Analyzing The Origin Of Adaptive Mutations In

Saccharomyces Cerevisiae Essay, Research Paper

Analyzing the origin of adaptive mutations in Saccharomyces cerevisiae

Abstract

Biological mutations play an integral role in the long-term survival of populations due to the potential benefit that the genetic variance provides. The main objective in this laboratory was to utilize the replica-plating technique to determine whether copper-tolerant mutants in Saccharomyces cervisiae arise spontaneously or if they are induced by their environment. This type of baker’s yeast is ideal for mutation experiments due to the specimens quick reproduction ability. Following several serial dilutions, individual yeast colonies were able to be produced on a agar medium. Through the technique of replica-plating, cells from these colonies were able to be transferred to three separate petri plates in the same spatial arrangement they were found on the original plate. One of these plates was prepared with a standard agar medium, while the other two contained a copper-based agar. The cultures were consequently refrigerated in order to prevent growth after the yeast reached 3 mm in diameter. The results of the experiment showed that the location of the mutant colonies were generally identical on all three petri plates. These findings suggest that mutations are pre-existing and occur spontaneously due to the fact that mutants of this nature would be found in positions identical with those on the first plate.

Introduction

The main objective in this laboratory was to utilize the replica-plating technique to determine whether copper-tolerant mutants in Saccharomyces cervisiae arise spontaneously or if the environment induces these mutations. This experiment is very significant because it exhibits the fact that mutations occur spontaneously at a

characteristic rate. The central focus in this laboratory was to explore the time that adaptive mutants arise in a population. If a mutation is pre-existing and occurs spontaneously, it is possible for the characteristic to be favored in an event that the environment changes. (Lab Manual; p. 27) On the other hand, induced mutations occur only after the cells are transferred to a different environment. Baker’s yeast, Saccharomyces cerevisiae, was an optimal specimen for the mutation experiment due to its quick reproductive ability. (Lab Manual; p. 28) However, due to their small size and tendency to grow in large groups, serial dilutions of yeast cells were prepared. Serial dilutions are made by adding 1 part stock solution to 99 parts of water. The goal of the technique is to obtain individual colonies on petri plates. Cells from these colonies could then be transferred to three separate petri plates in the same spatial arrangement by replica plating. In this procedure, a sterile velvet surface is utilized to obtain cells from colonies on an original plate that are then consequently transferred onto different growth mediums. (Hartl, Jones; p.561) One of the plates prepared in the experiment was a standard agar medium (control), while the other two contained a copper-based agar. The replica-plating technique plays an integral role in the analysis of the time that copper-tolerant mutants in yeast arise. If the mutants are pre-existing, their cells will be transferred onto the three replica plates. As a result, the location of the mutant colonies on the replica plates must be identical. Conversely, if the mutants were induced by their environment, the source of the mutation should not be found on the original replica plate.

Their colonies would be located on different parts of the replica plates. Following the results, a chi-square analysis can be performed to assess the goodness of a fit between a set of observed numbers and the theoretically expected numbers. (Hartl, Jones; p. 109)

Materials and Methods

The experiment follows the procedure described on pages 27-36 of the laboratory manual. The methods were as described except for the following deviations from the manual: 1) Three plates were prepared after the original yeast plate was produced. One of these plates served as the control and was prepared with a standard agar medium, while the other two contained a copper-based agar. 2) 1 L of the copper medium was prepared. 3) 500 mL of the original medium was prepared

Results

Table 1 exhibits the probabilities of different chi-square values for degrees of freedom from 1-50 when the expected hypothesis is true.

Table 2 shows the results obtained when cells from the original colonies were able to be transferred to three separate petri plates in the same spatial arrangement they were found on the original plate. Of the three plates that were analyzed, the primary control plate possessed all of the colonies found on the original plate. The + Copper I plate failed to produce any colonies at location 8, 12, and 20. Similarly, the + Copper II plate failed to produce any colonies at location 6, 16, and 21. The rest of the colonies (1-30) were visibly distinguished and were found in the same spatial arrangement in all three plates.

