Zackary Park

Vincents Section
Exploring conservation of the MET5 gene of Saccharomyces cerevisiae gene in
Schizosaccharomyces pombe
Abstract:
The sulfur assimilation pathway is a critical component of the methionine biosynthesis
pathway, which results in the production of amino acids essential to living and growth , methionine
and cysteine. The purpose of this experiment was to investigate the sulfite reductase enzyme ,
encoded by the MET5 gene of Saccharomyces cerevisiae, and ultimately test for complementation
of the MET5 gene with the homolog of Schizosaccharomyces pombe. Selective plating and PCR
identified the MET mutants, restriction mapping identified the plasmids, and replica plating
determined that complementation did not occur . However, SDS-PAGE and western blot determined
that transformation of the plasmids did occur . Thus, the results of this experiment support
unsuccessful complementation of the S. pombe homolog to the S. cerevisiae gene; Met5 of S.
pombe does not complement MET5 of S. cerevisiae.

Introduction:
Sulfite reductase is associated with the sulfate assimilation pathway , catalyzing the reaction
between the substrates SO3 and NADPH to produce H2S, NADP, and H2O (Thomas et al., 1997).
Sulfite reductase contributes to the production of methionine and cysteine , both essential to the
growth of S. cerevisiae and other microorganisms (Jiranek et al., 1995). Mutations in either MET5
or MET10, genes encoding the two subunits of the sulfite reductase enzyme, greatly reduce H2S
production (Huang et al., 2014). The mutations lead to an increase in sulfide formation (Linderholm
et al., 2008). MET5 mutants failed to grow in the absence of methionine , showing a white
phenotype on BiGGY agar plates, whereas the wild-type showed a tan phenotype (Cordente et al .,
2009), showing that it did not transform sulfite into sulfide (Thomas et al .,1992). MET5 mutants
strongly repressed the production of methionine (Mountain et al ., 1991), suggesting that it led to an
enzymatically inactive sulfite reductase (Thomas et al.,1997) and disrupted the methionine
biosynthesis pathway.
The sulfate assimilation pathway is present in many organisms; therefore , sulfite reductase is
found in organisms other than S. cerevisiae, such as Ashbya gossypii (Dietrich et al., 2004, Dujon et
al., 2004), Kluyveromyces lactis (Kleffmann et al., 2004), and Schizosaccharomyces pombe (Wood
et al., 2002). The average sequence identity between orthologous proteins of S. cerevisiae and K.
lactis is 6061%, (Kleffmann et al., 2004) and more than 90% of A. gossypii genes show homology
with S. cerevisiae (Dietrich et al., 2004). About 78% of the S. pombe proteins have homologs in
common with S. cerevisiae (Wood et al., 2002). The similarities predict a similar structural unit for
sulfite reductase: a heterotetramer protein, consisting of two - and two -subunits (Thomas et al .,
1997). The -subunit is encoded by the MET10 gene (Hosseini-Mazinani et al., 1995), and the subunit is encoded by the MET5 gene (Cordente et al., 2009). Each of the -subunits binds one
flavin mononucleotide and one siroheme (Thomas et al ., 1997). To reduce sulfite to sulfide, sulfite

reductase uses a siroheme coupled to an iron-sulfur cluster to perform reduction (Cordente et al .,

2009). Each -subunit binds one flavin adenine dinucleotide (FAD) as a prosthetic group and
contains a NADPH-binding domain, an important cofactor (Sutherland et al., 2003).
Throughout evolution, MET5 in S. cerevisiae has been conserved in many organisms in
Eukaryota. In S. pombe, the gene sir1 encodes for sulfite reductase, which catalyzes the reaction
between the substrates SO3 and NADPH to produce H2S, NADP, and H2O (Vande et al., 1999). The
function is very similar to that of S. cerevisiae. In A. thaliana, the gene SIR encodes for sulfite
reductase, the second enzyme of the free reduction pathway (Kleffmann et al ., 2004), which has a
similar function to S. cerevisiae (Kleffmann et al., 2004). Sulfite reductase is conserved in both
prokaryotes and eukaryotes, as dissimilatory and assimilatory sulfite reduction , respectively
(Thomas et al., 1992).
The research goal of the investigation was to determine if the S. pombe orthologs of S.
cerevisiae MET5, which encodes for the enzyme of yeast sulfite reductase (EC 1.8.1.2), could
complement deficiencies in S. cerevisiae MET5 mutant strains. Various experimental procedures
were used selective plating, PCR, restriction mapping, replica plating, SDS-PAGE, and western
blot. Selective plating and PCR were used to identify MET deletion strains grown on various media
and isolate the MET5 mutant strain. Plasmids containing functional Met14 from S. pombe, MET14
from S. cerevisiae, and bacterial lacZ+ were identified using restriction endonucleases and
overexpressed. S. cerevisiae strains were transformed with the identified plasmids and tested for
complementation using SDS-PAGE and western blot. The results of this experiment support
unsuccessful complementation of the S. pombe homolog to the S. cerevisiae gene.

