle, Briefings in Bioinformatics, 10, 4, pp. 424-434, Egel, R, Kohli, J, Thuriaux, P, & Wolf, K 1980, GENETICS OF THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE, Annual Review of Genetics, 14, pp. 77-108 Fedyanina, O, Mardanov, P, Tokareva, E, McIntosh, J, & Grishchuk, E 2006, Chromosome segregation in fission yeast with mutations in the tubulin folding cofactor D, Current Genetics, 50, 5, pp. 281-294 Hoose, S, Rawlings, J, Kelly, M, Leitch, M, Ababneh, Q, Robles, J, Taylor, D, Hoover, E, Hailu, B, McEnery, K, Downing, S, Kaushal, D, Yi, C, Rife, A, Brahmbhatt, K, Smith III, R, & Polymenis, M 2012, A Systematic Analysis of Cell Cycle Regulators in Yeast Reveals That Most Factors Act Independently of Cell Size to Control Initiation of Division, Plos Genetics, 8, 3, pp. 1-13, Howarth, S 2003, Mitosis and Meiosis, Journal of Biological Education (Society of Biology), 37, 3, p. 151 Knutsen, J, Rein, I, Rothe, C, Stokke, T, Grallert, B, & Boye, E 2011, Cell-Cycle Analysis of Fission Yeast Cells by Flow Cytometry, Plos ONE, 6, 2, pp. 1-8 Navarro, F, Weston, L, & Nurse, P 2012, Global control of cell growth in fission yeast and its coordination with the cell cycle, Current Opinion in Cell Biology, 24, 6, pp. 833-837 Piel, M, & Tran, P 2009, Cell Shape and Cell Division in Fission Yeast, Current Biology, 19, 17, pp. R823-R827 Pollard, T, & Jian-Qiu, W 2010, Understanding cytokinesis: lessons from fission yeast, Nature Reviews Molecular Cell Biology, 11, 2, pp. 149-155 Schmidt, M, Houseman, A, Ivanov, A, & Wolf, D 2007, Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe, Molecular Systems Biology, 3, 1, p. 79 Yurkovsky, E, & Nachman, I 2013, Event timing at the single-cell level, Briefings in Functional Genomics, 12, 2, pp. 90-98, Zhao, Y, Xiang, S, Dai, X, & Yang, K 2013, A simplified diphenylamine colorimetric method for growth quantification, Applied Microbiology & Biotechnology, 97, 11, pp. 5069-5077 APPENDIX Table 4: The absorbance of each of the samples. samples Absorbance 1 blank 0 2 0.073 3 0.186 4 0.254 5 0.276 6 0.092 7 0.101 8 0097 9 0.194 10 0.212 11 0.214 Figure 2: Standard graph showing absorbance and concentration Read More