Matt Markham Brian Nowlin T,Th 12-3 Conservation of MET3 between Saccharomyces cerevisiae and Schizosaccharomyces pombe in the

methionine biosynthesis pathway Abstract This studys goal was to determine the evolutionary conservation of MET3 between Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast in the phylum Ascomycota. S.cerevisiae met3 mutants were initially identified by growth on various sulfur sources. Once YMP 12 was tentatively identified as the S.cerevisiae met3 mutant, a PCR was run, which confirmed this assertion. A restriction digest with ScaI was used to identify yeast overexpression plasmids pBG1805-GAL1 : MET3, pYES2.1-GAL1 : Met3, and pYES2.1-GAL1 : lacZ, which transformed YMP 12 yeast. Following transformation, Met3p presence was confirmed with a Western blot, preceded by SDS-PAGE, which checked successful protein extraction. Once Met3p fusion protein expression was confirmed, yeast transformants were replica plated on glucose and galactose to test functional complementation. Expression of the S.pombe Met3 gene restored the wild type phenotype in S.cerevisiae met3 auxotrophs, suggesting that the MET3 gene was evolutionarily conserved between S.cerevisiae and S.pombe.

Introduction
S.cerevisiae and S.pombe yeast are believed to have diverged from a common ancestor within the phylum Ascomycota; our project goal was to determine the evolutionary conservation of MET3 across these two species. Individual open reading frames (ORFs) were replaced with the KANR cassette in the Saccharomyces Genome Deletion Project, following the Yeast Genome Project, in order to determine gene function (Winzeler et al., 1999). Although many genes were identified, the evolutionary conservation of gene function across different yeast species remains unclear. Distinct MET genes were deleted in S.cerevisiae yeast and the met auxotrophs were grown on various sulfur sources to determine the MET gene product and the location of the gene in the methionine synthesis pathway. Our gene of interest, MET3, was determined to encode a gene product involved with sulfate activation (Masselot & de Robichon-Szulmajster, 1975). S.cerevisiae met3 auxotrophs were unable to grow with extracellular sulfate, but could grow with 5-adenylysulfate (APS) and 3phospho-5-adenylysulfate (PAPS), suggesting a mutation upstream of MET14. The MET3 gene was eventually found to encode ATP sulfurylase (ATPS), which is involved in sulfate activation. S.cerevisiae met3 auxotrophs were able grow with an input of inorganic compounds such as sulfite and sulfide, suggesting that ATPS is involved in the processing of sulfate (Thomas & Surdin-Kerjan, 1997). S.cerevisiae enzyme ATPS and S.pombe enzyme, take inorganic sulfate and ATP as substrates and catalyze the production of adenosine 5phosphosulfate and diphosphate (Schiff & Hodson, 1973). ATPS is a hexameric homo-oligomer, consisting of six identical subunits arranged in two stacked rings in a symmetric assembly. This enzyme is encoded by the MET3 gene on chromosome X, and has no apparent cofactors. Highly conserved domains across S.cerevisiae and S.pombe have been identified in ATP sulfurylase such as the GRD-loop, the VGRDHAG module, which is involved in substrate binding, and the RNP-loop (Ullrich et al., 2001). The sC gene of the eukaryotic A.fumigatus fungi, which encodes for ATP sulfurylase, was