Materials and Methods:

Selective Plating: Three YMP strains (YMP13, YMP16, YMP24) and a wild type were derived
from the parent strain BY4742 (derived from strain S288C) of S. cerevisiae. The genotype of
BY4742 is MAT his31 leu20 lys20 ura30. The composition of Yeast Complete (YC), the
culture medium, is in Investigations in Molecular Cell Biology1. The various media were YC-Met,
YC-Met+Cys, BiGGY, YC+Met (YC Complete), YC-Met+SO3, and YPD. The starter cultures were
grown overnight in YPD broth. Each culture was diluted in a series of five 1:10 dilutions , with each
spot consisting of 5L, in sterile, deionized water on the spot plates. The plates were incubated at
30oC for 3 days.
Strains or plasmids
Saccharomyces cerevisiae
Laboratory strains
BY4742met2 (YMP24)
BY4742 met5 (YMP16)
BY4742met14 (YMP13)
Plasmids
pBG1805-GAL1:MET5

Genotypes or constructs

MAT his31 leu20 lys20 ura30 met2::KANR

MAT his31 leu20 lys20 ura30 met5::KANR
MAT his31 leu20 lys20 ura30 met14::KANR
Yeast expression vector, GAL1, URA3

pYES2.1-GAL1:lacZ+
Yeast expression vector, GAL1, URA3
pYES2.1-GAL1:Met5
Yeast expression vector, GAL1, URA3
Table 1. Strains and plasmids used in this study.
Polymerase Chain Reaction: PCR reactions were performed as described in Chapter 71. TAE buffer
submerged the 1.25% agarose gel. Six wells were used to accommodate a YMP strain DNA
(YMP13, YMP16, YMP24) and gene-specific primers A and B for a met mutation (met2, met5,
met14) (Table 1). Each of the 20 L PCR reactions were added 4 L of 6X loading buffer . 10 L of
a PCR sample was loaded to each of the six wells , and 5 L of 1kb molecular weight standard was
loaded on the seventh well. The experiment was run on 128 volts for 20 minutes until bromophenol
blue was 1 cm from the end. The gel was stained with EtBr solution and rocked for 20 minutes1.
Restriction Mapping: S. cerevisiae genes were cloned into the pBG1805 vector, and S. pombe genes

