- June 11, 2020
Thisexperiment aims to determine which genes from the adeninebiosynthetic pathways in the many anonymous yeast haploid strains aremutant. This research will specifically study the mutation pathway ofthe brewer’s yeast Saccharomycescerevisiae.In this experiment, the selection of mutagens will be made keenly tokeep the rates of mutation as low as possible because the majority ofmutations are deleterious (Lang, 2007). Despite the deleteriouseffect of the mutation, strains that possess high rates of mutationin a population can out-do strains containing a wild-type rate ofmutation (Botstein & Fink, 2011).
Thisstudy will follow the kinetics of mutagenesis and separate a group ofmutants having a specific phenotype. However, due to its small size,yeast can reproduce both sexually and asexually (Reed, 2012).Additionally, mutant yeast can easily be separated since yeast mightbe grown as a haploid (Kurtzman, Fell, Boekhout, & Robert, 2011).Notably, by mating the mutant yeasts, this experiment will perform agenetic analysis of the mutation. It is also crucial to note thatSaccharomycescerevisiaehas a complete sexual life cycle with the major stages represented bycells of definitely differentiated shapes (Botstein & Fink,2011). In order to understand the mutation of yeast, it is criticalto comprehend the haploid and diploid stages. These phases are stablewhile the transition from one phase to another is controllable by theenvironmental circumstances (Reed, 2012).
Thehaploid cell comprises one set of the 17 chromosomes noted by IN,while the diploid contains a pair of the 17 chromosomes denoted by2N. The haploid cell reproduces through budding a process that usesmitosis as the only source of reproduction and usually occurs quicklyas the typical S.cerevisiaehaploid cell disintegrates after every 40 minutes (Lang, 2007). Thediploids are created in the case where 2 haploids are mated. Again,the haploid only carries one copy of each gene it needs to survive.Typically, there are two kinds of mating haploid cells, namely Mat atype and Mat α. It is also important to note that identical yeasttypes are not capable of mating (Reed, 2012). However, when twodifferent cells come into contact with one another, fusion willeventually occur to create one cell with a complete chromosomes ofthe initial cells, thus, the 2N chromosomal notation. The resultantdiploids can either divide through budding or form spore consistingfour new haploid yeast when exposed to harsh conditions.
Sincethe yeast strain S.cerevisiaepossess both stable haploid and diploid states, the recessivemutations can easily be separated and manifested in haploid strains,and allow complementation tests to be performed in diploid strains(Botstein & Fink, 2011). In this experiment, an ultraviolet lightmutagen will be used to induce mutations that are not specificthroughout the genome of the yeast (Reed, 2012). Induced mutationsare believed to occur due to enzymatic processes that use DNA damageas a substrate (Lang, 2007). It is also critical to note that yeastscan either be planted onto a solid media made of agar on a plate orin liquescent media. Again, because yeasts have a wide range ofgrowth temperatures that can go as low as 120Cor as high as 420C,the experiment can be performed under a wide range of conditions(Botstein & Fink, 2011). The experiment will begin with an ade-2mutant that has a mutation in a gene which encodes enzymes needed foradenine formation. The outcome of this mutation is an adenineauxotrophy,implying that the yeast strain cannot live in the absence of adeninethroughout its growth pathway. Similarly, the ade-2leads to a clear observable phenotype, since the defects in thebiosynthesis of adenine results in the accumulation of a pathwayintermediate, which turns into a red pigment (Kurtzman, Fell,Boekhout, & Robert, 2011). To know whether a mutation hasoccurred, one simply needs to observe a red coloration on the mutantcolonies reared in a medium with adenine. The wild yeast cell typeswill remain white.
