RUNNING HEAD: Yeast: The Properties and Genetic Mutations Within

Transcription

1 RUNNING HEAD: Yeast: The Properties and Genetic Mutations Within Yeast: The Properties and Genetic Mutations Within BIOL Indiana University of Pennsylvania Meagan Lamar

2 Yeast 2 Abstract The many experiments performed were to help identify and characterize the fungi Saccharomyces cerevisiae. Saccharomyces cerevisiae is more commonly known as bread yeast (or baking yeast). Many types of experiments were performed on this fungi including: mating, UV light radiation, and temperature changes in growth. Most of the experiments were to find if the ADE1/ADE2 gene was involved with all the genetic mutations. It is found that the ADE1/ADE2 gene was involved with the formation of mutants.

3 Yeast 3 Introduction Yeast is most commonly used with the raising of bread or other baked food items. This fungi was picked to conduct experiments on due to the fact that it makes a great genetic model. It is safely handled as it is non-pathogenic, so it should not cause any type of great illness. Another excellent quality about it is that yeast has a rapid growth rate. It doesn t take very long for yeast to grow on plates especially for experiments that should only take ~one week to be completed. They can be maintained in a stable haploid or diploid state, so it is able to be studied and experimented on easily without out much worry about too many changes occurring over a period of days. Yeast also has an easy, understandable life cycle in which scientists can manipulate to form different types of yeast just from mating two different strands. The yeast that was used in the experiments were HA0 (wild type), HB0 (wild type), HB2 (mutant), HA1 (mutant). If labeled properly, the labels should tell the different strains of Saccharomyces cerevisiae by the A or B and the number associated to that type of strain. The H represents haploid cells. The A represents the A strain while the B represents the B strain. The 0 represents a wild-type, so it has no changes. The 1 represents a mutation in the ADE1. The 2 represents a mutation in the ADE2. These labels are important in characterizing the different types of bread yeast (Bedwell, 2000). The wild-types would grow white on a MV plate while a mutant would not grow at all. A wildtype will grow white on a YED plate while a mutant would grow red on the same exact plate (Hinrichsen, Diep & Croft, 2012). Though Saccharomyces cerevisiae is a spectacular model organism for experimentation, it needs to be studied to understand how it works with its mutations. For instance, baker s yeast has an adenine biosynthesis pathway that causes different characteristics of the yeast. It helps AIR turn to CAIR and CAIR to SAICAIR. This is important for the growth of the yeast.

4 Yeast 4 Figure 1. Adenine Biosynthesis Pathway The mutations of the adenine biosynthesis normally occur between one of the two different enzymes that are used as catalysts to produce adenine in the yeast. At least, these are the two that show the differences to spot a mutant. As stated above, the mutants will grow as a red color while the wild-types will become a white color as par normal. However, if another enzyme is affected, the color will not change, so it is not as easy to distinguish mutant from wild-type with them; however, they will stop the production of adenine, and they will not grow on MV plates. The enzymes, ADE, can be manipulated for experiments to find out which enzyme is affected. The known mutants from this pathway would have to be HB1/HB2 and HA1/HA2. The A and B are different strains that can be mated together to see if the mutations can be corrected. The numbers are the true indicated since they represent either ADE1 or ADE2. These enzymes are very important in the synthesis of adenine (as are all the others); however, they are easily told apart by morphology (Hinrichsen, Diep & Croft, 2012). The use of UV radiation in the experimentation of yeast has been used throughout the time and space that was used to research yeast as a genetic model. The experiment is termed UV mutagenesis. Basically, the UV lights should be able to turn a mutant into a wild-type, and it should turn a wild-type into a mutant, in a nutshell. UV doesn t kill yeast cells right away. In fact, it could cause a mutation before even killing the cells. The mutation occurs in the sequence of the DNA. It could change, for instance, the production of the enzyme ADE1 or ADE2. Of

5 Yeast 5 course, it can affect the whole range of ADE enzymes; however, the two mentioned are the most well-known of the whole Adenine Biosynthesis pathway. Of course, these mutations only occur if low doses of UV light are shown to the yeast cells (Banas, 1999). However, UV light is deadly to yeast cells. At high doses, the DNA can become extremely damaged. Meaning, no mutation occurs, and the yeast cell does something surprising. Instead of trying to live, it will kill itself. In a process called apoptosis, the yeast cell will start to digest itself. It won t grow, but rather, it will digest itself. Apoptosis is usually used to stop cancerous cells from growing and escaping to other cells. In yeast, it is for the cells to make room for healthier, more of a chance to survive, cells (Banas, 1999). So, in the course of all this information, this process is helpful in discovering the genetic properties of the DNA genome within a Baker s yeast cell. UV radiation in experiments is extremely important in the aspect of characterizing the DNA of a mutant yeast cell and a wild-type yeast cell. In all aspects of science, the flow of scientific logic is very important. The scientific flow of logic has a truth of word to it. Using the scientific method, one is able to go about forming an experiment to test to see if a hypothesis has a truth associated with it. The scientific method is a general way for one to work out a problem. Now, it does not have to be by the book; however, the method is there to make sure that everything is full proof as best as it could possibly be. There are steps to this flow of logic. The first step would be to ask a question about something that you had observed. Now, observing is an important aspect in all of the steps since it is the way to format results. After is to form a hypothesis. Finally, it is the process of creating the experiment. The important aspect is to find out the controlled variable and the changed variable. The controlled variable shouldn t change, and the changed variable should change.

