November 27, 2002 Bio251 Dr. Calhoon Verification of the Principles of Genetics by Manipulating Saccharomyces Cerevisiae and Observing Meiotic Products ABSTRACT The experiment was conducted to verify the principles of genetics set forth by Mendel by using several mutated strains of the haploid organism Saccharomyces cerevisiae. Three selective mediums were used: YED, MV and YEKAC. HA1 was crossed with HB1 resulting in cream-colored diploids which illustrated dominance of the cream-colored phenotype. By isolating diploids and inducing sporulation, we were able to verify segregation by mating red unknown colonies with two known strains: HA1 and HB1 and illustrating a 1:1 ratio of a and á mating types. Independent assortment was verified by the segregation of the mating types and by colony color (cream-colored vs. red color) which also segregated equally. Particulate inheritance is illustrated by the reappearance of the red phenotype in subsequent generations.
INTRODUCTION In the mid nineteenth century, Gregor Mendel, a monk in the Czech Republic, discovered through classical genetics several principles which have become the fundamentals of genetics today (Hartl and Jones, 2001). Prior to our usage of molecular genetics, the study of hereditary was facilitated through the analysis of phenotypes of various organisms. The experiment was set forth to verify the major principles of genetics through the use of Saccharomyces cerevisiae (baker's yeast). Yeast has several advantages. It is affordable and easy to grow. Mutant strains of S. cerevisiae in the adenine biosynthetic pathway are evident through their phenotype. Mutations in the ade1 and ade2 genes of the adenine biosynthetic pathway cause red pigmentation. The pigmentation in ade2 mutants is the cause of accumulation of phosphoribosylaminoimidazole (AIR) and ade1 mutants have a red pigment because of the accumulation of phosphoribosylaminoimidazole carboxylate (CAIR). Both mutations are detectable by the different intensities of pigmentation (Chaudhuri, Ingavale and Bachhawat, 1996, 77). Mutations in trp5 in the tryptophan 2
synthesis pathway cause a cream-colored phenotype. The strains used were HBT, HA1, HB1, HA2 and HB2. Several principles are being studied in this experiment: Dominance/recessiveness, particulate inheritance, segregation and independent assortment. Several media were used: YED for growing and crossing yeast, MV for eliminating haploid or diploid mutants that cannot synthesize adenine or tryptophan, and YEKAC to induce sporulation. Cream-colored diploids were produced by crossing HBT with HA2. The cream-color phenotype is dominant over red. This master plate with two reference streaks and a diploid center colony was used in replica plating to MV and fresh YED. Diploids were isolated on MV because the haploid strains HBT and HA2 cannot grow on MV which lacks adenine as is low in tryptophan. Diploids were placed on YEKAC to induce sporulation by eliminating a carbon source. A serial dilution was done with 2 X 10 2 cells of the asci found on YEKAC. The products revealed red and white colonies. After isolating and mating red unknowns (that could be a or á mating type), segregation was verified using a chi-square test which showed that our evidence had a statistically "good fit" for segregation. Independent assortment was illustrated with the segregation of both mating type and phenotype (cream- 3
colored and red colonies). Our results showed evidence for independent assortment to take place and rejected the possibility of linkage between mating type and mutations in the adenine biosynthetic pathway (which account for the phenotype). Particulate inheritance was illustrated by segregation and independent assortment which showed the meiotic products of the diploid to have the same phenotypes as the parental strains. MATERIALS AND METHODS Yeast strains, growth mediums and growth conditions: Four mutated strains of Saccharomyces cerevisiae (baker s yeast) were used for this experiment. The following table shows the information for each particular strain of yeast: Yeast Strain Genotype Phenotype Lacking Nutrient HBT á trp5 Cream colored Tryptophan HA2 a ade2 Red Adenine HB1 á ade1 Red Adenine HA1 a ade1 Red Adenine Table 1. -Strains of S. Cerevisiae. <http://www.phys.ksu.edu/gene/g1.html> Three selective media were used. YED (Yeast-Extract Dextrose Medium) was used to grow and cross yeast because they supplemented tryptophan and adenine. MV (Minimal with 4
Vitamins) allowed us to eliminating growth of haploid mutants. MV lacks adenine and is low in other chemical ingredients so that only wildtype cells for or diploid cells can grow. YEKAC (Yeast extract with potassium acetate) is a sporulation medium which induces sporulation in diploids by depriving them of nitrogen. Mating occurs when the strains are of opposite mating type (a and á). Growth was induced by putting petri dishes in the incubator at 31º C. Similarly, growth was slowed down by refrigerating the dishes to 4º C (Johnson, 1998). Creating and isolating diploids: Mutant strains HBT and HA2 are streaked using sterile toothpicks onto YED and crossed in the center for mating. The center shall be the source for sampling to be used subsequent experiments while the single streaks are reference streaks of each strain. The YED plate is then incubated for several days. We retrieved the master plate and used it for replica plating, transferring cells from the YED to MV and again to a fresh YED as a positive control. Sporulation: Cells that grew on MV were streaked onto a fresh YED as a precaution against contamination and to allow the cells to grow healthy again. After incubation, 5
