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. Strain YMP24 most likely has the met2 mutation; 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). Thus, YMP24 most
likely contains the met2 mutation. Strain YMP16 has the met5 mutation; 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).
Thus, YMP16 most likely contains the met5 mutation. Strain YMP13 has the met14
mutation; YMP13 is not present on YC-Met, but it is present on YC-Met+Cys, YC+Met, YCMet+SO3, and YPD (Figure 1). YMP13 is also lighter than the default wild type color on the
BiGGY plate (Figure 2). Thus, YMP13 must contain the met14 mutation. On the Methionine
Biosynthesis chart1, 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). 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. The data
confidently supports that YMP24 contains met2, YMP16 contains met5, and YMP13 contains
met14; however, these are only assumptions. A post-PCR experiment would determine the
correct met mutations with the appropriate YMP strain.
Deletions in met14 would result in the YMP13 strain to obtain sulfite or sulfide from a
sulfur source because sulfite would not be produced, therefore rendering the cycle from
completion. Deletions in met5 would result in the YMP16 strain to obtain sulfide from a
sulfur source because sulfide would not be produced, therefore rendering the cycle from
completion. Deletions in met14 would result in the YMP13 strain from completing the cycle
because there is no sulfur source following met2 in the cycle.
Strains YMP13, YMP16, and YMP24 were selectively paired with GSP-A and GSPB primers for mutations MET2, MET5, and MET14 based on conclusions from phenotype
observations of the selective plating experiment1. Thus, based on the results, it was
predicted that YMP24 contains MET2, YMP16 contains MET5, and YMP13 contains
MET14. Therefore, Well 2, Well 4, and Well 6 were predicted to have bands because the
MET genes would not have been deleted in the respective YMP strains (Figure 1). GSP-A
and GSP-B would have been able to create PCR products containing portions of each
specific MET gene. Likewise, Well 1, Well 3, and Well 5 were predicted to not have bands
because the MET genes would have been deleted in the respective YMP strains (Figure 2).
GSP-A and GSP-B would not have been able to create PCR products containing portions of
each specific MET gene. The observed results parallel the predictions for both the presence
of bands and the band lengths. 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 (Figure 1). 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 1, Figure 2). 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 (Figure 2). However, because the observed band lengths of the
other two MET mutation PCR products resembled their theoretical band lengths, the PCR
product of MET2 should also have a band length identical to its theoretical band length.
The bands resulted from GSP-A and GSP-B for its specific MET gene because the primers
were able to bind on the yeast chromosome and code to form complementary strands.
Bands did not appear when either one or both GSP-A and GSP-B for its specific MET gene
were not able to bind on the yeast chromosome and code to form complementary strands.
The experimental results confirmed the predictions that YMP24 contains met2, YMP16 contains
met5, and YMP13 contains met14.
One source of error was that in the three wells that did form bands, faint secondary
bands also formed around the1000 bp when the bands were predicted to be ~500 bp. One
explanation is that the PCR products did not go across the gel far enough, thus some of the
PCR products ended up at around the 1000 bp mark. Another explanation is that the
primers for both MET5 and MET14 resulted in the MET genes to create ~500 bp PCR
products that may have come together to form ~1000 bp PCR products.
AccI was selected and used as the RE for the restriction digest due to the variation of
sizes in the predicted number of restriction fragments for each plasmid, 1,2 and 11, which could
be identified either as pBG1805-MET5, pYES2.1-Met5, and pYES2.1-lacZ+ (Figure 1, Table 1).
This RE had the highest number of restriction fragment lengths that were unique to each
plasmid, making it easier to identify the plasmids1. 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 1). The reactions did not work; not even the undigested
plasmids were visible (Figure 1). Thus, Professor Warner provided the Master Digest, which
used ScaI-HF as the RE (Figure 2). This RE was most likely selected due to the variation of
sizes in the predicted number of restriction fragments for each plasmid (Table 2). The
estimated and predicted sizes of the restriction fragments due to ScaI-HF found on Table 2
supports the prediction that plasmid 1 is pYES2.1-Met5, plasmid 2 is pBG1805-MET5, and
plasmid 11 is pYES2.1-lacZ+ (Figure 2, Table 2). The predicted sizes of the undigested plasmids
were 10902 bp for pBG1805-MET5, 7695 bp for pYES2.1-Met5, and 8964 bp for pYES2.1lacZ+ (Figure 2). The observed sizes of the undigested plasmids corresponded to the predicted
sizes, supporting the prediction of the identities of the plasmids (Figure 2). For plasmid 1, the
predicted 6802 bp was close to the observed ~5000 bp for pYES2.1-Met5 (Table 2). For plasmid
11, the predicted 8071 bp was close to the observed ~8000 bp for pYES2.1-lacZ+ (Table 2). For
plasmid 2, both of the observed fragments, 1267 and 7115 bp, were similar in size to both of the
predicted fragments, ~1000 and ~7000 bp, for pBG1805-MET5 (Table 2). Thus, the data
supports that plasmid 1 is pYES2.1-Met5, plasmid 2 is pBG1805-MET5, and plasmid 11 is
pYES2.1-lacZ+ (Figure 2, Table 2).