Thematerials required in this experiment include four major mutantstrains D, K, M and CC of mating type a and other mutants whichincluded I, S, A, E, L, U, V, W, Z, AA, B, N, O, X, G, Y, H, R, T, C,F, and BB as well as other mutant strains 1, 31, 3, 6 , 12, 11, 18,20, 13, 22, 25, 30, 7, 32, 17, 24, 27, 29, 4, 8, 9, 14, 5, 15, 16,23, 28, 21, and 26 of mating type α. Other required materials are 2YPD agar plates (1YPD and 2YPD), sterile toothpicks, and a 30oCincubator. After obtaining all the relevant materials, make labels onthe YPD plates with name, date, mating type and strain number orletter. Place an agar plate and streak the four mutant strains ofsimilar mating type along the lines across the agar plate. Atoothpick is used to pick the strains into the master plate. Again,prepare a similar agar plate with the second group of the fourmutants and incubate both the plates at 300Cfor one day. At this stage, a UV light is used to induce mutations inyeast cells that are plated onto the agar plates (Kurtzman, Fell,Boekhout, & Robert, 2011). The YPD plate represent ample mediaplate that offer the yeasts with the necessary nutrients they need tosurvive irrespective of any mutation in the pathway of biosynthesis.Similarly, in this experiment, selective media is used as a tool foridentification of mutant phenotype in the genetic screening. Further,the mutants will be selected on the basis of a positive selectionscheme that reveals the existence of white diploids. Again, thisstudy requires large number of cells to assist in unveiling all thegenes involved in the phenotype.
Usinga sterilized toothpick, obtain little amounts of the strain Aprepared from one of the plates prepared and slur it on the circleapproximately the magnitude of a dime onto YPD plate. The next stepis to pick another toothpick and obtain little amounts of the 1strain and once again, slur it onto the original slur made earlier.Mix the two strains together into one patch for them to mate. Thisprocedure is repeated for the strains B, C, and D each mated witheach of mutants 1, 2, 3, and 4 using the other 2YPD plate. Theseplates are saved, and the results are sixteen patches on the plate,with each patch being a representative test cross that is exposed toUV light for 15 seconds. Afterward, these plates are then incubatedthroughout the night at 30oC to prevent photo-repair. The process isrepeated using the other two test cross until 3 YPD plates areobtained, with each being a representative of one test cross. Again,the plates are incubated, but this time for two days at 30oC.
Aftertwo days, the isolated colonies should be observable inside thestreaks. The colonies’ colors should be noted and verify whetherthey have changed from the initial strains (Kurtzman, Fell, Boekhout,& Robert, 2011). Save the plates. Now, get 4-Adeplates. Within 3 of these plates, segment into fourths and put labelscorresponding to test crosses that were conducted earlier. Forinstance, on a particular 1YPD plate, label A-1. The second YPD platelabel B-1 and the rest label in a similar manner. It is important tokeep in mind that the plates do not contain adenine in their media.Thus, any yeast which is grown on the plates should acquire their ownadenine through the pathways illustrated earlier. Finally, smear thedistinct colonies onto the -Adeplate found on the test cross YPD streak plates. Streak threecolonies with white coloration and one red colony from each plate,unless there are only colonies made of only one color, that is, redcolor only. Again, these plates need to be incubated for two days at300C.Each cross is given a score for the existence of growth or without. Once the mutants have been isolated, figure out if the identifiedtrait is dominant or recessive. Mate each mutant with a wild-typecell.
Afterthe mutagenesis, eight colonies were observed. Similarly, after thescreening process, the entire eight streaks on both the 1YPD and the1YPD plates. The eight streaks on the 1YPD plate were conspicuous,even though streaks two and four were the most visible streaks on the1YPD plate. The resulting colonies from the yeast crosses had whitecoloration, although there existed some crosses that did not havecolonies. These were marked as unknown phenotype. The cross with A,1a yeast and 4, Aα crossed with B, 2a yeast did not yield colonies.Again, the plates with A, B, C, D, M, AA, and BB 2a yeast had beencontaminated. The diploid streaks had many crosses that indicatedcomplementation, no complementation, no growth, or unanimous growth.The crosses B-1, B-4, C-4, D-1, D-2, D-3, and D-4 also did notindicate complementation. On the other hand, there was nocomplementation seen with A-4, B-3, C-3, D-3, D-4, E-4. The unknownphenotype were observed with A-1, and A-3. Further there were no anygrowth in A-2, B-2, C-1, C-2, D-1, D-2, and all AA, and BB crosses.
