Matt Markham

Brian Nowlin

T,Th 12-3

Conservation of MET3 between Saccharomyces cerevisiae and Schizosaccharomyces pombe in the methionine biosynthesis pathway

Abstract

This study’s 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 3’-

phospho-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 5’-

phosphosulfate 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 eukaryote’s 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.cerevisiae’s MET3, the negative

control, E.coli’s lacZ, or the experimental gene, S.pombe’s 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 (O’Connor). 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 Genotypes or constructs

BY4742∆met3 Mat α his3∆1 leu2∆0 ura3∆0 met3::KANR

BY4742∆met2 Mat α his3∆1 leu2∆0 ura3∆0 met2::KANR

BY4742∆met17 Mat α his3∆1 leu2∆0 ura3∆0 met17::KANR

pBG1805-GAL1 : MET3 Yeast overexpression plasmids, URA3ª, MET3*

pYES2.1-GAL1 : Met3 Yeast overexpression plasmids, URA3ª, Met3*

pYES2.1-GAL1 : lacZ Yeast overexpression plasmids, URA3ª, lacZ*

*Each of these genes was under the control of the galactose-inducible system of yeast overexpression plasmids’ GAL1 promoter. ª 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 (O’Connor, 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 0.5µM of KAN primer B as reverse primer, and

0.5µM of MET2 primer A, MET3 primer A, or MET17 primer A as forward primer. The

PCR conditions were: 2 min at 95˚C, (30s at 55˚C, 30s at 72˚C, and 1 min at 72˚C) x 35,

and 10 min at 72˚C. 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 (O’Connor, 2012).

Restriction digest of overexpression plasmids

Overexpression plasmids were isolated from three different E.coli bacterial cultures using

a standard alkaline lysis method; only 50µL of elution buffer was used to elute plasmid

DNA. The A260 of each plasmid strain’s DNA was measured using a NanodropTM

spectrophotometer (O’Connor, 2012). 0.5µg 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 (O’Connor, 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 0.4µg miniprep plasmid DNA (O’Connor). 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 30˚C, while rotated, for two days.

Each yeast sample was then split equally between YC +glu+met–ura 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 (O’Connor). Yeast and

media samples were incubated at 30˚C 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% 2-

mercapethanol, 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 (O’Connor, 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 (O’Connor, 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+met–ura+glu, YC–met–ura+glu,

YC–met–ura+gal, and BiGGY agar media (O’Connor, 2012). Again, transformant

cultures were incubated at 30˚C 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.pombe’s 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.cerevisiae’s 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 30˚C 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 strainsYMP Strain Mutation

12 met319 met224 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 : lacZ*

pYES2.1-GAL1 : Met3

pBG1805-GAL1 : MET3

Predicted 8071bp, 893bp 6526bp, 893bp 5529bp, 2577bpObserved 4500bp, 850bp 3500bp, 830bp 2500bp, 1500bp

*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 densityOverexpression Plasmids Used to

Transform YMP 12 yeast & Growth Conditions

OD600 reading

pYES2.1-GAL1 : lacZ, grown in glu* 0.052ApYES2.1-GAL1 : lacZ, grown in gal 0.046A

pYES2.1-GAL1 : Met3, grown in glu* 0.052ApYES2.1-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 it’s the cell’s 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 HRP-conjugated rabbit anti-mouse secondary antibodies, and visualized with 3,3’5,5’-tetramethyl 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 proteinsLane Overexpression

Plasmids & Growth Conditions

Expected Bands Actual Bands

A pYES2.1-GAL1 : lacZ + gal

117kD 140kD, 37kD

B pYES2.1-GAL1 : lacZ + glu

None 140kD, 37kD

C pYES2.1-GAL1 : Met3 + gal

65kD 140kD, 55kD, 37kD

D pYES2.1-GAL1 : Met3 + glu

None 140kD

E pBG1805-GAL1 : MET3 + gal

77kD 140kD, 75kD, 37kD

F pBG1805-GAL1 : MET3 + glu

None 140kD, 37kD

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 Met3’s 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.

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