the cells were transferred to YEKAC so that they may sporulate and produce asci (if they were diploid). Analysis of meiotic products: The asci present on YEKAC were used to do a serial dilution of 2 x 10 2 cells equivalently, 200 cells in a turbid solution. The turbid mixture was poured into YED and incubated. In order to establish the mating type of the red colonies, they were streaked onto fresh YED for future use. Determination of mating type: Red unknowns were crossed with both HA1 and HB1. Those which did not mate were of the same mating type, and those which did were of opposite mating type. RESULTS Verification of mating type of strains HBT and HA2: Mutations in the adenine synthesis pathway of S. cerevisiae reveal a red pigment whose shade is defined by the place of the mutation (i.e. HB1 strains are lighter red than HA2 strains). HBT mutants are á mating type and HA2 mutants are a mating type. Thus, their progeny consists of creamcolored cells. (see Figure 1). 6
HA2 H B T White progeny Figure 1. Cross between HA2 and HBT on YED reveal white progeny. YED Isolation of diploids: Replica plating shows that cells grew in the center of MV while HBT and HA2 reference streaks did not grow. Sporulation and analysis of meiotic products. The sampling of cells taken from the center of the MV plate was induced to sporulate when plated onto YEKAC. Analysis of the cells confirmed the presence of asci (See Figure 2). Figure 2. Ascus observed with four ascospores. A serial dilution of the asci allowed us to observe a ratio of cream-colored colonies versus red colonies on YED. Cream-colored colonies were found in higher numbers than 7
red colonies. Only three red colonies were obtained from the dilution. Obtaining mating types of red colonies: Red unknowns that were crossed with HB1 and HA2 mated in a 1:1 ratio. Of the three unknowns I had available, only two were salvageable due to contamination. Of the two, one red unknown mated with HB1 and the other red unknown mated with HA1. Thus, CET1 has is a mating type and CET2 á mating type (See Figure 3). White colony CET1 CET2 HB1 HA1 Figure 3. Red unknowns mated with HB1 and HA1. White colony Number of mating type a Number of mating type á Red diploids Lost data Total 38 37 24 8 Table 2. -Total results from mating red unknowns with HA1 and HB1. 8
Chi-Square test verification. Using the Chi-Square method to test if our results statistically prove our hypothesis (or disprove it), we obtain the following results: Chi-Square value =0.0133 Critical Value (C.V.) = 3.841 Level of significance á = 0.05 Degrees of freedom = 1 Therefore, we failed to reject the null hypothesis. DISCUSSION The purpose of this experiment was to verify the principles of genetics. We were able to derive testable hypotheses by learning Mendelian genetics. Gregor Mendel was a monk at the monastery of St. Thomas, located in the Czech Republic (Hartl, 88, 2001). His experiments were conducted from 1856-1863. Through the use of pea plants and phenotypic analysis, Mendel was able to create certain laws on which geneticists have based their experimentation. There are certain terms that we have become familiar with in describing certain types of parents or progeny. 9
"Wildtype" refers to a phenotypically normal organism, whereas mutant refers to any mutation noticeable in the phenotype. A testcross is performed by crossing a heterozygote with a homozygous recessive mutant. Testcrosses allow us to determine if an organism is heterozygous or homozygous and test the ratios of segregation that Mendel discovered. Traits in a particular organism are passed down from generation to generation, although they may not be expressed in a particular generation. The theory of particulate inheritance states that genes are particles that retain their ability to be expressed even if they are not seen in a generation (Kambhampati 2002). Thus, we can expect the ascospores in yeast to be either red or white and a or á mating types without a rearrangement or blending. Mendel's first law, the Principle of Segregation, states that two alleles are equally likely to be present in each gamete. Thus, for each parent that has two copies of each allele, the offspring will have one copy from each parent. The diploids that were created in a simple cross were induced to sporulate. They formed asci that contained four ascospores. According to the principle of 10