sequenced and compared to similar versions of the same protein in eukaryotes and prokaryotes. The gene product amino acid sequence matched 59% of the S.cerevisiae equivalent, 55% of the S.pombe equivalent, and 43% of the closest prokaryote, the Aquifex aeolicus bacteria (De Lucas et al., 2001). These conserved domains contain blocks of amino acids that participate in the binding of MgATP2 and SO4 (Jaramillo et al., 2012). ATP sulfurylase was also found to be ubiquitous among eukaryotes, with minor differences, and in some lineages this enzyme is fused with APS kinase into the PAPS synthetase protein. This fusion was likely the result of a merging in ancestral eukaryotes proteins, which is supported by the fusion of this protein in diverse lineages (Patron et al., 2008). In this study, met3 auxotrophs were identified through selective plating on various sulfur sources and PCR. Restriction enzyme digests were used to identify yeast overexpression plasmids, which all contained the galactose-inducible promoter, GAL1. These overexpression plasmids contained the positive control, S.cerevisiaes MET3, the negative control, E.colis lacZ, or the experimental gene, S.pombes Met3. Once identified, each plasmid was used to transform BY4742-derived S.cerevisiae met3 auxotrophs (WT), which were grown in glucose or galactose to repress or induce the GAL1 promoter. To determine protein expression, SDS-PAGE was used to analyze total protein extracts prepared from transformant yeast. A western blot was carried out to analyze Met3p protein expression in yeast transformed with overexpression plasmids. Once Met3p expression was determined, transformants were replica plated on inducing (galactose) or repressive (glucose) media to determine functional complementation. This study suggests that YMP 12 is the met3 mutant and that the S.pombe Met3p is a functional homolog of S.cereivisae Met3p. This result suggests that MET3 was evolutionarily conserved between S.cerevisiae and S.pombe.

Materials and Methods

Microorganisms and culture conditions The yeast strains and plasmids used in this study are listed in Table 1. All auxotrophic strains were obtained from the Yeast Genome Deletion Project, and maintained on YPD media plates prior to this study (Winzeler et al., 1999). Yeast strains were transformed with plasmids using the LiAc/SS carrier DNA/PEG method as previously described by Geitz & Schiestl (OConnor). Yeast complete media (YC) + raffinose uracil (ura) was used to select for transformants, which were replica plated on YC + methionine (met) + glucose (glu) or galactose (gal) to test functional complementation.

Table 1. Strains and plasmids used in this study

Strains or plasmids BY4742met3 BY4742met2 BY4742met17 pBG1805-GAL1 : MET3 pYES2.1-GAL1 : Met3 pYES2.1-GAL1 : lacZ Genotypes or constructs

Mat his31 leu20 ura30 met3::KANR Mat his31 leu20 ura30 met2::KANR Mat his31 leu20 ura30 met17::KANR
Yeast overexpression plasmids, URA3, MET3* Yeast overexpression plasmids, URA3, Met3* Yeast overexpression plasmids, URA3, lacZ*

*Each of these genes was under the control of the galactose-inducible system of yeast overexpression plasmids GAL1 promoter. The URA3 gene served as a selectable marker.

Identification of met auxotroph strains S.cereivisiae met2, met3, met17, and WT yeast were each spot plated onto four minimal media plates with the addition or absence of a sulfur source: +met, - met, +cysteine (cys), + sulfate (SO3). Mutants were also grown on a separate YPD plate as a positive control. These plates were then incubated at 30 C for 72 hours (OConnor, 2012).

Yeast colony PCR PCR was carried out on genomic DNA (gDNA) from YMP 12, 19, and 24. The forward primers used in each reaction were specific for native or recombinant MET genes. In each reaction, the primer mixture contained 0.5M of KAN primer B as reverse primer, and 0.5M of MET2 primer A, MET3 primer A, or MET17 primer A as forward primer. The PCR conditions were: 2 min at 95C, (30s at 55C, 30s at 72C, and 1 min at 72C) x 35, and 10 min at 72C. Amplified gDNA was loaded with 6x bromophenol blue dye and resolved on a 1.25% agarose gel in TAE buffer for 30 minutes at 120V. The amplified gDNA was then stained with 0.0005mg/mL ethidium bromide (EtBr), and visualized with a UV transilluminator (OConnor, 2012). Restriction digest of overexpression plasmids Overexpression plasmids were isolated from three different E.coli bacterial cultures using a standard alkaline lysis method; only 50L of elution buffer was used to elute plasmid DNA. The A260 of each plasmid strains DNA was measured using a NanodropTM spectrophotometer (OConnor, 2012). 0.5g of each plasmid was digested with 1.0 U of ScaI and NEBuffer2; digested sample mixtures were incubated at 37 C for 2 hours. Plasmid and a second reaction mixture containing the undigested plasmid were resolved on a 1% agarose gel in TAE buffer for 30 minutes at 123V. This gel was washed with deionized water, stained with 0.0005mg/mL ethidium bromide, and visualized with a UV transilluminator (OConnor, 2012). SDS-PAGE and western blot analysis of cell extracts YMP12 yeast was transformed with the identified plasmids, pBG1805-GAL1 : MET3,
pYES2.1-GAL1 : Met3, or pYES2.1-GAL1 : lacZ, in a transformation master mix: 382mM

