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Analysis of gene conservation of MET17 in Saccharomyces cerevisiae and Neurospora crassa Eric Echeandía-Lube Boston College Investigations in Molecular Cell Biology Sam Dyckman, Group B May 13, 2017

Abstract Experiments were performed to determine if the function of the MET17 gene in Saccharomyces cerevisiae was evolutionarily conserved in Neurospora crassa. Yeast cultures were conserved on YPD agar plates. The yeast strains were first cultured on different media to confirm whether or not they had met mutations. PCR colonies of S. cerevisiae mutant strains were then created and electrophoresed to observe for specific met mutants in the strains. Because these strains were to be transformed with the plasmids pBG1805-MET17, the N. crassa homolog pYES2.1-Met17, or pYES2.1-lacZ, restriction digests were prepared to distinguish which plasmids contained open reading frames (ORFs) of S. cerevisiae or N. crassa. When the plasmids and ORFs were determined, the yeast strains were transformed with the plasmids and replica plated onto selective media. Afterwards, SDS-PAGE and western blot were performed to confirm the presence of proteins, specifically the protein of interest. Successful transformation did not result, which indicates that the N. crassa ortholog did not complement the mutated S. cerevisiae MET17 gene. This suggests failure of conservation throughout evolution and no functional equivalency.

Introduction The methionine biosynthetic pathway is a series of important reactions that are necessary for the growth of S. cerevisiae, particularly for the production of methionine in the yeast. The pathway begins with the reduction of sulfite to sulfide, which is catalyzed by the enzyme sulfite reductase. Downstream in the pathway, MET17, the gene of interest, plays a key role in the pathway by encoding for the enzyme homocysteine synthase. This enzyme catalyzes the reaction between sulfide and O-acetyl homoserine to form homocysteine, which is the precursor to methionine. A mutation in the gene of interest would result in failure to produce homocysteine and, thus, methionine (which is essential to for protein synthesis in the cells.) The methionine biosynthetic pathway in S. cerevisiae is divided into three major parts: synthesis of a four-carbon skeleton, sulfur assimilation, and the creation and attachment of methyl groups (Cherest et al. 1970). All of these steps are essential for yeasts that lack the amino acid methionine. Several genes, such as MET3, MET5 and MET10, among others, regulate the different parts of the pathway (Thomas et al., 1992). Specifically, the MET17 gene in S.

cerevisiae encodes for the bifunctional enzyme, O-acetylhomoserine sulfhydrylase or homocysteine synthase (Thomas and Surdin-Kerjan, 1997). The reaction catalyzed by this enzyme involves O-acetyl-L-homoserine and H2S as the reactants and homocysteine and acetate as the products (Spiropoulous and Bisson, 2000). Mutations, such as deletion of nucleotides in this gene, can lead to a greatly diminished transcription for the Met25p (Thomas and Surdin-Kerjan, 1997). This can ultimately result in a reduction of up to 90% of the activity of the homocysteine synthase (Thomas and Surdin-Kerjan, 1997). Because MET17 (or MET25) is essential for the biosynthesis of methionine, it is important to observe whether this gene has a mutation or not. If there is a met mutant in this gene, the final goal of the pathway cannot be attained; the production of methionine, or ultimately AdoMet is not possible. In this experiment, the group observed the functions of the MET17 gene in the methionine biosynthesis pathway. It also observed the function of the MET17 homologue in N. crassa and compared their functions, which are very similar to that of S. cerevisiae because it has similar enzymatic activity and byproducts (Radford, 2004). Even though both species have convergences methionine biosynthesis pathways, the experiment showed that the function of MET17 was not conserved through evolution of the S. cerevisiae and N. crassa.

Materials and Methods Strains or plasmids Saccharomyces cerevisiae

Genotype or constructs MATα his3-∆1 leu2∆0 lys2∆0 ura3∆0

BY4742 YMP30

MATα his3-∆1 leu2∆0 lys2∆0 ura3∆0 met3::KANR

YMP36

MATα his3-∆1 leu2∆0 lys2∆0 ura3∆0 met4::KANR

YMP37 Plasmids

MATα his3-∆1 leu2∆0 lys2∆0 ura3∆0 met17::KANR Commercial cloning vector

pYES2.1

Yeast overexpression vector, pYES2.1-GAL1:Met17

pYES2.1-Met17

pYES2.1-LacZ

Yeast overexpression vector, pYES2.1-GAL1:LacZ Cloning vector

pBG1805 Yeast overexpression vector, pBG1805-GAL1:MET17 pBG1805-MET17 Table 1. Yeast strains and plasmids used in the experiment. The yeast strains utilized in the study are denoted in table 1.