Afterconsolidating all class data and rearranging them, the potentialrevertants included (D, K, M, CC). The potential complementationgroups included (1, 31, I, S), (2, 6, 12, 13, 18, 22, 25, 30, 32, A,E, L, U, V, W, Z, AA), 3, 17, 24, 27, 29, B, N, O, X), (4, 8, 9, 14,19, G, U), AND (21, 26, BB). From the experiment above, four whitecolonies were obtained after the process of mutagenesis had occurred.Thus, the haploids did not mate. It is also observable that thevariation in the ade-2 genes makes its gene products non-functional,thus leading to the accumulation of a byproduct, which is has aunique red color (Lang, 2007). Further, it is also noted that a whitestrain from the ade-2 mutant bears a mutation in a gene which had itsproducts involved in the preliminary stage of the adeninebiosynthetic pathway, such as the ade-7, which would further deterthe creation of molecule “Y” preventing the red byproducts not tomount up. The complementation process can be summarized in the tablebelow:
Figure1: RawClass Complementation for Diploid mutants.
Themethod that was established to select the yeast mutants that aredefective in the UV-induced mutation was useful since it does notincorporate the presumption that all such mutations are sensitive toUV light (Lang, 2007). Hence, the mutants that are extracted from theuse of this method helps in answering the questions like a) Are allmutations defective on UV light mutagenesis often accompanied by arise in UV light sensitivity? b) Is it that such mutants aresensitive to other forms of mutagenic agents? c) Is it thatUV-induced mutations controlled by a wide or a limited number ofgenes? From the above complementation test, one would determine thewhite strains that carry a mutation in a known gene whose product hadfunctioned earlier than the ade-2 in the adenine synthesis.
Theobservations above imply that the UV-induced mutations in yeast aregenerated by the pathways, which have shared phases with pathwaysthat are capable of repairing lethal UV damages (Lang, 2007). In theexperiment, four non-reversion mutants were created to represent onlyone gene. This observation indicates that yeast UV mutability iscontrollable by a small number of genes. It can also be deduced thatmajority of the UV-sensitive yeast mutations either do not affect ormaybe improve the UV mutability, while only a small number tend tolower the UV mutations (Kurtzman, Fell, Boekhout, & Robert,2011). The yeast test cross carried out was useful as it helped inthe determination of the unknown mutations after the mating of thehaploid yeast to form the diploids (Kurtzman, Fell, Boekhout, &Robert, 2011).
Inconclusion, the test showed that the yeast strain containing theade-2 mutation, which has the effect of disrupting the adeninebiosynthetic pathways are not capable of growing on a media thatlacks adenine. Similarly, mating haploids of a different geneticcomposition will result into wild-type yeast that has both the genesand thus, completing the biosynthetic pathway (Reed, 2012). However,mating haploid mutants of the same kind such as ade-2mutants would not result to an ade-2prototroph since the biosynthetic pathways would still be obstructed.Finally, the experiment revealed that mutagenesis is a random processand that majority of the mutations could have not effects on thephenotype under screening.
Botstein,D., & Fink, G. R. (2011). Yeast: an experimental organism for21st Century biology. Genetics, 189(3),695-704.
Kurtzman,C. P., Fell, J. W., Boekhout, T., & Robert, V. (2011). Methodsfor isolation, phenotypic characterization and maintenance ofyeasts. Theyeasts, a taxonomic study, 5th edn. Elsevier, Amsterdam,87-110.
Lang,G. I. (2007). Mutationrate variation in the yeast, Saccharomyces cerevisiae.Harvard University.
Reed,G. (Ed.). (2012). Yeasttechnology.Springer Science & Business Media.