The effects of an Unknown Drug on S. Cerevisiae Strain of Yeast Cell Growth Cycles March 1, 2006 Theresa Collins Ana Caputo Scott Cathcart

Transcription

1 The effects of an Unknown Drug on S. Cerevisiae Strain of Yeast Cell Growth Cycles March 1, 2006 Theresa Collins Ana Caputo Scott Cathcart

2 Abstract: The purpose of this lab was to determine if the unknown drug, Nocodazole, had an affect on the cell growth cycle of the yeast cell S. cerevisiae and test for the viability of the cell. The drug was tested to see if it affected the cells viability and growth cycle. The different concentrations of the unknown drug were used in this experiment. It was determined that this drug would affect a cell cycle of S. cerevisiae but at which cell cycle was unknown because the drug was unknown. The cells were viewed under a microscope before and after to determine its growth stage and the percentage of cells within each cell cycle. The samples were also diluted by a factor of 10-6 and plated on YPD plates. These plates helped us evaluate if the drug affected the viability of the cells. The results yielded showed that the drug Nocodazole was not capable of killing viable cells but may have had an affect on the growth of the yeast cells in the G2/M cell cycle phase. This was determined because the amount of cells in the G2/M phase was the largest after a twohour incubation period. Introduction: The goal of this experiment was to determine if a larger concentration of the drug administered to the yeast cells, Saccharomyces Cerevisiae, would have a greater affect on the growth cycle and the viability of the cells. The different cell cycle stages are G1 which is the separate cells followed by the initiation of DNA synthesis, and then S which shows the emergence of a bud. The last stages observed were G2/M, which is the nuclear migration, the formation of a septum between cells, and followed by late nuclear division. After determining this, it would be possible to identify the unknown drug. The unknown

3 drug was Nocodazole, which is an anti- neoplastic agent, which exerts its effect by depolymerizing microtubules. i The interference with microtubules allows the cell to go into cell cycle arrest in G2/M phase of cell cycle. The microtubules can not separate appropriately if the microtubules are depolymerized. This drug would be to slow down the growth of cancerous cells. The experiment included an assessment of yeast growth through a technique of serial dilutions in which the yeast and the drug, Nocodozale, were diluted to a 10-6 dilution and YPD plates were used to test viability of the cells after inoculated with the drug. Two separate experimental drug concentrations were used, one being 1 mg/ ml and the other 5 mg/ml. These two concentrations were chosen to get a wide range of the drug. A 10 μl sample of the drug and yeast were taken initially to view under the microscope. The sample was then diluted and incubated for two hours. After the diluted samples of 1 mg/ml and 5 mg/ml were incubated for two hours, 250 μl was used to plate to test cell viability. Also a view of the cells under the microscope after incubation would help us determine the cell cycle arrest affect the drug would have on a yeast cell. 10 μl were taken from each sample and placed on a slide to view under the microscope to look at the cell cycle. These were then compared to the observations taken from pre incubation. The results from these procedures alluded to Nocodozal not being able to affect the viability of a cell but rather its cell growth. The cells did not die but the majority of them around 70-75% when viewed under a microscope showed to be in the G2/M phase. On the plates there was no significant difference between the before and after plates to determine that the drug was affective in killing viable cells.

4 Materials and Methods Two different concentrations of an unknown drug were tested to see their effects on a yeast culture of S. cerevisiae. The first desired concentration was 1 μg/μl and the second one was 5 μg/μl. Since the unknown drug provided had a concentration of 0.5mg/mL, a serial dilution was needed in order to obtain the desired concentration of drug for each sample. For the 1 μg/μl sample, 20 μl of the drug were added to 80μL of distilled (d.) water. 10 μl of this diluted sample were added to another 90 μl of d. water. Finally, 10μL of this dilution were added to 90μL of yeast. Ten μl of drug were added to 90 μl of d. water in order to prepare the 5μg/μL solution. Then 10 μl of this diluted sample were added to 90μL of yeast. In order for the diluted solutions to be homogenous, they were pipetted and discarded several times into the same tube in order to mix them. After this, a serial dilution of 10^-6 was made from each mixture with varying concentration. This was achieved by adding 10 μl of the desired mixture to a test tube and then adding 90 μl of d. water. This same procedure was repeated another five times, each time taking 10 μl of the further diluted solution and adding 90 μl of d. water. Out of the dilution for each concentration (1 μg/μl and 5 μg/μl), a 10μL sample of each was used to create a slide and were looked at under the microscope. The total number of cells were counted and it was noted how many cells were at a different stage of the cell cycle (G1 phase, S phase, or G2/M phase). 250μL were used of each concentration and plated on two different agarose plates containing all the necessary materials for the yeast to grow. The remaining sample was incubated for two hours.