Transformants were created using plasmids pYES2.1-Met5, pYES2.1-lacZ+, and
pBG1805-MET5, and replica plating was conducted to determine if complementation had
occurred. Complementation occurs if the Met5 protein is conserved between the two yeast
species, pYES2.1-Met5 (S. pombe homolog) and pBG1805-MET5 (S. cerevisiae gene). Cells
transformed with pYES2.1-lacZ+ served as the negative control. According to the data,

complementation was not observed (Figure 1, Table 1). 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 1, Table 1). These transformed cells were expected to grow
on plates containing galactose because galactose is a carbon source that induces high levels
of GAL1 expression. Galactose results in the expression of the GAL1 gene to increase about
1000-fold above the level observed in glucose1. The GAL1 promoter controls the
transcription of MET and lacZ+ genes, encoded by plasmids, in the transformed cells, which
determines the growth of transformants on selective media1. In contrast, glucose represses
transcription, so little to no growth was expected for the transformants on plates containing
glucose. No growth was observed for both pYES2.1-Met5 and pBG1805-MET5 on the YC-MetUra+Glu media (Figure 1, Table 1). However, unexpected growth was observed on the YC-MetUra+Glu and YC-Met-Ura+Gal plate with the pYES2.1-lacZ+ transformant, which could be due
to contamination of particles because pYES2.1-lacZ+ transformants are not expected to
survive in those media (Figure 1, Table 1). The efficiency of the transformation can be
determined by the transformation efficiency value. The transformation efficiency of
pBG1805-MET5 is 1.10 and pYES2.1-Met5is4.18,whichindicatesthatabout4timesmore
cellsweretransformedforthepBG1805-MET5 transformants than the pYES2.1-Met5
transformants(Table1).Thus,theevidencesupportsthatpYES2.1-Met5transformantsshould
havebeenobservedintheYC-Met-Ura+Gal media but was not observed because
complementation most likely did not occur (Figure 1). One possibility of complementation not
being observed is that the transformed cells could have produced excessive amounts of 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. This
experiment should be repeated multiple times to ensure that complementation not
occurring is not due to errors in the experiment. 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 MET5 gene in the mutant (Figure 1,
Table 1). Thus, S. pombe Met5 is not functionally equivalent to S. cerevisiae MET5.
Analysis of the SDS-PAGE gel indicates a poor amount of proteins present on the
gel (Figure 5). Bands indicate the presence of proteins for its specific plasmid, and
the intensity of the band is directly proportionate to the amount of protein present
in the band. 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).
The presence of galactose results in the expression of the GAL1 gene to increase
about 1000-fold above the level observed in glucose 1. The GAL1 promoter controls
the transcription of MET and lacZ+ genes, encoded by plasmids, in the transformed
cells1. In contrast, glucose represses transcription1. Induced bands were predicted to
be of high intensity to represent an abundant amount of proteins. Bands were
predicted to be in all of the lanes; however, the data does not support the prediction. The
bands are not very clear and only a few bands are present, indicating an error.
Perhaps, the gel was not run long enough because an abundance of blue dye is still

present in the wells. It was not possible to detect fusion proteins in the SDS-PAGE
gel because multiple protein species could be in the bands1.
Analysis of the western blot reveals that the predictions did not match the
observations because protein is absent in Lane 3 (Figure 5). The western blot indicates
that protein expression is possible in the S. cerevisiae MET5 strain transformed with
pBG2805-MET5 and with p2.1YES-Met5 (Figure 5). Lane 1 has an intense band at a size
~180 kDa, similar to the predicted 177.73 kDa (Table 1). Lane 5 has an intense band at a
size of ~168 kDa, similar to the predicted 167.13 kDa (Table 1). The induction of the
GAL1 promoter is demonstrated in Lanes 1 and 5, which were mixed with galactose.
Protein expression did not occur in Lane 3 in the western blot because
perhaps not enough protein was transferred from the gel to the membrane or,
although unlikely, an air bubble was trapped between the gel and the membrane in
Lane 3, resulting in a lack of the protein of interest in Lane 3. Another possible
reason, although very unlikely, that protein expression was not observed in Lane 3 is
that the primary antibodies did not bind to the proteins encoded by pYES2.1-lacZ+
because perhaps the blocking solution saturated both the non-specific and specific
binding sites for the proteins in Lane 31. One possible explanation of the multiple
bands in Lanes 1 and 5 is the insufficient blocking of non-specific protein binding
sites on the transfer membranes or the degradation of proteins because all of the
bands appeared simultaneously1.
The findings enrich the complementation findings because previous data
supported that complementation did not occur since pYES2.1-Met5 transformants were
not observed in the YC-Met-Ura+Gal media; however, fusion proteins are evident in the
western blot, indicating that transformation did occur. Thus, this indicates that the
expressed fusion proteins resulted in the loss of function of the original proteins,
resulting in the death of the cells.