Selective Plating The group was assigned three met deletion strains (YMP30, YMP36 and YMP37) that were derived from parental strain BY4742 (genotype: MATα his3-∆1 leu2∆0 ura3∆0). First, the given strains were serially diluted before transferring them to the media plates. The group worked with 6 different media: YPD, YC+Met, YC–Met, YC–Met+SO 3, YC–Met+Cys and BiGGY. Both YPD and YC+Met were positive control media because all of the strains were expected to grow in them. YC–Met was a negative control medium because, besides from the parental strain, all

other strains are not expected to propagate in the absence of methionine. Both YC–Met+SO3 and YC–Met+Cys were utilized to observe whether the strains had mutants depending on the growth of the cultures. Lastly, the BiGGY agar plate was used to determine the amount of hydrogen sulfide (H2S) that each strain produced. The darker the strain grew in the BiGGY agar plate, more H2S was produced; the lighter the strain grew, less H2S was produced.

For the dilutions, the group had to follow the procedure to create 5 1:10 dilutions with sterile water for each of the strains (O’Connor, 2017.) After all of the strains were serially diluted, 5µL of each dilution were transferred to each medium. When the culturing was done, the plates were incubated at 30˚C until yeast colonies were sufficient to count.

Lane Strain Primer A (GSP-A) Primer B (GSP-B) 1 MET3A MET3B YMP30 2 MET3A KANR 3 MET8A MET8B YMP36 4 MET8A KANR 5 MET17A (25A) MET17B (25B) 6 MET17A (25A) KANR YMP37 7 MET8A MET8B Table 2. Primers that were utilized for each PCR reaction, with their respective strain. Yeast Colony PCR The group prepared PCR reactions and agarose gels to separate the DNA molecules produced in the PCR reactions. First, the reactions were prepared by taking the primers (shown in Table 2) and creating a 10 µL mixture (5 µL of each primer.) Then, the group transferred a small amount of yeast colony to the primer mixture. To lyse the yeast cells, the tubes were placed in the thermocycler for 15 minutes at 98˚C. After the time elapsed, 10 µL of PCR master mix were added to each tube (O’Connor, 2017.) A 1.25% agarose gel was ten made following the procedure in the laboratory manual (O’Connor, 2017.) Thereafter, the samples for electrophoresis

were prepared by adding 4 µL of 6X loading buffer to the PCR reaction tubes. When these were ready, the members proceeded to fill the electrophoresis tank with TAE buffer to submerge the agarose gel. Following this, they pipetted 10-12 microliters of one sample to each of the wells (numbered 1 through 7) that were created with the comb. On the lane labeled “ladder,” the group pipetted 5 µL of molecular weight standard. The group continued by placing the lid on the tank and applying 130 V at first and then 140 V to separate the PCR products more quickly. After this procedure was done, the group stained the gel with Ethidium Bromide (Et-Br,) which is a fluorescent dye that binds to the DNA and absorbs fluorescent light. The members then examined the agarose gel under UV light (reference Figure 1.) (O’Connor, 2017)

Restriction Mapping The group planned the restriction digest. Because the group was assigned MET17 gene, they worked to purify the plasmids using the Zyppy Plasmid Miniprep Kit and calculate the length of the plasmids (pBG1805, pYES2.1 and pYES2.1-lacZ,) using the NEBCutter tool and the procedures in the Laboratory notebook (O’Connor, 2017.) Afterwards, the group created a summary for the restriction maps. When finishing this, the members used the data to deduce the best restriction enzyme to use according to the least number of fragments produced for the plasmids. Thereafter, the group worked to set up the restriction digests. On different tubes, the group was supposed to prepare the digests by adding 7 µL of the plasmid, 1 µL of CutSmart 10X buffer and 2 µL of the restriction enzyme. Nonetheless, the group committed an error: instead of adding the plasmid 3, 4 and 17 with the restriction enzyme, they added the pBG1805-MET17 with the restriction enzyme, pYES2.1-Met17 with restriction enzyme, and pYES2.1-lacZ with the restriction enzyme. Additionally, control groups were developed with three undigested

plasmids containing 10 µL of the plasmid, 5 µL of deionized water and 3 µL of 6X sample buffer. After doing this, the group incubated the samples at 37˚C for at least 2 hours and were later stored in the freezer. For the last part of the experiment, the group used agarose gels to analyze the restriction digests. To prepare the gel, the group followed the guidelines stated in the Laboratory notebook and prepared 1% agarose gel in 300mL of TAE buffer (O’Connor, 2017.) Afterwards, the group electrophoresed the agarose gel at 151 V and stained it with 5 µL Et-Br. Finally, the group placed the gel in the transilluminator to analyze it.