5 After two hours elapsed, 10 μl of each sample were used to look at under the microscope at a magnification of 100x. Again, the total number of cells was counted and it was determined how many cells were at the various staged of the cell cycle. 250μL of each sample were plated on two other agarose plates and allowed to grow for a week along with the previously plated samples. The following week the plates were looked at and the total number of colonies in each were counted. A control sample was diluted and plated in the same way, except water, instead of drug was used because there was no drug at all in this sample. A 10 μl sample was looked at under the microscope and 50μL were plated on an agarose plate. The sample was incubated for two hours. After incubation 10μL were used to look at under the microscope. The total number of cells and how many cells were at each stage were once again counted. 250μL were plated on an agarose plate and allowed to grow until the following week. Results Dilution G1 S G2/M Cell Total 1x Pre x Post x Pre x Post Control pre Control post >200 Table 1: The total number of cells along with the number of cells at each phase of the cell cycle at a 1x and 5x concentrations before (pre) and after (post) incubation along with a control.

6 Dilution G1 (%) S (%) G2/M Cell (%) Total 1x Pre x Post x Pre x Post Control pre Control post >200 Table 2: Percent of cells along with the number of cells at each phase of the cell cycle at a 1x and 5x concentrations before (pre) and after (post) incubation along with a control. In the 1 μg/μl dilution, before incubation, a total of 28 yeast cells were seen under the microscope for the 1 μg/μl dilution. Out of these 5 were in the G1 phase (17.8%), 9 were in S phase (14%), and 14 were in G2/M phase (50%). After two hours of incubation of the 1 μg/μl dilution, a total of 10 cells were counted under the microscope, out of which 2 were in G1 (2%), 1 was in S phase (10%), and 7 were in G2/S phase. For the 5 μg/μl dilution, a total of 54 cells were seen under the microscope prior to incubation. 9 cells were in G1 phase (16.7%), 17 were in S phase (31.5%), and 28 were in G2/M phase (51.9%). After two hours of incubation, a total of 20 cells were counted under the microscope. Three were in G1 phase (15%), 2 were in S phase (10%), and 15 were in G2/M phase (75%). For the control, prior to incubation, a total of 52 cells were counted. Five of these were in G1 phase (10%), 17 were in S phase (33.3%), and 30 were in G2/M phase (57%). After incubation there were over 200 cells. These were too many cells to accurately count exactly how many were at each stage, but it was determined that around 5% were in G1 phase, 20% were in S phase and 75% were in G2/M phase.

7 Figure 1: The cell cycle stages as seen under the microscope at 100x magnification. Concentration Colonies CFU 1x pre 2 8 x xpost 2 8 x x pre x x post x 10 7 control pre 2 8 x 10 6 control post x 10 7 Table 3: Number of yeast colonies grown on plates and the number of colony forming units from the solution prior to incubation (pre) and the solution after the incubation (post) of the 1x and the 5x drug concentration along with the drugless control. After a week of incubation, the colonies on the plates were counted and the CFU were calculated based on the colonies. In the plate containing the 1x dilution prior to the 2 hour incubation 2 colonies grew. This is the equivalent of 8 x 10 6 colony forming units (CFU). In the plate with the same concentration of the drug after being incubated 2 colonies grew as well, accounting for the same number of CFUs. In the 5x concentration plate there were six colonies both before and after incubation. This was equivalent to 2.4 x 10 7 CFUs. In the control, 2 colonies grew in the plate before the solution was incubated

8 which is the same as 8 x 10 6 CFUs, and in the plate after the incubation there were nine colonies, equivalent to 3.6 x 10 7 CFU. Figure 2: The plates containing the culture of yeast. The yeast colonies are the big, yellow circles. The small, green circles are probably bacterial growth. Low concentration (μg/μl) High concentration (μg/μl) Table 3: The low concentration and high concentration of drug added to each yeast culture by each group. Each group added two different concentrations of drug to their cultures. 1 added 1 μg/μl and 5 μg/μl of the drug. two added 0.1 μg/μl and 1 μg/μl, group