lithium acetate, sterile 38.15% PEG-3350, 0.8% 2-mercapoethanol, 0.03mg salmon sperm DNA, and 0.4g miniprep plasmid DNA (OConnor). Three samples of liquid YC

media + raffinose ura were each inoculated with a culture of S.cerevisiae YMP 12 yeast to select for transformants. Yeast were incubated at 30C, while rotated, for two days. Each yeast sample was then split equally between YC +glu+metura and YC+glu+met ura. Equal volumes of each of the six samples were removed, and the OD600 of the transformed cells was determined with a spectrophotometer (OConnor). Yeast and media samples were incubated at 30C while rotated, four hours prior to the following procedure. Yeast were pelleted, treated with 0.2M NaOH, and incubated at room temperature for 5 minutes. Transformant YMP 12 yeast cells were pelleted again, lysed in 2X SDS-PAGE loading buffer (120 mM Tris/HCL, 10% glycerol, 4% SDS, 8% 2mercapethanol, and 0.004% bromophenol blue). Total cell lysates were resolved on a 12% SDS-PAGE polyacrylamide gel at 203V for 1 hour with a Precision Plus Protein Standard Bio Rad Kaleidoscope as a standard. The gel was stained with Simply Blue and rocked overnight. A second 12% SDS-PAGE polyacrylamide gel was made for a western blot, and samples from this gel were transferred onto a polyvinylidene diflouride (PVDF) membrane at 127V for 30 minutes. The PVDF membrane was blocked with 5% nonfat milk in TBS-T, and incubated at 4 C for 24 hours. The PVDF membrane was rinsed with TBS-T buffer and probed with primary monoclonal antibodies specific for the V5 epitope at 4 C for 24 hours. Primary antibodies were detected with horseradish-peroxidase (HRP) conjugated, rabbit anti-mouse, polyclonal secondary antibodies, and visualized with 3,3'5,5'-tetramethyl benzidine (TMB) as the chromogen (OConnor, 2012). Replica plating of transformed strains Transformed YMP 12 yeast was plated on yeast complete (YC) media +glu +met ura, incubated at 30 C for 2 days, and spot plated on YPD media (Amberg et al., 2005).

Finally, transformed yeast was replica plated on YC +met ura +glu, YC +gal ura met, and YC +glu ura met, for a total of nine replica plates. These plates were incubated at 30 C for 2 days (OConnor, 2012). This experiment was repeated, but YMP 12 yeast was transformed with pYES2.1-GAL1 : Met8 instead of pYES2.1-GAL1 : lacZ as a negative control. Transformants were each spot plated onto YC+metura+glu, YCmetura+glu, YCmetura+gal, and BiGGY agar media (OConnor, 2012). Again, transformant cultures were incubated at 30C for 2 days. Results In order to determine the conservation of MET3 between S.cerevisiae and S.pombe, met3 auxotrophs were first identified in a mutant screen. S.cerevisiae met mutants were grown on various sulfur sources to reveal the location of the MET gene mutation in the methionine biosynthesis pathway (Fig. 1). Auxotrophs were tentatively identified according to their ability to grow on different sulfur sources (Table 2). The +met and YPD media were used as a positive control, which supplied all the nutrients met3 mutants needed to display the wild type growth phenotype. The met media served as the negative control, while +cys and +SO3 were the experimental media. YMP 12 was identified as the met3 mutant because it grew with cysteine, but not sulfite, suggesting a mutation upstream of the sulfite reduction pathway. YMP 19 and YMP 24 were assigned met17 and met2, respectively, because both were unable to grow with the addition of cysteine or sulfite. To clarify mutant identities, a PCR was run, and reaction products were visualized with a 1.25% agarose gel (Fig. 2). YMP 24 was amplified with MET2 Primer A as forward primer, and KAN Primer B as reverse primer, which formed a 524bp amplicon. MET17 Primer A as forward primer and KAN Primer B as reverse

