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UV is one of the many types of radiation considered to be harmful due its effect on cellular organisms at protein levels. This form of radiation has potential deleterious effects on organisms, irrespective of its dosage. It´s mechanism functions through directly targeting the DNA, where minor damages to the genetic composition are mainly intensified after the cell undergoes replication and growth (Pringle, 1997). Although, UV has an impact only on specific molecules, its concentration makes it as destructive as other forms of radiations. Therefore, the mechanisms for repairing DNA damage play a vital role in its survival and existence. (Friedberg, Cellular Responses to DNA damage, 1983)
All these systems work distinctively on different organisms keeping in account the degree of UV damage, often resulting in either complete or partial damage repair, or a failed one depending upon the mutation. Photoreactivation, being one of the earliest processes of all, uses light energy in the form of a photon and enables the use of a specific protein called photolyase from the cell for the repair of dimerised pyrimidines in the DNA. Excision repair is another method whereby the faulty DNA lesion is removed by the cells within, and is replaced by DNA synthesis using the undamaged strand as a template. Although this method is more complex and utilizes more enzymes, it can be used for DNA repair caused by agents other than UV. (Friedberg, DNA Repair and Mutagenesis 2nd Ed, 2006). In Error-prone repair, the gap opposite the site of DNA damage is directly filled in by newly synthesized DNA. However, the repair is quite inaccurate and consists of numerous mutations due to the template damage. Recombinational repair uses unaffected parental strand or other DNA molecules to undergo genetic recombination to fill in the gaps created by blocking of DNA replication by the dimers. (Lodish, 2000)
Repair mechanisms also depend on the chance that DNA has undergone repair before getting replicated. Since the probability of cell dying is proportional to the amount of radiation absorbed, we predict that diploid cells have better chances of survival than the haploid ones as they contain two copies of DNA in each cell (Nickoloff, 1998).
Taking into account the aforementioned data, the diversity in organisms has almost null effects on the differences of repair processes between organisms. Yeast cells have proved to be potentially useful in UV irradiation studies (Guthrie, 2002). They contain specific genes (RAD9 in case of S. cerevisiae) that function for cell cycle checkpoint. These genes function for the repair of damaged DNA, and their presence blocks the continuation of the cell cycle to the M phase, providing more time for the repairing of damaged DNA in G2 phase (Jefton, 1998).
Here, we aimed to investigate the effect of UV irradiation upon different strains of S. cerevisiae: Haploid(HB0), Diploid(D02), wild-type(D02) and homozygous rad9 mutant(D02- Δrad9/Δrad9), carried out the comparison between their mutations and tested their sensitivities by measuring the % survival and mutation rates.
Preparation of overnight broth cultures for the different S. cerevisiae strains.
This experiment mainly consisted of two synergetic parts and was carried out in pairs synonymously. Whilst one pair had to attain the required data on the effects of UV light on haploid (HB0) versus diploid (D02) strains of S. cerevisiae, the other pair attained the required data on the effects of UV light on wild-type (D02) versus homozygous rad9 mutant (D02-rad9).
Counting Yeast Cells using a Haemocytometer
Preceding the experiment, the work space was made sterile by swabbing the bench with detergent and paper towels. Each bench was provided with a Bunsen burner. The entire experiment was performed close to the flame for sterility. A wire loop provided was first sterilized with the help of Bunsen flame by gradual heating of the wire in the hottest region of the flame. Once cooled down, the colony of different S. cerevisiae strains was transferred from the plate into the two 10ml bottles of YED broth. These were cultivated at 30ºC water bath overnight with shaking.
The next day, the cultures were taken and the number of cells were counted. 20-300 cells were needed for the results to be statistically significant and haemocytometers were used. These were placed on the bench and covered with a cover slip. With a pipette, 100 µl of yeast from the culture was removed and the tip was immediately placed in contact with the edge of the cover slip allowing the counting chamber to quickly fill by capillary action. The haemocytometer was observed under a microscope at 4x magnification to find the ruled lines. To do this, the condenser diaphragm was closed. The magnification was increased to 10x to count the cells more easily and recorded. Cells of only large corner squares (0.1mm3 each) were counted and cells on the top and left boundaries were included but right and bottom were excluded. The equation 10000(x/4) where ‘x' is the number of cells in the 4 large squares was then used to calculate the cell density (cells/ml).
Upon having a cell density, the samples were diluted to obtain the density wanted of 2.5x10^7 cells/ml. The dilution ratio needed was calculated using the equation: density wanted / density achieved. This was the amount in ml of sample which was added to distilled water in eppendorf tubes and made up to 1ml. Dilution was continued by removing 0.1ml of the sample and adding 0.9ml of distilled water into a new eppendorf tube and this was done 6 times successively till the right amount was reached. In the last dilution, 0.2ml of the sample was removed and 1.8ml of distilled water was added so as to provide enough sample to produce six plates of 200 µl.
Plating the Yeast Cells
YED plates for irradiation were prepared by labeling 6 plates with their corresponding UV-C intensities of 0, 10, 20, 30, 60 and 80 J m-2. 200 µl of culture was removed and pipetted around the YED plate evenly. Using a sterile plastic spreader, the liquid was spread evenly but not towards the edges as the UV-C does not reach there. The plates were then placed in the UV-C chamber and were irradiated.
Once irradiated, the colonies on each plate of the cultures were counted and the score was recorded. Even the number of colored mutants were counted and the different types of strains were compared.