Figure 1 illustrates the observations of three plates (+ Cu I, + Cu II, Primary plate) after the yeast cells were incubated and consequently refrigerated in order to prevent the cells to growth past 3 mm in diameter. All the plates possessed a crowding group of cells toward the middle of the plate. Furthermore, each plate had several individual colonies circling the mass. The plates showed that their was significant similarity in location of the individual colonies.

Supplemental Calculations:

X2 = ? (O-E)2 O= Observed number in class

E E= Expected number in class

X^2 = (24-30) = -0.2

30

Degrees of Freedom=

Probability Value=

Discussion

Based on the results of the experiment, mutations are pre-existing and occur spontaneously in the environment. In the experiment, three plates were prepared utilizing two different types of growth medium. The first plate consisted solely of a standard agar medium. This plate served as the control due to the fact that the experiment called for its comparison to the two copper-based plates. Furthermore, the first plate exemplified the non-selective medium, whereas the copper containing agar plates served as the selective medium.

When the three plates were incubated and consequently refrigerated to prevent further growth, observations were able to be made concerning the origin of adaptive mutants. The cells of the yeast culture that was produced on the master plate were replica plated onto a non-selective medium then transferred to the two copper-based plates. All four plates possessed a crowded mass of yeast cells in the middle their respective plate. (Refer to Figure 1) Several possible explanations for this occurrence can account for this observation. Firstly, it is possible that not enough serial dilutions were performed in order to dilute the concentration of yeast cells. Furthermore, the yeast cells may have not been evenly distributed over the master plate. Lastly, the cells may have been incubated for a prolonged period of time. Aside from the mass of crowded cells in the center of the plates, individual colonies of cells were observed. Their location on each plate correlated with their respective locations on other plates. This result suggests that the mutation existed prior to the cell’s exposure to the copper medium. The pre-existing mutation was favored by selection and the new variation tended to dominate. This genetic variation did not exist on all the yeast cells. There were however, six colonies that were not present on all three mediums. (Please refer to Table 2) It is possible in these cases that there was an incomplete transfer of cells or contamination was present. Furthermore, it is also probable that a small number of the mutants have mutations that randomly occurred during the growth of the transferred colonies and advocated growth on copper.

Some of the data obtained in this experiment contradicted the expected results. There should have been approximately ten preexisting mutants on each of the replica plates. However, 24 colonies were found in identical locations on their respective plates. A possible explanation for this occurrence may be traced to the agar medium itself. If the copper based agar medium was not concentrated enough, a significant number of cells may still be able to reproduce and grow. As a result, similar colonies may be regarded as possessing the genetic variation. This can account for the high number of individual colonies. However, most of the results that were obtained in this experiment coincided with the expected results.

The chi-square analysis would not be a very effective in estimating the frequency of an observed deviation because there wasn’t enough colonies to make a generalization. Thus the probability value obtained in the results section, is an inaccurate representation of the frequency of the observed deviation.

Conclusion

The main objective in this laboratory was to utilize the replica-plating technique to determine whether copper-tolerant mutants in Saccharomyces cervisiae arise spontaneously or if they are induced by their environment. Furthermore, another aims of the lab included the ability to identify mutant cells and make bacterial plates and cultures. The results of the experiment show that mutations in cells are pre-existing and occur spontaneously in an environment due to the fact that mutants were found to be in positions identical with those on the first plate. Furthermore, the experiment exhibited that replica-plating is an effective technique in testing when adaptive mutants arise in populations. Other experiments that can be performed in the future for further comprehension of the origin of mutations include: exposing the yeast cells to different environments in order to observe if other similar pre-existing mutations exist and subjecting different types of cells to various environments in order to test the similarity between specimens. The experiment was effective in providing a good foundation for learning and observing the origin of adaptive mutants.