9 3 added 0.5 μg/μl and 3 μg/μl, group 4 used the same concentrations as group 3, and group five added 1 μg/μl and 15 μg/μl of drug. Concentration Low Pre Post High Pre Post Table 4: Classroom results showing number of yeast colonies growing in each plate at different concentrations and the number of colony forming units. Concentration Low Pre 8.0 x x Post 8.0 x x x 10 7 High Pre 2.4 x x x Post 2.4 x x x x 10 7 Table 5: Colony forming units (I ncfus) for each group at a high concenentration and low concentrarion of drug either prior to (pre) or after incubation. Each group plated 250 μg of the two varying drug concentrations before incubating the sample, and after incubating the samples for 2 hours. 1 saw 2 colonies (8.0 x 10 6 CFUs) on the low concentration plate before incubation and 2 colonies (2.4 x 10 7 CFUs) on the same concentration after the incubation. For the high concentration, this same group saw 6 colonies (2.4 x 10 7 CFUs) for the solutions prior and after incubation. two saw no colonies (0 CFUs) for the low concentration solution either before or after incubation. For their high concentration solution, 8 colonies (3.2 x 10 7 CFUs) grew on the plate with the solution before incubation and 1 colony (4.0

10 x 10 6 CFUs) after incubation. 3 had 44 colonies (1.76 x 10 8 CFUs) growing on their low concentration/pre incubation plate and 22 colonies (8.8 x 10 7 CFUs) for their low concentration/ post incubation plate. This same group had 4 colonies (1.6 x 10 7 CFUs) on the high concentration plate/ pre incubation, and 10 colonies (4.0x 10 7 CFUs) for their post incubation plate. There were 89 colonies on the low concentration/ pre incubation plate for group 4, and 151 colonies for the high concentration/post incubation plate. For their high concentration plates, 4 had 69 colonies pre incubation and 256 colonies post incubation. 5 counted 60 colonies in their pre incubation/ low concentration plate and 104 colonies (4.2 x 10 7 CFUs) for their post incubation/ low concentration plate. For the high concentration, 5 counted 58 colonies on the plated with solution before incubation and 82 colonies (3.2 x 10 7 CFUs) for the plate with the solution after incubation. Discussion At the outset of the experiment, it was hypothesized that the drug would effect yeast cell growth by a arresting the cell cycle in one of the three phases, G 1, S, or G 2 /M, however, the drug would not be toxic to the cells.. In order to determine the initial effect of the drug on the yeast cultures, we examined samples at each drug concentration (1μ/ml and 5μ/ml) under the microscope. The data illustrated in first two charts (Tables I and II) represent the data for both the 1 and 5 μg/ml concentrations of the unknown before and after the two hour incubation period.. A sample of each culture was taken and placed on a slide to be observed under a microscope. The information obtained from the slides prepared immediately after drug administration is compared to the information obtained from the slides prepared after incubating the yeast with the drug for a two hour period.

11 From Table II one can see that about half the cells for each culture (50% and 51.9%) were in G 2 /M phases, about one-third (32.2% and 31.5%) in S and a small percentage (17.8% and 16.3%) in G 1. Both data sets were expected to be similar because the drug, at any concentration, would have had little to no effect on the culture after such a short interval after the administration. In Table II one sees an increase in the amount of cells observed to be in G 2 /M phase (70% and 75%). This information was taken after the two cultures were incubated with the 1 and 5 μg/ml concentrations of the drug. The drug is known to affect the cells by arresting their growth in a certain stage of the cell cycle. From what has been observed it is reasonable to conclude that the drug stops cell growth in G 2 /M phase. It was hypothesized that a higher the drug concentration administered to a culture of yeast, will result in a higher number of cells arrested in a certain stage of the cell cycle. In other words, the cells in the culture exposed to a higher concentration of the drug will be affected or arrested sooner. Since the two concentrations of the drug used in this experiment were 1 and 5 μg of drug per 1 ml of yeast culture, we expected to observe a higher number of cells in one stage (the arrested stage) for the 5 μg/ml concentration than the 1 μg/ml concentration. Although the charts display similar numbers, there is a slightly higher number of cells in G 2 /M phase in the 5 μg/ml culture than there are in the 1 μg/ml culture, 75% and 70%. The results up to this point support the hypothesis put forth at the beginning of the experiment. It appears that the cells in the culture subjected to a higher concentration of the drug are observed being arrested slightly more effectively in G 2 /M phase.