6 Yeast 6 Throughout the whole experiment, it has been found that the two enzymes that seem to change the most are the ADE1/ADE2 enzymes. It has been assumed that the two enzymes that can change the easiest into a mutant or wild-type would have to be the two enzymes that are at the very end of the adenine pathway. Through everything, the experiment seemed to be successful in the characterization of yeast in terms of genetics. Methods and Materials The materials used in the experiments were vast; however, most of them were used over and over. The strains of yeast were used constantly. The strains include the HA0 wild-type, the HB0 wild-type, the HA1 mutant, and the HB2 mutant. These strains were always used to perform all experiments. The plates that were used would be the YED, MV and YAC. The YED plate has all the nutrients for all types of strains to grow including the ones lacking processed adenine. The MV plate does not have all these nutrients, and the mutant strains will not grow on it since it does not contain adenine. The YAC plate has no nutrients, so it will starve the yeast to force them into their haploid forms to allow spores to form within the yeast. The other materials that were used were toothpicks to help spread the yeast, Bunsen burners that were used to keep contamination to a minimum, spreaders to spread yeast evenly and pipettes to help equally distribute the yeast and the water used to help spot and dilute the yeast. The first experiment was to mate the HA1 and the HB2 strains of yeast together. To accomplish this, the strains should be placed in 50 ul of water in microtubes. The yeast cells should be transmitted to the water through the use of a toothpick. After that is done, the tubes should be vortexed to mix the yeast cells thoroughly throughout the water. The mix tube should have both the HA1 and the HB2 strain. As soon as this is completed, the YED plate should be clearly labeled with HA1, HB2 and mix. The mix should be in between the two parent strains. As

7 Yeast 7 soon as the labeling is done, and the YED plate heats to room temperature, the yeast should be added to their respective label. 10 ul of the water/yeast mixture should be placed underneath the label that was written on the plate. As soon as this is completed, the strains should air dry with the lid on the plate, then it should be placed back into a 30 degree refrigerator. ~ 24 hour period, the newly formed yeast should be transferred to an MV plate. Careful observations should be taken before the move takes place. As the MV plate is warming, it should be labeled the same as the YED plate before it. As soon as all the plates are done warming to room temperature, using a toothpick, the cells should be mixed, once again, with 50 ul of water. After it has been vortexed, 10 ul of the yeast/water mixture should be transferred to the MV plate. As soon as the water dries within the plate, the plate should be placed back in the refrigerator. After a few days, the MV plate should be removed from the refrigerator. Careful observations should be taken during this time. A new YED plate should also warmed to room temperature. The mix strain should be transferred to 50 ul of water in a tube. A lot of the cells should be placed on the toothpick. Afterward, 10 ul of the mixture should be placed on the new YED plate. The HA1 and HB2 should not be needed. It can be discarded. After a few days, the yeast should be observed underneath a microscope. For the next experiment, a UV-mutant yeast stock was given to use for mutagenesis. The stock should have been used with the dilution technique. The dilution technique is to reduce the number of yeast that is present in the solution as to be able to count the yeast on the plate. The dilution technique is to take a certain amount of the stock and mix it with water. 100 ul of the diluted solution should be spread around on the two YED dates that have warmed to room temperature. These controlled plates should be placed under UV light for 0 and 10 seconds. As

8 Yeast 8 soon as this part is complete, the next part of the experiment can be continued. HA0 wild-type yeast cells should be diluted using the dilution technique. Six YED plates should be warmed to room temperature before the diluted mixtures are to be spread on the plates. 100 ul of the mixture should be spread on the YED plates. The spreader should be sterilized with each and every use during the course of preparing the plates. The plates should be labeled with their respective time for being under the UV light. The times should be: 0, 20, 25, 30, 35 and 40 seconds. After everything is set, the plates should be placed under the UV light for these specific amounts of time. After, the plates should be returned the 30 degree refrigerator. After 3-4 days, the colonies should be counted on each of the plates. Afterward, a survival curve should be created to find out the survival rate during each time under the UV light. The last experiment encompasses all the other experiments before it. The HA0 strain given by the instructor should be diluted using the dilution technique. Again, it is when a certain amount of stock is mixed with water to dilute the yeast cells, so it is easier to count the yeast on a plate once incubated. Eight YED plates should warm to room temperature. 100 ul of the dilution solution should be placed on the eight YED plates. The spreader, used to spread the yeast evenly, should be sterilized between every spread to make sure contamination is reduced to ~0. Using the survival curve found from the last experiment, the UV that gives the 10% survival rate should be used. All eight should be placed under the UV light for the time that was found from the 10% survival curve. As soon as this is completed, the same techniques should be used for the HB2 strain. However, instead of eight YED plates, only 4 YED plates should be used for this part of the experiment. Let the yeast incubate for over a period of three days. Afterward, they should be recorded to either have changed white to red or red to white.