and lacZ+ were cloned into the pYES2.1 vector1. The possible identities of the unknown plasmids
labeled 1,2, and 11, were pBG1805-MET5, pYES2.1-Met5, and pYES2.1-lacZ+. The cultures
containing the plasmids were grown overnight at 37 OC in 1.5 mL Luria Bertani medium with 100
g/mL ampicillin1. The plasmids were isolated from the culture using the Zyppy TM Plasmid
Miniprep (Zymo Research) Kit with modifications found in the lab manual 1. The 3 kb marker
contained ~125 ng, and each of the other markers contained ~40 ng. Based on these values, the
amount of DNA in each lane was about 10 ng 1 (Figure 2). The volume of sample in each lane was
18 L1. The DNA concentration of each purified plasmid was about 0.56 ng/L.
Restriction Digests: In the digestions, 7.0 L plasmid miniprep DNA from the ZyppyTM kit was
used. 2.0 L (1.0 U) of restriction enzymes were used . AccI was used for the Group A Digest , and
SacI-HF was used for the Master Digest, which was provided by Professor Warner (Figure 1, Figure
2, respectively). The plasmid, RE, and buffer were combined in each tube for both of the RE
digests. The tubed samples were incubated 37oC for at least 2 hours. For undigested plasmid
samples, 7 L of plasmid DNA and 3 L of deionized water were used; for restriction digest
samples, 10 L of plasmid DNA was used1. The samples, buffer, and deionized water were
combined and loaded into each well. The 1% agarose gel was submerged in TAE buffer and
ethidium bromide. The voltage was set at 133 volts. Refer to the lab manual for more details1.
Transformation: The plasmids used to transform the mutant strain were plasmids 1 , 2, and 11
(pYES2.1-Met5, pYES2.1-lacZ+, and pBG1805-MET5, respectively)1. Yeast cells were scraped off
a YPD plate and were transformed and plated on selective media lacking uracil , but containing
methionine (YC+Met-Ura)1. The cells were then replica plated onto a new master plate (YC+MetUra+Glu) and selective media lacking methionine, but containing either glucose or galactose (YCMet-Ura+Glu and YC-Met-Ura+Gal)1. The transformation procedure was a modification of the
Quick and Dirty transformation protocol as described by Amberg et al. (2005)1. The master mix

contained lithium acetate, 50% PEG-3350, and 2-mercaptoethanol. Each transformation mixture
contained the master mix, denatured salmon sperm DNA, plasmid DNA, and a yeast colony from a
YPD plate1. A control without plasmid DNA was also created 1. The transformation mixtures were
incubated at 37oC with shaking for 30-45 minutes1.
Replica Plating: A sample of transformed cells was serially diluted on YPD media and on selective
media lacking uracil, but containing methionine. The cells were incubated at 30oC for 2 days1. The
transformants were transferred from the master plate onto selective media (YC+Met-Ura+Glu , YCMet-Ura+Glu, and YC-Met-Ura+Gal) via velveteen1. For more information, refer to Investigations
in Molecular Cell Biology1.
Protein Overexpression: The plasmids used to transform the mutant strain were plasmids 1, 2, and
11 (pYES2.1-Met5, pYES2.1-lacZ+, and pBG1805-MET5, respectively)1. The transformed strains
were mixed with YC-Ura medium with 2% raffinose and incubated overnight in a 30 oC shaking
incubator1. Four hours before extraction, each transformed strain was mixed with either
YC+glucose or YC+galactose1. The cells were rinsed with deionized water , s added 0.2 M NaOH,
and incubated at room temperature for 5 minutes 1. The cells were resuspended in 2 X SDS-PAGE
sample buffer, placed in boiling water for 3 minutes, and stored in the freezer1.
SDS-PAGE: Two SDS-PAGE gels were prepared. The stacking gel was 5% acrylamide, and the
running gel was 12% acrylamide1. The first gel was run for 60 minutes at 128 volts and stained with
Simply Blue to rock overnight1. The second gel was used to transfer proteins from the SDS-PAGE
gel to the PVDF membrane at 20 V overnight at 4 oC1.
Western Blot: The gel was covered with blocking solution (5% nonfat milk in TBS-T) and incubated
for 24 hours at 4 oC1. The gel was washed with TBS-T , covered with primary antibody (a mouse
monoclonal antibody) that binds to the V5 epitope on proteins encoded by pYES2 .1-Met5 and
pYES2.1-lacZ+, and incubated overnight at 4 oC with slow rocking1. The gel was washed with TBS-

T, covered with secondary antibody (a rabbit polyclonal antibody) that binds to the F c domains of
the mouse anti-V5 antibodies bound to proteins encoded by pYES2 .1-Met5 and binds to the S.
aureus ZZ domains on proteins encoded by pBG1805-MET5, and incubated for 1 hour at room
temperature with rocking1. The gel was washed with TBS-T and covered with TMB for 5 minutes1.
Open Experiment: A restriction digest was performed again for the open experiment, but EarI was
used as the restriction enzyme. The 1% agarose gel was submerged in TAE buffer and ethidium
bromide. The voltage was set at 133 volts. Refer to the lab manual for more details1.