Calculation of Data
Firstly, the % survival and mutation rate was calculated by recording the number of cells from the plates. The amount obtained was divided by the total number of colonies (at 0J m-2) and then multiplied by 100 to convert it to a percentage. A two sample t test was performed. These tests are usually carried out to test whether two sets of unpaired data are different from each other. From the data collected over the past10 years, the mean and standard error for each dose of UV-C were calculated. The mean was calculated by summing up all percentages and then dividing it by the total number of values. The standard error was calculated by dividing the standard deviation of the mean by the root of total samples of observations. Once the data was obtained, the t value was calculated by using the equation: Mean(1)-Mean(2) / √((SE1)2 +(SE2) 2). The t values for each were compared to the critical t value at 5% significance level and 18 degrees of freedom. The degrees of freedom were calculated by using (N+M)-2 where N and M were 10. The critical t value came out to be 2.1.
Description of Data
By performing the t test, it was observed that between the survival rates of DO2 and DO2-Δrad9/ Δrad9 strains, there was no significance difference at any dose of UV-C but between their mutation rates, a significance difference was seen. Between the DO2 and HBO strain, there was significance at all doses but 40J m-2 which was viewed as an anomaly. For the mutation rates between the same strains, the significance level was discarded as it was lower than the critical t value of 2.1.
Presentation of Data
When analysing the class results, it was demonstrated that the DO2- Δrad9/ Δrad9 strain's survival rate was lower compared to the DO2 strain. Usually, during the cell cycle, DNA repair is coordinated by the RAD9 gene which is used as a checkpoint. The G2 phase is halted until the DNA has been repaired which allows the cell to enter the M phase of the cell cycle. (Reece, 2005). If this gene is mutated or damaged for some cause, the cell cannot undergo DNA repair and the cell continues to the M phase regardless of whether the DNA has been repaired or not. (Martini, 2006) (Smith P. J., 1999). This may explain as to why the DO2- Δrad9/ Δrad9 strain had a lower survival rate as the DO2- Δrad9/ Δrad9 strain may not have been able to produce the required enzyme by the RAD9 gene for the cell to be able to repair the DNA. Therefore all the mutations would have been left unchanged, causing a lower survival rate (Evans, 1996).
The class data also showed that the mutation rate in the DO2 was lower compared to the DO2- Δrad9/ Δrad9 strain. This may be due to the lack of the checkpoints present therefore allowing all mutations to replicate and not be fixed (Walton, 1989).
The comparison between the D02 and HB0 strain showed that the survival rate wasn't significantly different. As predicted earlier, the diploid cells would have a higher survival rate because of the fact that the cells carry a pair of chromosomes which would allow the cell to survive even if one chromosome were to be mutated (Smith S. A., 1988). This was not seen to be the case because if only one chromosome is mutated, the diploid cell cannot survive in most cases, and if it does survive, the daughter cells produced result in death. (Guthrie, 2002) The haploid cells survival rate was as predicted as cells only carry one chromosome. Therefore if the haploid cell is mutated, it has less chances of survival (Lodish, 2000).
When looking at the mutation rate between the 2 strains, it was found that the mutation rate in the diploid cells was significantly higher compared to the haploid cells (Gitsham, 2003). This observation was similar as predicted as the diploid cells contain 2 chromosomes which cause them to become more susceptible to mutations then the haploid cells which only carry one chromosome.
The ADE2 yeast genes encodes for phosphoribosylamino-imidazole-carboxylase, an enzyme in the biosynthetic pathway of adenine. Ade2 mutants, but no other ade- mutants, produce a red pigment that is apparently derived from the polymerization of the intermediate phosphoribosylamino-imidazole. The DO2 carries 2 copies of the ADE2 locus and is heterozygous (ade2/ADE2) (Jefton, 1998). Therefore, DO2 will be expected to make white colonies. HBO on the other hand, has only one copy of the ADE2 locus and therefore would also make white colonies.
Red sectors sometimes arose in white colonies due to the fact that when a cluster of yeast cells was close to undergoing mitotic division, only a few of the cells got mutated when exposed to UV-C radiation therefore producing the red colour where as the other cells which were unaffected, remained white (Smith S. A., 1988).
There were fewer number of red colonies in the HBO strain than the DO2 or the DO2 Δrad9/ Δrad9. This is because the survival rate of the HBO strain is lower than of the diploids as it only carries one chromosome which, if mutated, can be lethal.
There were certain limitations observed in the experiment which could affect the reliability and accuracy of the results obtained.
Due to the use of a Haemocytometer to count the number of cells throughout the experiment, numerous sources of error could have occurred such as non uniform suspensions, improper filling of chambers, failure to adopt a convention for counting cells in contact with boundary lines or each other and statistical errors. A more accurate way of counting cells could be done by using a Petroff Hausser counting chamber or a CD counter which are more accurate.
As the experiment was performed in pairs, two people were involved in counting the number of yeast cells. This process being quite subjective, human error could have occurred. This again could have been prevented by either making sure only one person was counting or using a Petroff Hausser counting chamber.
When UV-C light was exposed to the YED plates, the cover slips of the plates had to be removed as the light could not penetrate the cover. This could have led to contamination of the plates and affected our results.
To increase the accuracy of the results, the same experiment could be repeated again three times to get an average result for this experiment.
Further work could be done to improve the data on this experiment by repeating the experiment with other types of yeast cells or even other organisms such as amoeba or E. coli.
It would be interesting to see if UV-A and UV-B have an effect on the yeast cells as UV-C does. Also, investigating the effect on other RAD genes on the chromosomes could give us more insight as to how the cell works.
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