segregation, half of the spores should be a mating type and half should be á mating type. Mendel's second law, the Principle of Independent Assortment, involves two pairs of alleles (or two different genes). Excluding the possibility of linkage in the case that two genes may be closely juxtaposed, Mendel stated that two different pairs of alleles will segregate independently of one another. The diploids that segregated for mating type should also segregate for the ade2 and trp5 genes. The possible genotypes are ade2/a, ade2/á, trp5/a and trp5/á (Hartl and Jones, 2001). When two strains of the opposite mating types a and á are mixed together on a petri dish, they produce pheromones which are able to diffuse through the agar and respond to one another. The cells stop dividing and produce pearshaped cells called shmoos. The shmoos come together at their small ends to form a zygote and the diploids begin dividing by budding once again. Due to its life cycle, yeast is easy to study because several reactions can be induced by controlling the media which it is grown on (Johnson, 1998). By analyzing the meiotic products of yeast, we can determine if the two traits (mating type and nutrient mutation) segregated independently and verify segregation 11
and independent assortment. For this experiment, the primary strains used were HA2 and HBT. First, HA2 was crossed with HBT to produce diploid cells. The diploid cells were cream-colored which demonstrates dominance and recessiveness. Although two colors represent two different genotypes, cream-colored is dominant and masks the red phenotype marked by the ade2 gene. Alleles are either dominant or recessive; in diploids, the dominant allele masks the phenotype of the recessive allele (in the case of the cream-colored diploids, red was masked because it is recessive). In haploid organisms like yeast (before they are mated), only one allele is present and that phenotype is expressed, regardless if it is dominant or recessive in a diploid organism. Diploids were confirmed by replica plating the master plate (containing two reference streaks of HBT and HA2 with a colony that was mated in the center) to MV and to YED again as a control. The only growth on MV was positioned at the same location as the center colony which was a mating between HBT and HA2. Only diploids can grow on MV because MV lacks adenine and is low in other chemical ingredients. The HA2 strain has a mutant gene ade2 which blocks the adenine synthesis pathway. HA2 mutants need a source that contains adenine to grow. HBT mutants have a 12
mutant gene trp5 which blocks the synthesis of tryptophan. MV does not contain enough tryptophan for HBT mutants to grow. Therefore, haploid mutants cannot grow on MV. Diploids and wildtype haploids can grow on MV if they can make their own adenine and tryptophan. Using the cells from YEKAC, we made a serial dilution of 200 cells/ml and poured the turbid solution into a petri dish. By spreading the solution over the agar with a cell spreader, we were also able to break up asci so that we may examine their meiotic products. Due to segregation, mating type should be found equally among the four ascospores. The products of meiosis are haploid cells. After the serial dilution of the asci revealed many more white colonies than red. There are several possibilities for why this occurred. Perhaps some asci did not break, thus giving rise to more white colonies. Some haploid cells that were in close proximity could have also mated, forming white colonies since white is dominant. White strains of opposite mating type could have also mated, forming white diploid mutants for trp5. Due to the ambiguity of white colonies, red colonies were tested to verify independent assortment. The red colonies could be either a or á mating type. In order to determine the 13
mating type of our red "unknowns," we decided to use two different strains HA1 and HB1 to mate with our red unknowns. HA1 and HB1 are both red. Prior to mating our red unknowns, we mated HA1 and HB1 with HA2 and HB2 as a control test, expecting a 1:1 ratio of red:white colonies. The white colonies represent complementation (thus, successful mating) while the red colonies are haploids that did not mate. In this control experiment, HA1 mated successfully with HB2 and HB1 mated successfully with HA2. Red unknowns were now mated with these two strains to determine the mating type. There is also the possibility of red diploids which is evident in the data. Cells of opposite mating type but the same gene (ade2) could have mated because they were in close proximity. These matings form red colonies that are mutant diploid. Red diploids were found where complementation did not occur. Out of two red unknown colonies which were used in the mating, one mated with HA1 resulting in a white diploid colony and the other mated with HB1 resulting in a white diploid colony. Thus, one red unknown was a mating type and the other á mating type. The hypothesis is that the mating type gene segregated independently from the nutrient lacking gene. Particulate inheritance is proven by the results which showed red 14
progeny. The meiotic products which were recovered had the same phenotypes as the parent strains, demonstrating how genes are carried from generation to generation without amalgamating. Using the chi-square test, we are able to determine if in fact we proved a 1:1 ratio in the progeny. If the two genes that accounted for mating type and mutation in the adenine synthesis pathway were linked, red colonies of a mating type would be expected. However, if it is proven that red colonies are half a and half á mating type, then there is evidence for independent assortment. Results from the chi-square test show that the data is a statistically "good fit." Therefore, the null hypothesis was failed to be rejected. 15