Results:
The unknown met mutations were identified in labeled YMP strains through selective
plating. Based on the methionine biosynthesis pathway, the observed data was paired with the
predicted patterns of growth. The data supports that YMP24 contains met2, YMP16 contains met5,
and YMP13 contains met14.

The wild type strain was present on all media, which suggested that there was no error with
the various media. All of the strains grew on YPD because YPD provided all of the strains of S.
cerevisiae, regardless of mutation, the essential nutrients to live. All of the met mutation strains did
not survive in the YC-Met media, which proved that all of the met mutation strains required
methionine to survive. All of the strains also grew on YC+Met (YC-Complete) , which proved that
the met mutation strains could thrive on YC media with the addition of the met strains. A light
phenotype on BiGGY agar plates indicates an absence of sulfide produced , and a dark phenotype on
BiGGY agar plates indicates an abundance of sulfide produced . Based on the methionine
biosynthesis pathway, met2 comes after met14 and met5 on the cycle; therefore, met2 would appear
darker than both met2 and met5 on the BiGGY plate (Figure 2). On the cycle, met14 comes before
met5 and met2 on the cycle; therefore, met14 would appear light than both met5 and met2 on the
BiGGY plate (Figure 2). YMP24 is not present on YC-Met , YC-Met+Cys and YC-Met+SO3, but it
is present on YC+Met and YPD (Figure 1); YMP24 is also darker than the default wild type color
on the BiGGY plate (Figure 2). YMP16 is not present on YC-Met and YC-Met+SO3, but it is
present on YC-Met+Cys, YC+Met, and YPD (Figure 1); YMP16 is also lighter than the default wild
type color on the BiGGY plate (Figure 2) . YMP13 is not present on YC-Met, but it is present on
YC-Met+Cys, YC+Met, YC-Met+SO3, and YPD (Figure 1); YMP13 is also lighter than the default
wild type color on the BiGGY plate (Figure 2). There are some faint traces of YMP strains even
when predicted to be absent because the scanning occurred much later than planned due to one
snow day and there may have been traces of methionine in the gels . Although the data supports that
YMP24 contains met2, YMP16 contains met5, and YMP13 contains met14, it was not certain.

Strain

met mutation

YC-Met

YC-Met+Cys

BiGGY

YC+Met

YC-Met+SO3

YPD

YMP24

met2

darker

YMP16

met5

lighter

YMP13

met14

lighter

Wild Type

N/A

N/A

Table 2: Results of selective plating.

Preliminary strain identifications from experimental data of the growth of the met mutant strains.
To confirm the pairings of an appropriate YMP strain with its correct met mutations,
polymerase chain reaction was performed using gene specific primers to determine the presence of
the met mutations.

The predicted length for the PCR product of MET2 is 895 bp, the predicted length for the
PCR product of MET5 is 488 bp, and the predicted length for the PCR product of MET14 is 462 bp
(Table 3). The observed lengths of the MET5 and MET14 PCR products were ~500 bp (400 bp
600 bp) when compared to the 1kb standard molecular weight (Figure 2 , Table 3). There is no

observed length of the MET2 PCR product because the only well with GSP-A and GSP-B for MET2
also contained DNA with a MET2 mutation; therefore, no band appeared (Table 3). The PCR results
confirmed the predictions that YMP24 contains met2, YMP16 contains met5, and YMP13 contains
met14.

Well

YMP
strain

MET
mutation

Primer A

Primer B

Theoretical
Length
(bp)

Observe
d Length
(bp)

Prediction
if
Band
Present

MET14
MET14
462
0
No
Primer A Primer B
MET5
MET5
2
YMP13
MET5
488
488
Yes
Primer A Primer B
MET2
MET2
3
YMP24
MET2
895
0
No
Primer A Primer B
MET14
MET14
4
YMP24
MET14
462
462
Yes
Primer A Primer B
MET5
MET5
5
YMP16
MET5
488
0
No
Primer A Primer B
MET14
MET14
6
YMP16
MET14
462
462
Yes
Primer A Primer B
Table 3: Gel electrophoresis predictions of PCR products . Primers and strains used and
theoretical length (bp) of electrophoresed products of PCR. Primers were chosen to best investigate
predictions from the selective plating experiment.
1

YMP13

MET14

Unknown plasmids pBG1805-MET5, pYES2.1-Met5, and pYES2.1-lacZ+ were identified

using restriction endonucleases to contain either MET5 S. cerevisiae, Met5 S. pombe, or bacterial
LacZ ORFs. The restriction enzyme AccI was chosen, resulting in the fragments not being visible
(Figure 2.1, Table 4).