9 Yeast 9 To characterize if the mutant changes were due to the ADE gene or not, a variety of techniques were incorporated. For one, the red yeast was placed on a YED plate to grow as was the white yeast. Both yeast were placed on a YED plate to grow, because the red colored yeast was the mutant, and the white colored yeast was the wild-type; therefore, both had to be used to conduct the rest of the experiments. When they were grown, both were mated with other strains to see if the mutation corrected itself or not. Finally, they were placed on a MV plate to see if the mated strains would survive or not. Temperature was used to see if the yeast would grow better with higher or lower heat. This was another way to determine if the change was DNA change or not. The experiment is complete after characterization of the yeast is complete. Characterization of the yeast was accomplished by utilizing every experiment that had lead up to this point except for the UV one. The yeast strains were mated to see if they would correct themselves through the mating process. They were placed on MV plates to see if they would grow or not as a sign that the mutation was corrected through the process of mating the two, separate strains. Afterward, the strains of yeast were thrown away as per protocol as they were not needed anymore for experimentation. Results The results of the experiments were to par with the predictions made by the group members. When the two strains of mutant yeast were successfully mated, the result was that the mix had turned white instead of being red like its parents. The mixture hasn t turned exactly white like the wild-type mutant should be when put on a YED plate. It is kind of a creamish/red color; however, there seems to be some change since the mix is different from the HB2 and the HA1. Picture 1, below, has shown this exact pattern.

10 Survival Rate (%) Yeast 10 Graph 1. UV Lethality Curve. UV Light Effect on Yeast Bacteria The results collected from the UV light experiment seemed to point toward the fact that UV light can infact mutatn yeast in the Time (s) opposite direction. The lethality curve that was gathered seemed to pinpoint in what way the time exposed to the yeast can affect the yeast the greatest. According to Graph 1, the lethality of the UV light is rather positive when trying to determine the survival of the yeast cells within a particular time frame. It has been concluded that the survival rate ~10% would have to be the time of 25 seconds. Placing the yeast cells under the UV light for around 25 seconds, the yeast will yield around a 10% survival rate which would be the mutants that were formed during the use of the UV light. The zero second plate had around 1,000 yeast cells. The 20 second plate had 222 yeast cells. The 25 second plate had 59 yeast cells. The 30 second plate had 12 yeast cells. The 35 second plate had 1 yeast cells. The 40 second plate had zero yeast cells. The strain shown in Picture 2 had managed to mutant some of the yeast to have them be colored red. Picture 2 shows the whole plate after being incubated after being under the UV light for 25 seconds. This results in the fact that UV light had indeed changed the wild-type into a mutant type that could potentially be related to the ADE1/ADE2 mutant change. The HB2 strain had the same exact result except in the opposite direction. The red cells were turned to white as the mutant was mutated back to the wild-type state. This has happened when the yeast cells are placed under a UV light for 25 seconds to have only the mutant cells

11 Yeast 11 survive and grow within the next couple of incubated days. Picture 4 shows the plate after it has been incubated a couple of days. The picture is to tell that the red mutants have indeed begun to mutate back to their wildtype forms. It is shown that the UV light is effected in even reversing a mutation and placing it back into the wild-type form. Though, it is unknown whether the mutation is caused by the ADE1/ADE2 missing enzyme which helps make adenine for the yeast cells. It is through multiple experiments that will allow those to characterize whether the mutations are caused by normal means or unknown means. When the two mutant strains are mated with other strains, it forms a very interesting result. The two mutants, when mated with other strains, will either correct themselves or will stay mutants in which it would suggest that the mutant is based on the ADE 1/ ADE 2 process, or if it is not corrected, the mutation is based upon something else entirely. Picture 5 seems to suggest that the mutated strains are in fact related to those of the ADE1/ADE2 enzymes. When the two mutants are mated, the result is that the mix has turned white. Obviously, they have corrected themselves to become the wild-type once again. The HA0 mutant was placed with an HB0 wild-type, and the results were a white patch of yeast which suggests that the change was not within the normal realm of ADE1/ADE2 since the patch has remained white even though the HA0 is a mutant. When a wild type and a mutant are to be mated, normally the mutant will show; however, it had not shown during this mating process. The same with the mutant HA0 being crossed with a mutant HB2; the cells had retained their white color. The HA0 mutant and the HB2 parent (not being mutated) also kept the same white color. Though if it was due to those two particular enzymes, all of them would be white.