primer were used as the control group for YMP24. YMP12 was amplified with MET3 Primer A as forward primer and KAN Primer B as reverse primer, which formed a 604bp amplicon. MET2 Primer A as forward primer and KAN Primer B as reverse primer were used as the control group for YMP12. YMP 19 was amplified with MET17 Primer A as forward primer and KAN Primer B as reverse primer, which formed a 481bp amplicon. MET3 Primer A as forward primer and KAN Primer B as reverse primer were used as the control group for YMP 19. In preparation for complementation analysis, yeast overexpression plasmids were digested with ScaI and resolved on a 1% agarose gel (Fig. 3). Plasmid 4 was digested with ScaI, and formed 4500bp and 850bp restriction fragments that approximately matched those expected from pYES2.1-GAL1 : lacZ (Table 3). Plasmid 14 undigested ran as a negative control for plasmid 14 digested, and produced a restriction fragment with a size of 3700bp. Plasmid 10 was digested with ScaI, and formed 3500bp and 830bp restriction fragments that approximately matched those expected from pYES2.1-GAL1 : Met3. Plasmid 10 undigested ran as a negative control for plasmid 10 digested, and produced a restriction fragment with a size of 3530bp. Plasmid 14 was digested with ScaI, and formed 2500bp and 1500bp restriction fragments that approximately matched those expected from pBG1805-GAL1 : MET3 (Table 2). Plasmid 14 undigested ran as a negative control for plasmid 14 digested, and produced a restriction fragment with a size of 3600bp. Once identified, transformant yeast was selected for in uracil-deficient media. The OD600 of transformants was measured and yeast were grown with galactose or glucose to induce or repress, respectively, the GAL1 promoter. Total cell lysates from yeast were loaded into a 12% SDS-PAGE gel, and stained with Simply Blue to visualize

protein extracts (Fig. 4). A low total protein complementation, ranging in size from 25kD to 250kD, was observed from met3 auxotrophs transformed with pBG1805-GAL1 : MET3, grown in gal. This low total protein concentration was the result of a low initial cell density, revealed by a low OD600 of 0.037A (Table 4). High total protein concentration was observed from met3 mutants transformed with pYES2.1-GAL1 : Met3, grown in glu. A total protein concentration was observed from the same mutants, grown in gal. The latter yeast had a lower OD600 of 0.046A relative to the yeast grown in glu, which had an OD600 of 0.052A. A high total protein concentration was observed from met3 mutants transformed with the negative control, pYES2.1-GAL1 : lacZ, grown in glu. A lower total protein concentration was observed from the same mutants, grown in gal. The latter yeast had a lower OD600 of 0.046A relative to the former grown in glu, which had an OD600 of 0.052A. Once protein expression was confirmed, a Western blot was used to determine if Met3p was expressed from transformant yeast (Fig. 5). Protein was observed from yeast transformed with pYES2.1-GAL1 : lacZ, with sizes of 37kD and 140kD, in both glu and gal. In addition to this nonspecific protein, a 55kD protein was observed from yeast transformed with pYES2.1-GAL1 : Met3, which approximately matched the expected size of S.pombes Met3p, 65kD (Table 5). Nonspecific protein, with a size of 140kD, was observed from the same transformants grown glucose. Protein was observed from yeast transformed with pBG1805-GAL1 : MET3, with sizes of 37kD and 140kD, in both glu and gal. Also, protein was observed with an approximate size of 75kD in these transformants, which approximately matched the expected size of S.cerevisiaes Met3p, 77kD.