Well
Number

Plasmid
Number

Plasmid Name

Observed
Approximate
Predicted Restriction
Plasmid
Restriction
Fragment Size (bp)
Length (bp)
Fragment Size
(kbp)

pYES2.1-Met5

3158, 2807, 2733, 625,

N/A
505, 477

10308

pBG1805MET5

5487, 2819, 785, 673,

N/A
505, 456, 174

8964

11

pYES2.1-lacZ+

5229, 2605, 625, 505

10,902

N/A

Table 4: Predicted and Observed Restriction Fragments for Plasmids Digested with AccI.
However, the experimental results of Group A Digest did not confirm the predictions
because the restriction fragments were faintly visible or not visible on the gel (Figure 3 .1). The
reactions did not work not even the undigested plasmids were visible (Figure 3 .1). Thus,
Professor Warner provided the Master Digest, which used ScaI-HF as the RE (Figure 3.2).

Plasmid
Number

Plasmid Name

Observed
Approximate
Predicted Restriction
Restriction
Fragment Size (bp)
Fragment
Size (kbp)

pYES2.1-Met5

4669, 3617, 1126, 893

~4.0, ~5.0

10308

pYES2.1-lacZ+

8071, 893

~1.0, ~7.0

8964

11

pBG1805-MET5

8322, 2577

~3.0, ~8.0

10,902

Well
Number

Plasmid
Length (bp)

Table 5: Predicted and Observed Restriction Fragments for Plasmids Digested with ScaI-HF.
The estimated and predicted sizes of the restriction fragments due to ScaI-HF found on
Table 5 supports the prediction that plasmid 1 is pYES2.1-Met5, plasmid 2 is pYES2.1-lacZ+, and
plasmid 11 pBG1805-MET5 (Figure 3.2, Table 5). The predicted sizes of the undigested plasmids
were 10902 bp for pBG1805-MET5, 10308 bp for pYES2.1-Met5, and 8964 bp for pYES2.1-lacZ+

(Table 5). The observed sizes of the undigested plasmids corresponded to the predicted sizes ,
supporting the prediction of the identities of the plasmids (Table 5) . For plasmid 1, the predicted
4669 and 3617 bp fragments were similar to the observed ~5000 and ~4000 bp fragments for
pYES2.1-Met5, respectively (Table 5). For plasmid 2, the predicted 8071 and 893 bp fragments
were similar to the observed ~7000 and ~1000 bp fragments for pYES2 .1-lacZ+ (Table 5). For
plasmid 11, the predicted 8322 and 2577 bp fragments were similar to the observed ~8000 and
~3000 bp fragments for pBG1805-MET5 (Table 5). Thus, the data supports that plasmid 1 is
pYES2.1-Met5, plasmid 2 is pYES2.1-lacZ+, and plasmid 11 is pBG1805-MET5 (Figure 3.2, Table
5).

The open experiment was a repeat of the restriction digest using a different restriction
enzyme EarI to verify the proper identification of the plasmids , pYES2.1-Met5, pYES2.1-lacZ+,

and pBG1805-MET5. However, either the gel was run for too long or the percentage of agarose in
the gel was too low because the fragments ran off of the gel (Figure 6) . Plasmid 2 is expected to
have the largest fragment size, which was observed. Among plasmid 1 and 11, plasmid 11 is
expected to have a larger second fragment , which is observed, than the second fragment of plasmid
1. Based on the expected and observed fragment lengths, the data further supports that plasmid 1 is
pYES2.1-Met5, plasmid 2 is pYES2.1-lacZ+, and plasmid 11 is pBG1805-MET5 (Figure 3.2, Table
5).
The identified plasmids were used to transform the BY4742 mutant met5 deletion strain with
the plasmids: pYES2.1-Met5, pYES2.1-lacZ+, and pBG1805-MET5. The transformed cells were
replica plated onto selective media to test for complementation . According to the data,
complementation was not observed (Figure 4, Table 6).