Complementation Analysis The strain YMP37, with a met deletion, was used to clone MET17 orthologues into two different gene overexpression plasmids: pBG1805-MET17, pYES2.1-Met17, and pYES-LacZ. Afterwards, the group followed the procedure in the laboratory notebook to prepare a transformation master mix (O’Connor, 2017.) This mix, prepared in a microcentrifuge tube, contained 100 μL of 2 M lithium acetate, 400 μL of 50% PEG-3350 and 4 μL of mercaptoethanol. For each individual transformation, 15 μL of denatured salmon sperm DNA, 5μL of miniprep plasmid DNA, and a large yeast colony were mixed into a new microcentrifuge tube labeled with the name of the plasmid of interest. The transformation mixtures were incubated at 37˚C with shaking for 35 minutes (O’Connor, 2017.) The reactions were resuspended and serially diluted at a ratio of 1:10 and cultured on YPD media. Finally, the remainder of the transformed cells were cultured on selective media lacking uracil and incubated at 30˚C, until colonies were detected. Thereafter, the group proceeded with replica plating and complementation. They cultured the colonies into media containing YC-Met-Ura+GLU, YC-

Met-Ura+GAL, and YC+Met-Ura+GLU, following the procedure in the laboratory manual (O’Connor, 2017.)

SDS-PAGE and Western Blot Analysis Cells were prepared for protein extraction by inoculating six tubes of 1mL YC-Ura medium with 2% raffinose and a single yeast colony of each transformant, taken from YPD media. Cultures were incubated at 30˚C overnight. For each transformant, 1 mL of YC+glucose was added to one culture and 1 mL of YC+galactose was added to the other. (O’Connor, 2017)

Proteins were separated on a 12% polyacrymalide SDS-PAGE gel and electrophoresed at 134V for 50 minutes as described in the laboratory manual (O’Connor, 2017) SDS-PAGE gels were stained using simply blue. Proteins from the gel were transferred to a PVDF membrane by electrophoretic transfer at 100 V for 1 hour (O’Connor, 2017) The group utilized 5% nonfat milk in TBS-T to block for nonspecific protein binding sites on the PVDF membrane. Thereafter, the immunodetection was performed as detailed in the laboratory manual. (O’Connor, 2017.) Western blots were incubated with a mouse anti-V5 primary antibody, a monoclonal antibody that binds to the V5 epitope on the proteins expressed from pYES2.1 plasmids. The blot was incubated for 24 hours at 4˚C cold room with slow rocking. Thereafter, the group incubated the western blot with a polyclonal HRP-conjugated rabbit anti-mouse secondary antibody for one hour at room temperature. This antibody was prepared against Fc domains of mouse IgGs and binds to the ZZ domain in the large C-terminal tag of pBG1805-MET17 from Staphylococcus aureaus proteins A (O’Connor, 2016.) The blot was washed, according to the steps in laboratory

manual. (O’Connor, 2016) Finally, the group covered the blot with 1 mL of TMB and allowed the membrane to develop for about 6 minutes (neutralized with deionized water.)

Results

Figure 1. Selective plating of parent type and mutant yeast strains. Parent Type (denoted P in figure), YMP 30, YMP 36, and YMP 37 strains of Saccharomyces cerevisiae were serially diluted and cultured on YPD, YC + Met (YC Complete), YC – Met, YC – Met + Cys, YC – Met + SO3, and BiGGY. Afterwards, the plates were incubated three days at 30° C.

Selective Plating

YPD

YC+Met

YC - Met

YC - Met + Cys

YC - Met + SO3

BiGGY

30

+ growth

+ growth

- growth

+ growth

+ growth

lighter

met 3 36

+ growth

+ growth

- growth

+ growth

- growth

lighter

met 8 37

+ growth

+ growth

- growth

+ growth

- growth

darker

met 17 Table 2. Expected results of the strains that were cultured in the different media. YPD

YC+Met

YC - Met

YC - Met + Cys

YC - Met + SO3

BiGGY

30

+ growth

- growth

- growth

+ growth

+ growth

lighter

met 3 36

+ growth

- growth

- growth

+ growth

- growth

lighter

met 8 37

+ growth

- growth

- growth

+ growth

- growth

darker

met 17 Table 3. Observed results the strains that were cultured in the different media. For this experiment, a selective plating procedure was utilized to determine if S. cerevisiae strains YMP30, YMP36 and YMP37 had mutations in the MET3, MET8 and MET17 genes, respectively. As denoted in Table 2, all of the strains were expected to grow in the YPD medium, given that it had all the nutrients necessary for growth. Effectively, all strains grew in said medium, as seen in Figure 1. Additionally, it was anticipated that all strains would grow in the YC+Met medium because, in the presence of methionine, all strains should be able to proliferate. Nonetheless, as observed in Figure 1 and Table 3, none of the strains propagated. In the YC-Met