12 The results obtained from the second part of the experiment were contrary to those of the first. It was assumed that the higher the drug concentration that was administered to the yeast, the greater the effect, however, the number of colonies counted for the 1 μg/ml culture versus the 5 μg/ml culture contradicted this assumption. In the presence of a 5 μg/ml concentration of the drug, six colonies or CFU s were observed, while only two were counted for culture exposed to a 1 μg/ml concentration of the drug. These numbers suggest that the drug is more effective at the lower concentration. A possible reason for this may be that a high concentration of the drug results in a complete shut-down of a certain pathway. Another pathway may then be activated that accomplishes the same task. In this mechanism, the effects of the drug would be offset by the activation of this alternative pathway, explaining the higher CFU count in the 5 μg/ml culture. The plates representing the pre- and the post-two hour incubation cultures with a 1 μg/ml concentration of the drug (Figure 2) yielded the exact same results, namely two colonies on each. The same was observed for the plates of the 5 μg/ml culture, each with six colonies. The fact that, in both cases, the plates from before and after the incubation were the same leads us to believe that at the two concentrations it was administered, the drug was not lethal. After the accepted two hours required for the drug to become active, the same number of CFU s were present as there were initially. The viability of the cell was not affected by either the 1 μg/ml or 5 μg/ml concentration of the drug. If the experiment was run again, we would perform the 10-6 dilution of the yeast culture following the two hour incubation, instead of prior to it. The fact that the dilutions were performed before the incubation period may have led to skewed data. The diluted culture

13 may not have grown in the same manner as it would have if both the drug and yeast cells were present in respectively higher concentrations. This however, was unavoidable because the sample of drug we were given was not large enough to perform a pre and post incubation microscopy observation for each culture. The class data, when compiled, also proved very inconclusive. As represented in Tables 4 and 5, half the class (s 4, 5, and half of 3) observed much fewer cells before the two incubation and many more after. s 2 and 3 both had a culture in which they observed a marked decrease in the number of CFUs following the incubation period, which contradicts the others. For this reason, no definitive conclusions could be made concerning the effectiveness different drug concentrations. The data regarding the viability of the cells in the presence of different concentrations is also erroneous. s 3 and 4 both administered the drug at a concentration of.5 μg/ml, but both showed a different number of viable cells following the two hour incubation. For 3 more viable cells observed before the incubation, while 4 observed an increase in CFUs following the incubation. In the same fashion, the class was split concerning the results of the differing concentrations. s 3 and 5 showed a higher number of CFUs for the culture with lower concentration suggesting that a higher concentration if more effective. s 1, 2, and 4 counted a higher number of CFUs for the culture with higher concentration of the drug, which conversely suggests that a lower concentration is more effective. The control containing yeast in the present of no drug was consistent with the latter, with 2 CFUs prior to incubation and 9 after. This makes sense because without the drug the yeast was able to flourish and the population grew during the two hour incubation period.

14 Conclusion As previously alluded to, no definitive conclusions can be drawn from the data collected through the course of this experiment. The goal of this experiment was to observe the effects of an unknown drug on cells cycles of cultures of S. cerevisiae. It was hypothesized that the administration of the unknown drug would inhibit cell growth by arresting the cell cycle, but it would do so without proving lethal to the cells. Two concentrations were tested, 1 μg/ml and 5 μg/ml, and at both these concentrations, both the effectiveness of the drug and its toxicity level were evaluated. From the microscope observations made before and after the two hour incubation period, a slightly higher number of cells appeared to be arrested in G 2 /M phase in the 5 μg/ml culture than in the 1 μg/ml. This suggests a slightly higher effectiveness for the higher drug concentration in arresting the cell cycle in G 2 /M phase. However, upon examination of the cultures, it was found that a larger number of CFUs grew on the plate with the higher concentration of the drug, suggesting the complete opposite, that the lower concentration is in fact more effective. The class data proved just as inconclusive. Some group displayed data that supported a higher effectiveness for the lower drug concentration, while the data from others pointed toward the opposite conclusion. In the future, it would be necessary to conduct these tests with a large number of cultures so that a broader pool of data may be compiled. From this general tendencies can be observed and in turn definitive conclusions can be drawn. Also more concentrations of the drug should be tested and in greater variation. This will hopefully

15 allow us to determine the relative effectiveness s of the drug at a variety of concentration, and also, perhaps more importantly, the toxicity levels. In testing many different concentrations, we will be able to find the concentration at which the drug becomes toxic to the yeast cells. Also we would make sure that a large enough sample of the drug was obtained to allow us to perform the serial dilutions on the cultures after the two hour incubation period.

16 References 1. 1 Wikipedia: The Free Encyclopedia Accessed February 26, 2006, Nocodazole, pg Genetic laboratory Module Packets 1 and 2, Michael Piatelly Accessed February 24, 2006, pgs. 3-9.