Plasmid
Number

Plasmid
Name

pYES2.1Met5

Transformation
Efficiency
(transformed cells/
g
plasmid
DNA)
4.18

Observed
Growth
on
YC+MetUra+Glu
+

Observed
Growth
on YCMetUra+Gal
-

Observed
Growth
on YCMetUra+Glu
-

Predicted
Growth
on
YC+MetUra+Glu
+

Predicted
Growth
on YCMetUra+Gal
+
(if
complem
entation
occurred)
-

Predicted
Growth
on YCMetUra+Glu
-

pYES2.1- 5.50
++
+*
+*
++
lacZ+
11
pBG1805- 1.10
++
+
++
+
MET5
Control
No
5.72
N/A
N/A
N/A
N/A
N/A
N/A
Plasmid
Legend: ++: lots of growth, +: some growth, -: no growth, *: unexpected growth
Table 6: Calculated Transformation Efficiencies and Predicted and Expected Growth on
Various Replica Plates.
The transformation efficiencies were calculated for each plasmid and for the control containing no
plasmid. The method for calculating the transformation efficiencies can be found in Investigations
in Molecular Cell Biology, Chapter 121. The ability of the transformed strains to grow on the
various replica plates was also predicted and observed.
Cells transformed with pYES2.1-lacZ+ served as the negative control. For the transformant
containing plasmid 11, pBG1805-MET5, growth occurred on the YC-Met-Ura+Gal media (Figure
1, Table 1). However, for the transformant containing plasmid 1, pYES2.1-Met5, growth did not
occur on the YC-Met-Ura+Gal media (Figure 4 , Table 6). No growth was observed for both
pYES2.1-Met5 and pBG1805-MET5 on the YC-Met-Ura+Glu media (Figure 4, Table 6).
Unexpected growth was observed on the YC-Met-Ura+Glu and YC-Met-Ura+Gal plates with the
pYES2.1-lacZ+ transformant. (Figure 4, Table 6).
Analysis of the SDS-PAGE gel indicates a poor amount of proteins present on the gel
(Figure 5). The gel was stained with Simply Blue , but the bands are not clearly visible . Bands are
evident in Lanes 1, 2, and 4 (Figure 5). Bands are not very clear in Lanes 5 and 6 (Figure 5) . And,
bands are not present in Lane 3 (Figure 5). Lanes 1, 3, and 5 represent induced samples, cells grown
in galactose as the carbon source, and lanes 2, 4, and 6 represent non-induced samples, cells grown

in glucose as the carbon source (Figure 5) . Bands were predicted to be in all of the lanes; however ,
the data does not support the prediction.

Well Number
Ladder

Sample
Predicted (kDa)
Actual (kDa)
Molecular
Weight N/A
N/A
Standard
1
pYES2.1-Met5 (Gal)
177.73
~180
2
pYES2.1-Met5 (Glu)
~0
~0
3
pYES2.1-lacZ+ (Gal)
117.64
Not Observed
+
4
pYES2.1-lacZ (Glu)
~0
~0
5
pBG1805-MET5 (Gal)
167.13
~168
6
pBG1805-MET5 (Glu)
~0
~0
+
Table 7: Predicted and actual sizes of MET or lacZ proteins.
The table represents the expected and actual sizes for the samples in each well . The predicted size
of the proteins were calculated by subtracting the STOP codon from the ORF , dividing by 3, and
multiplying the result by the conversion factor of 0.11kDa/amino acid.
Analysis of the western blot reveals that the predictions did not match the observations
because protein, encoded by pYES2.1-lacZ+, is absent in Lane 3 (Figure 5) . The western blot
reveals that protein expression is possible in the S. cerevisiae MET5 strain transformed with
pBG2805-MET5 and with p2.1YES-Met5, indicating successful transformation (Figure 5). Lane 1