medium, a negative control, none of the strains (except for the parental strain BY4247) should have grown (as seen in Table 2.) It was observed that none of the mutant strains grew because, in the absence of methionine, none of the strains are supposed to proliferate. In the YC-Met+Cys, it was expected that all strains would propagate; this expectation was confirmed in the experiment, as seen in Figure 1. Pertaining to the medium with YC-Met+SO3, only strain YMP30 was expected to grow. In Table 3, it is observed that YMP30 was the one that propagated in this medium. Finally, for the BiGGY agar, it was anticipated that YMP30 and YMP36 would result in a lighter colony color than YMP37, which was expected to have a darker colony color. The results obtained for this medium were also confirmed. As seen in Figure 1 and Table 3, all strains grew with the color that was expected. This data supported the idea that YMP30, YMP36 and YMP37 had mutations in the MET3, MET8 and MET17 genes, respectively.

Yeast Colony P

Figure 2. Colony PCR of Saccharomyces cerevisiae mutant strains, YMP 30, YMP 36, and YMP 37 with gene specific primers (GSP) and kanamycin resistance primers (KANR). PCR products were separated using electrophoresis on 1.25% agarose gel. The sizes of molecular weight standards are shown in lane “Ladder.” Lane 1: YMP30 with MET 3A and MET 3B primers; Lane 2: YMP30 with MET 3A and KANR B primers; Lane 3: YMP36 with MET 8A and MET 8B primers; Lane 4: YMP36 with MET 8A and KANR B primers; Lane 5: YMP37 with MET 25A and MET 25B primers; Lane 6: YMP37 with MET 25A and KANR B primers; Lane 7: YMP37 with MET 8A and MET 8B primers.

Lane

Strain

1

Primer A (GSP-A)

Primer B (GSP-B)

MET3A

MET3B

MET3A MET8A

KANR MET8B

MET8A MET17A (25A)

KANR MET17B (25B)

YMP30 2 3 YMP36 4 5

YMP37

Predicted lengths (bp) No PCR product expected 605 No PCR product expected 631 No PCR product

Observed lengths (bp) N/A

None N/A

~700 N/A

expected 6 MET17A (25A) KANR 482 None 7 MET8A MET8B 825 ~600 Table 4. Primers that were utilized for each PCR reaction, with their respective strain. Following the results from the Selective Plating experiment, the group decided to create the primer mixtures denoted in Table 4. The PCR reactions and the electrophoresis works the following way: if a combination of primers GSP-A and GSP-B for gene X yields a product, then gene X does not have a met mutant; if a combination of GSP-A and KAN R yields a product, then gene X has a met mutant. Given this information, the group anticipated the solutions in lanes 2 (because YMP30 had a met3 mutant,) 4 (because YMP36 had a met8 mutant,) 6 (because YMP37 had a met17 mutant,) and 7 (because YMP37 did not have a met8 mutant) to generate PCR products. Nonetheless, not all of these anticipated results were observed in the experiment. As seen in Figure 2, lanes 2 and 6 did not have PCR products. Furthermore, lanes 4 and 7 yielded a PCR product of about 700 and 600 base pairs, respectively, as seen in Figure 2. The group also expected no PCR products for lanes 1, 3 and 5 (as seen in Table 4.) Effectively, in lanes 1, 3, and

Figure 3. Restriction enzyme digests of pBG1805-MET17, pYES2.1-MET17 and pYES2.1-lacZ. Saccharomyces cerevisiae genes were cloned into the pBG1805 vector and Neurospora crassa and LacZ genes were cloned into the pYES2.1 vector. Plasmids were digested using the ScaI restriction enzyme. Lane 1 contains digested pBG1805-MET17, lane 3 contains digested pYES2.1-MET17, and lane 5 contains digested pYES2.1-LacZ. A molecular weight standard was used as a control in lane “Ladder” to determine the size of the products. Undigested plasmids were also used as a control group: lane 2 contains plasmid 3, lane 4 contains plasmid 4, and lane 5 contains plasmid 17. All plasmids were run on a 1% agarose gel at 151 V and stained with Ethidium Bromide.

Predicted length (bp)

Observed length (bp)

Lane 1- pBG1805-MET17 with RE

5328, 2577

5300*, 2600*

Lane 2- Undigested plasmid 3

Anomalous bands

9000

Lane 3- pYES2.1-Met17 with RE

6364, 893

5300*, 2600*

Lane 4- Undigested plasmid 4

Anomalous bands

7900

Lane 5- pYES2.1-lacZ with RE

8071, 893

5300*, 2600*

Lane 6- Undigested plasmid 17

Anomalous bands

7200

Table 5. Predicted vs. Observed lengths of the restriction digests. *These observed lengths are due to error. Lanes 1, 3, and 5 were loaded incorrectly. The Restriction Mapping experiment was done to identify and distinguish which plasmids (seen in Figure 5) contained S. cerevisiae or N. crass...