After Met3p expression was confirmed, complementation was tested (Fig. 6). All transformants were grown on YC + glu + met ura as an inductive (positive) control, YC + glu met ura as a repressive (negative) control, and YC +gal met ura as the experimental condition. Yeast transformed with pYES2.1-GAL1 : lacZ only grew on YC +glu +met ura. Yeast transformed with pYES2.1-GAL1 : Met3 grew on both YC +glu +met ura and YC +gal met ura, but not on YC +glu met ura. Yeast transformed with pBG1805-GAL1 : MET3 also grew on both YC +glu +met ura and YC +gal met ura, but not on YC +glu met ura. S.cerevisiae met3 transformants expressing MET3 or Met3 fusion proteins were spot plated with WT S.cerevisiae on galactose inducible, YPD, and BiGGY agar media (Fig. 7). Instead of pYES2.1-GAL1 : lacZ, the pYES2.1-GAL1 : Met8 overexpression plasmid was used to transform met3 auxotrophs as a negative control. Unlike the replica plating observations, yeast did not grow on YC +gal met ura, the experimental media. However, all yeast grew on YPD media, the positive control. All yeast grew on BiGGY agar media; a tan phenotype was observed in WT yeast, while a brown phenotype was observed in the transformants. No yeast grew on YC +glu met ura, the negative control media.

Figure 1: Growth of S.cerevisiae met auxotrophs on various sulfur sources to identify mutants. WT and mutant yeast were plated on a minimal medium (MM), with or without an extracellular sulfur source, at 30C for 72 hours. A. Growth was observed on MM + methionine (met) for YMP 12, 19, 24, and WT. B. No growth was observed on MM met for YMP 12, 19, 24, and WT. C. Growth was observed on MM + cysteine (cys) for YMP 12, 19, and WT. D. Growth was observed on MM + sulfite (+SO3) for YMP 12 and WT, but not for YMP 19 and 24. E. Growth was observed on YPD for YMP 12, 19, 24 and WT. Table 2: Predicted MET mutation of YMP 12, 19, and 24 strains YMP Strain Mutation 12 met3 19 met2 24 met17

Figure 2: Gel electrophoresis of met auxotroph PCR products to confirm mutant identities . PCR products were loaded with bromophenol blue dye and analyzed on a 1.25% agarose gel in TAE buffer. The gel ran for 20 minutes at 120V. The amplified DNA was stained with 0.0005 mg/mL ethidium bromide and visualized with a UV Transilluminator. A. Standard ladder digest of Lambda phage EcoRI + Hind III B. Genomic DNA from YMP 19 amplified with WT specific primer, MET3 Primer A, and KAN Primer B. No bands were present. C. Genomic DNA from YMP 19 amplified with WT specific primer, MET17 Primer A, and KAN Primer B. A 481bp band was present. D. Genomic DNA from YMP 12 amplified with a WT specific primer, MET2 Primer A, and KAN Primer B. No bands were present. E. Genomic DNA from YMP 12 amplified with a WT specific primer, MET3 Primer A, and KAN Primer B. A 604bp band was present. F. Genomic DNA from YMP 24 amplified with a WT specific primer, MET17 Primer A, and KAN Primer B. No bands were present. G. Genomic DNA from YMP 24 amplified with a WT specific primer, MET2 Primer A, and KAN Primer B. A 524bp band present

Figure 3: Gel electrophoresis of restriction endonuclease digests to identity unknown yeast plasmids. Yeast overexpression plasmids 4, 10, and 14 ran on a 1% agarose gel in TAE buffer at 123V for 30 minutes. The gel was stained with 0.0005mg/mL ethidium bromide (EtBr) and visualized with a UV transilluminator. A. Standard ladder: digest of lambda phage EcoRI + HindIII B. Plasmid 14 digested with ScaI. Bands present at 2500bp and 1500bp. C. Plasmid 14 undigested. A band was present at 3600bp D. Plasmid 10 digested with ScaI. Bands present at 3500bp and 830bp. E. Plasmid 10 undigested. A band was present at 3530bp F. Plasmid 4 digested with ScaI. Bands present at 4500bp and 850bp. G. Plasmid 4 undigested. A band was present at 3700bp.