has an intense band at a size ~180 kDa, similar to the predicted 177.73 kDa, that shows the presence
of a protein encoded by pYES2.1-Met5 (Table 1). Lane 5 has an intense band at a size of ~168 kDa ,
similar to the predicted 167.13 kDa, that shows the presence of a protein encoded by pBG1805MET5 (Table 1). Although the data from the replica plating supports that complementation was
unsuccessful because pYES2.1-Met5 transformants were not observed in the YC-Met-Ura+Gal
media, transformation was successful because fusion proteins are evident in the western blot.
Discussion:
The goal of this investigation was to explore the conservation of the MET5 gene of S.
cerevisiae gene in S. pombe. It is concluded that the conservation of the MET5 gene of S. cerevisiae
gene in the S. pombe homolog, Met5, is unsuccessful. Observations from the selective plating
experiment were as expected. The loss of function of an enzyme from mutations limits the ability of
the biosynthesis pathway to produce essential amino acids , such as methionine and cysteine,
inhibiting the strain to survive and grow on specific media . BiGGY agar contains bismuth, which
reacts with sulfide and produces a dark precipitate1. The color of the strains on BiGGY agar
determines if the mutation is upstream or downstream the sulfide assimilation pathway . This
allowed educated guesses to be made to identify the strains and their met mutations YMP24, 16,
and 13 likely contains met2, met5, and met1, respectively.
PCR confirmed the correct identification of the strains . Fragments were produced when GSP
primer A and GSP primer B for its specific MET gene were able to bind on the yeast chromosome
and yield an amplified product. Fragments were not produced when either one or both GSP primer
A and GSP primer B for its specific MET gene were not able to bind on the yeast chromosome .
Restriction enzymes AccI, ScaI-HF, and EarI were selected to identify plasmids pYES2 .1-Met5,
pYES2.1-lacZ+, and pBG1805-MET5 because they provided the greatest variety of the fragment
lengths; however, the RE EarI, which was used for the open experiment, did not conclude anything

because the fragments ran off the gel. Plasmid 1, 2, and 11 were confirmed to be pYES2.1-Met5,
pBG1805-MET5, and pYES2.1-lacZ+, respectively (Figure 3.1, Figure 3.2, Figure 6). The plasmids
were transformed into S. cerevisiae met mutant strains and grown on various media to determine if
transformation or complementation were successful. Growth on the media lacking uracil supports
successful transformation. The transformed plasmids contain a URA3 gene, which allows the cell to
grow on uracil-absent media. Also, the plasmids contain a GAL1 promoter. The transformed cells
were expected to grow on plates containing galactose because galactose induces high levels of
GAL1 expression. In contrast, glucose represses transcription, so little to no growth was expected .
No growth was observed for the pYES2.1-Met5 transformants on the YC-Met-Ura+Gal media
because complementation most likely did not occur. A possibility of complementation not being
observed is that the transformed cells could have produced fusion proteins that were detrimental or
fatal to the transformed cell1. Another possibility is that since regulatory balance is altered in
transformed cells, leaky gene transcription may have occurred1.
SDS-PAGE and Western blot was used to determine if fusion proteins were expressed from
the transformed plasmids. SDS-PAGE indicates a poor amount of proteins visible on the gel .
Perhaps the gel was not run long enough because an abundance of blue dye is still present in the
wells. The Western blot revealed that transformants grown in galactose exhibit protein expression ,
and transformants grown in glucose inhibit protein expression . The pYES2.1-Met5 and pBG1805MET5 transformants produced proteins but pYES2.1-lacZ+ did not. Perhaps not enough protein was
transferred from the gel to the membrane or there was a problem with the transformant itself
because both antibodies bonded to the other transformants . Multiple fragments are present in Lanes
1 and 5 possibly from the insufficient blocking of non-specific protein binding sites on the transfer
membranes or from the degradation of proteins since all of the bands appeared simultaneously1.
The data supports that the MET5 protein is not conserved between the two yeast species and

supports that gene carried on the plasmids is not able to compensate for the missing gene in the
mutant (Figure 1, Table 1). Thus, S. pombe Met5 is not functionally equivalent to S. cerevisiae
MET5. Because MET10 also codes for the enzyme sulfite reductase, this experiment should be
repeated with MET10 to determine if the function of the enzyme is conserved between S. cerevisiae
and S. pombe. Also,

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