12 Yeast 12 According to the Picture 6, the mutations have to do with other significant ideals at work. The yeast was placed on an MV plate where nutrient lacking adenine yeast should not be able to grow. However, the HA0 mutant was able to grow quite well on the MV plate; the mix was able to grow quite well on the MV plate as well. This indicates that the mutation is not within the adenine biosynthesis pathway as predicted early before the experiment. Discussion The results of the experiment have concluded that the fungi, yeast, is able to have multiple mutations, and not everything falls onto one mutation. For instance, the results of the latest experiment have proven that the adenine biosynthesis pathway has not been altered since the yeast are still white and they grow on the MV plates that were obtained. The adenine lacking yeast should not be able to grow on the MV plates as well as the mutants from the UV light experiment have done (Hinrichsen, Diep & Croft, 2012). The results mean that something else is at work within these types of mutant yeast. If the adenine pathway was not altered, then another process must be at work since the adenine is still being produced even though the white yeast is still a mutation. Other factors can be in play about these mutations instead of just one factor which is the adenine pathway that was discussed. What the mutation has done to the yeast? That is unknown at the present moment; however, it could be easily found through more experimentation. It could be found through more experimentation to see if the mutant yeast can be mated back to their original wild-type in which it can show if the adenine pathway was truly affected to see if another part of the pathway was mutated not just the last two needed to produce the adenine. The findings that were found thorough the experiments seem to suggest that another mutation has occurred rather than the ADE1/ADE 2 mutation that commonly occurs in Baker s

13 Yeast 13 Yeast. It seems to prove that there is more than meets the eye to these tiny, fungi creatures that help to make bread raise when it needs to be made. It just gives a higher opportunity for geneticists to find out more about the yeast to help understand the more complex creatures like that of the humans. The mated strands of mutants had corrected themselves since the wild-type genes had override the mutant genes. The wild-type genes are the dominant genes. When a mutant and a wild-type were mated, the offspring could have either been a mutant or a wild-type yeast; however, most had been seen as the wild-type yeast. When the UV experiment had been completed, the yeast have shown that they can survive under certain times when under a UV light. They have shown to repair their DNA to keep reproducing new cells until ~40 seconds when all the yeast had died on the plate. It was proven that the 40 seconds under the UV light had damaged the cells beyond repair. However, the ones that managed to survive at the lower times under the UV light had gone through a change. The white colored yeast had turned to red, and the red colored yeast had turned to white. The UV light had managed to mutate them from wild-type to mutant and from mutant to wild-type. Through experimentation with these changed yeast, it has given rise to the fact that it might not be the adenine enzymes that are mutated. It could be some other source; however, it could just be an enzyme higher up in the adenine pathway being affected which wouldn t turn the yeast a different color (to red), but it would still not produce adenine to survive in an MV plate. The results tie into other work by allowing them all to connect in one way. Everything single experiment that was performed was to try and show that mutations occur in yeast; however, it shows that not all mutations occur within the same concept as one another. It ties into the information in lecture that something is not always just one way. It ties in with classes like Microbiology that work on finding the different types of microorganism throughout the world.

14 Yeast 14 Finally, the work ties into other work by allowing the other work to expand from the knowledge gained from the experiments performed on this yeast. The next step would be to conduct more experiments to find the mutation that has occurred in the UV exposed yeast cells. Why did the HA0 go from white to red? Why did the HB2 go from red to white even though the mutation doesn t seem to associate with the adenine biosynthesis pathway? Well, the adenine could have been corrected; however, why did the mutants grow on an MV plate then? Adenine lacking yeast cells should not be able to grow on the MV plates since they are lacking adenine in the nutrients of the plate. More experiments can be tested to find the other mutations that can occur between the different yeast strains throughout the world.

15 Yeast 15 Picture 1. Yeast Parents and Mating Strain Picture 2. HA0 strain after being under the UV light. Picture 3. The red mutant within the sea of white wildtypes.

16 Yeast 16 Picture 4. The red mutants turning white. Picture 5. The mating crosses of the mutants with other strains. Picture 6. The MV plate for the HB0, HA0 mutant and the mix.

17 Yeast 17 Reference Page Banas, T. (1999). The Effects of Ultraviolet Radiation on Yeast. In ehow. Retrieved March 19, 2013, from Bedwell, D. M. (2000). Genetics Model System: Yeast. Retrieved March 23, 2013, from Hinrichsen, R., Diep, C., & Croft, D. (2012). Laboratory Manual: Genetics BIOL 263 (pp. 7-17). Indiana, PA: Biology Department.