Table 3: Predicted and observed restriction fragments for yeast overexpression plasmids pYES2.1-GAL1 : pYES2.1-GAL1 : pBG1805-GAL1 : lacZ* Met3 MET3 8071bp, 893bp 6526bp, 893bp 5529bp, 2577bp Predicted 4500bp, 850bp 3500bp, 830bp 2500bp, 1500bp Observed *pYES2.1-GAL1 : lacZ served as the negative control of the PCR.

Figure 4: Simply Blue stain of protein extraction from transformed S.cerevisiae yeast with SDS-PAGE. YMP 12 S.cerevisiae met auxotrophs were transformed with pBG1805-GAL1 : MET3, pYES2.1-GAL1 : Met3, or pYES2.1-GAL1 : lacZ, and grown in YC + gal + met media, or YC + glu + met media. Protein extracts from transformed yeast were resolved on a 12% SDS-PAGE polyacrylamide gel at 203V for 1 hour, and stained with Simply Blue. A. Protein extract from yeast transformed with pBG1805-GAL1 : MET3, grown in gal. Low concentrations of protein were present that ranged in size from 20kD to 250kD. B. Protein extract from yeast transformed with pYES2.1-GAL1 : Met3, grown in glu. Bands were present that ranged in size from 20kD to 250kD. C. Protein extract from yeast transformed with pYES2.1-GAL1 : Met3, grown in gal. Bands were present that ranged in size from 20kD to 250kD. D. Protein extract from yeast transformed with pYES2.1-GAL1 : lacZ, grown in glu. Bands were present that ranged in size from 20kD to 250kD. E. Protein extract from yeast transformed with pYES2.1-GAL1 : lacZ, grown in gal. Bands were present that ranged in size from 20kD to 250kD. L. Bio Rad Kaleidoscope Precision Plus Protein Standard.

Table 4: Determination of transformed YMP 12 yeast cell density Overexpression Plasmids Used to OD600 reading Transform YMP 12 yeast & Growth Conditions pYES2.1-GAL1 : lacZ, grown in glu* 0.052A pYES2.1-GAL1 : lacZ, grown in gal 0.046A pYES2.1-GAL1 : Met3, grown in glu* 0.052A pYES2.1-GAL1 : Met3, grown in gal 0.046A pBG1805.1-GAL1 : MET3, grown in gal 0.037A *Relatively greater cell densities were expected from yeast transformants grown in glucose because its the cells preferred carbon source.

Figure 5: Western blot of Met3p expression from transformed S.cerevisiae yeast. Proteins from the SDS-PAGE gel of transformed S.cerevisiae YMP 12 yeast were electrophoretically transferred to a PVDF membrane at 127V for 30 minutes. Primary antibodies specific for the V5 epitope of Met3p fusion proteins were bound by HRPconjugated rabbit anti-mouse secondary antibodies, and visualized with 3,35,5tetramethyl benzidine (TMB). L. Bio Rad Kaleidoscope Precision Plus Protein Standard. A. Protein extract from yeast transformed with pYES2.1-GAL1 : lacZ, grown in gal. Bands were present at 37kD and 140kD. B. Protein extract from yeast transformed with pYES2.1-GAL1 : lacZ, grown in glu. Bands were present at 37kD and 140 kD. C. Protein extract from yeast transformed with pYES2.1-GAL1 : Met3, grown in gal. Bands were present at 37kD, 55kD, and 140 kD . D. Protein extract from yeast transformed with pYES2.1-GAL1 : Met3, grown in glu. Bands were present at 37kD and 140kD. E. Protein extract from yeast transformed with pBG1805-GAL1 : MET3, grown in gal. Bands were present at 37kD, 75kD, and 140kD. F. Protein extract from yeast transformed with pBG1805-GAL1 : MET3, grown in glu. Bands were present at 37kD and 140kD.

Table 5: Identification of transformed YMP 12 yeast fusion proteins Lane Overexpression Expected Bands Actual Bands Plasmids & Growth Conditions pYES2.1-GAL1 : 117kD 140kD, 37kD A lacZ + gal pYES2.1-GAL1 : None 140kD, 37kD B lacZ + glu pYES2.1-GAL1 : 65kD 140kD, 55kD, 37kD C Met3 + gal pYES2.1-GAL1 : None 140kD D Met3 + glu pBG1805-GAL1 : 77kD 140kD, 75kD, 37kD E MET3 + gal pBG1805-GAL1 : None 140kD, 37kD F MET3 + glu

Figure 6: Replica plating of S.cerevisiae YMP 12 yeast transformants on galactose inducible media. YMP 12 strain yeast were transformed with pYES2.1-GAL1 : Met3, pYES2.1-GAL1 : lacZ, or pBG1805-GAL1 : MET3 yeast overexpression plasmids. Transformed yeast strains were plated on YC +glu +met ura, and incubated at 30 C for 2 days. These plates were replica plated onto galactose inducible media and incubated at 30 C for 2 days. A-C. YMP 12 yeast transformed with pYES2.1-GAL1 : lacZ. A. YC

+met ura + glu, growth was observe. B. YC met ura + gal, no growth was observed. C. YC met ura + glu, no growth was observed. D-F. YMP 12 yeast transformed with pYES2.1-GAL1 : Met3. D. YC +met ura + glu, growth was observed. E. YC +met ura + gal, growth was observed. F. YC met ura + glu, no growth was observed. G-I. YMP 12 yeast transformed with pBG1805.1-GAL1 : MET3. G. YC +met ura + glu, growth was observed. H. YC met ura + gal, growth was observed. I. YC met ura + glu, no growth was observed.

Figure 7: Complementation and BiGGY agar analysis of S. cerevisiae yeast transformants. S.cerevisiae met3 mutants were transformed with pBG1805-GAL1 : MET3, pYES2.1-GAL1 : Met3, or pYES2.1-GAL1 : Met8. A. BY4742 (WT) and transformant yeast strains, grown on YC +met +glu ura media. Growth was observed in transformants, no growth was observed in WT yeast. B. Yeast grown on YC met ura +gal media. No growth was observed in any strain. C. Yeast grown on YPD media. Growth was observed in all strains. D. Yeast was grown on BiGGY Agar media. Growth was observed in all strains. A tan phenotype was observed in WT, while a brown phenotype was observed in the transformants. E. Yeast was grown on YC met ura +glu. No growth was observed in any strain. Discussion Our project goal was to determine the evolutionary conservation of MET3 across S.cerevisiae and S.pombe yeast. Expression of S.cerevisiae MET3 and S.pombe Met3 in S.cerevisiae met3 mutants restored WT phenotype. These transformants both grew on media containing methionine as a positive control, but failed to grow in repressive media lacking methionine as a negative control. Expression of the S.cerevisiae MET3 and the S.pombe Met3 restored WT growth. This observation suggests that the S.pombe ortholog,

Met3, functionally complements the met3 auxotrophy. This also suggests that that MET3 was evolutionarily conserved between S.cerevisiae and S.pombe. The expression of Met3p was confirmed, and therefore complementation most likely occurred because of this fusion protein as opposed to an unknown, external factor. Protein was present from yeast transformed with pYES2.1-GAL1 : Met3 with a size of 55kD, close to the expected Met3p size for S.pombe of 65kD. Also, protein was present from yeast transformed with pBG1805-GAL1 : MET3 with a size of 75kD, close to the expected Met3p size for S.cerevisiae of 77kD. In terms of unexpected results, a protein from yeast transformed with pYES2.1-GAL1 : lacZ was expected with a size of approximately 117kD. This protein was absent most likely because its size was too large for the Western blot; the SDS-PAGE and Western blot experiments could be repeated to test this hypothesis. The identity of YMP strains 12, 19, and 17 was confirmed as met3, met2, and met 17 respectively. The identity of the yeast overexpression plasmids was also confirmed; plasmid 4, 10, and 14 were confirmed as pYES2.1-GAL1 : lacZ, pYES2.1-GAL1 : Met3, and pBG1805-GAL1 : MET3 respectively. S.cerevisiae MET3 and S.pombe Met3s ability to restore wild type growth in YMP 12 yeast indicates that these identifications were correct. Asides from a growth phenotype, the effect of transformation on the methionine biosynthesis pathway was also determined using BiGGY agar media. This media provided a readout of sulfide production, which is an intermediate of methionine biosynthesis; sulfide synthesis was confirmed in all of the transformant strains. This revealed that not only did the S.pombe Met3 restore methionine biosynthesis to S.cerevisiae met3 auxotrophs, but also that its transforamants expressed a greater

concentration of sulfide relative to wild type yeast. Transformants displayed a brown phenotype on BiGGY agar media, while wild type yeast only displayed a tan phenotype. This brown phenotype occurred in all of the transformants because BiGGY agar media contains sodium sulfate, which is produced downstream of the MET3 gene in the methionine biosynthesis pathway. Therefore, met3 mutants are still able to produce sulfide even though the S.pombe Met8 was not expected to functionally complement the S.cerevisiae met3 mutant. The darker the color of colonies on BiGGY agar, the greater the level of sulfide, which suggests yeast transformants produced a greater concentration of total protein than WT yeast. According to this data, all of the yeast transformants have normal enzymatic activity as measured by their ability to produce sulfide in BiGGY agar media. To further test the evolutionary conservation of Met3p function between S.cerevisiae and S.pombe, replica plating on various media (Fig. 6) could be repeated with S.pombe met3 mutants instead of S.cerevisiae mutants. If MET3 is conserved between S.pombe and S.cerevisiae, then S.pombe met3 mutants transformed with pBG1805-GAL1 : MET3 should grow on inductive YC + gal met ura media.

References De Lucas J, Dominguez A, Higuero Y, Martinez O, Romero B, Mendoza A, GarciaBustos J, Laborda F (2001) Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase. Archives of Microbiology 176: 106-113 Jaramillo M, Abanto M, Quispe R, Calderon J, del Valle L, Talledo M, Ramirez P (2012) Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli. Bioinformation 8.15: 695-704 Masselot M & de Robichon-Szulmajster H (1975) Methionine Biosynthesis in Saccharomyces cerevisiae. MGG Molecular & General Genetics 139.2: 121-132 OConnor C (2012) Investigations into Molecular Cell Biology. Plymouth: HaydenMcNeil. Print. Schiff, J & Hodson R (1973) The Metabolism of Sulfate. Annual Review of Plant Physiology 24: 381-414 Patron N, Durnford D, Korpriva S (2008) Sulfate assimilation in eukaryotes: fusions, relocations and lateral transfers. BioMed Central Evolutionary Biology 8: 39 Thomas D & Surdin-Kerjan Y (1997) Metabolism of sulfur amino acids in Saccharomyces cerevisiae. Microbiol Mol Bio R 61: 503532 Ullrich TC, Blaesse M, Huber R (2001) Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation. EMBO Journal 20.3: 316-29 Winzeler E, Shoemaker D, Astromoff A, Liang H, Anderson K, Andre B, Bangham R (1999) Functional Characterization of the S. cerevisiae Genome by Gene Deletion and Parallel Analysis. Science 285: 901