Transforming, Tagging, and Identifying PRE2 in Saccharomyces Cerevisiae Using GFP

Author: Brenna Moya

Lab Partners: Narissa Lamont and Michael Marshall

I. Abstract

S. cerevisiae is an extensively studied and commonly used model organism for various

scientific fields due to being an easily accessible tool. Since the entire genome of S.

cerevisiae has been sequenced, S. cerevisiae is an ideal candidate for observing genetic

manipulation via the use of protein tags, such as GFP. By tagging a specific gene with

GFP, researchers are able to locate, identify, and determine the function of the products

that are produced by that specific gene. In this experiment, yeast cells were given an

unknown piece of transformant DNA that contained GFP, and were transformed via

homologous recombination. The newly transformed cells underwent DNA extraction,

polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing analysis in

order to discover the identity of the unknown, tagged gene. After being successfully

transformed and sequenced, the unknown gene that was tagged was correctly identified

as PRE2. PRE2 is an essential gene that is a member of beta-type proteasomal gene

family, and is responsible for the chymotryptic activity of proteosomes. PRE2 can be

found in the many species in the kingdom of animalia, such as humans where the gene

functions to enable peptidase activity and can act upstream of, or within proteolysis.

Also, in studies mutated forms of PRE2 have been found to be a homolog of RING10,

which is a gene that is utilized in the production human MHC II. Other similar studies

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 may be conducted to further the knowledge of the function and possibly other potential

functions of PRE2.

II. Introduction

Saccharomyces cerevisiae, commonly known as baker’s yeast, is an extensively studied

model organism that has been utilized in various scientific fields and a large range of

other applications. In 1996, during the Genome Project, the entire genome of S.

cerevisiae was sequenced, which provided a main reference for comparing all eukaryotic

genomes that have been sequenced, such as humans. Out of the 5,780 protein coding

genes in S. cerevisiae, more than 80% have been functionally identified. S. cerevisiae is

an unrivaled model organism for studying fundamental cellular processes, including

metabolism, DNA replication, cell division, and the cell cycle. Also, S. cerevisiae, along

with other yeast models systems, are advantageous model organisms due to being an

easily available tool, having the ability to grow rapidly, growing in simple, inexpensive

media, simple manageability, can be safely used, and can be cultivated and reproduced

with ease. S. cerevisiae continues to be one of the most extensively used model organism

for its basic structure and genome compatibility (Tulio, 2022).

In the early 1960s, researchers discovered the first green fluorescent protein (GFP)

while studying the Aequorea victoria jellyfish’s bioluminescent properties (Kremers, et

al, 2011). GFP since has become a widely used tool in the tagging of protein due to the

unique characteristic of not needing any additional cofactors or substrates to be functional

(Kaishima, et al, 2016). The ability to engineer GFPs derived from jellyfish and other

organisms have thus allowed for the production of numerous useful fluorescent proteins

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 in a variety colors, such as cyan and yellow mutant (Kremers, et al, 2011). Engineering

has also allowed for the creation of GFPs that are specific for certain organisms. Over the

years, GFPs have commonly been used in model organisms, such as S. cerevisiae, to

study the localization of proteins via genomic transformation (Kaishima, et al, 2016).

In this experiment homologous recombination, which is when horizontally

transferred DNA is integrated into the recipient organism’s genome, was utilized to

incorporated the GFP in the genome of S. cerevisiae to identify an unknown gene ( Kung,

et al, 2013) At the conclusion of the experiment the unknown gene was identified as

PRE2, which is a member of beta-type proteasomal gene family. PRE2 is deemed as

essential for life, since the gene codes for the protein that is responsible for the

chymotryptic activity of proteosomes. It is predicted that PRE2 has an amino acid length

of 287 and molecular mass of roughly 31.6 kDa (Heinemeyer, et al, 1993). Also, the

PRE2 has been found in many kingdoms of animalia, such as Homo sapiens, Mus

musculus, and Drosophila melanogaster.

PRE2 has been utilized in various studies, such as those involving human major

histocompatibility complexes (MHC). When the PRE2 gene undergoes two missense

mutations, it becomes homologous to the RING10, which is a gene in humans that is

encoded in MHC class II region. The high percentage of homology, which is 60%

identity, between the PRE2 and the RING10 suggest that RING10 produces a protein that

central in the chymotryptic activity of MHC II. Also, this homology suggests that it is

possible to replace RING10 with mutated PRE2, and vice versa. This theory of

replacement could become very beneficial as both PRE2 and RING10 are essential for

cellular survival (Heinemeyer, et al, 1993).

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 The purpose of this experiment was to transform, tag, and identify an unknown

gene. Techniques, such as transformation via homologous recombination, DNA

extraction, polymerase chain reaction (PCR), gel electrophoresis, and DNA sequencing,

were utilized to correctly identify the PRE2 gene.

III. Materials and Methods

A. Transformations

A small number of yeast cells were prepared in a YPD media and were allowed to grow

while being shaked at 30C. The OD600 of the culture was checked using a spectrophometer

and then the culture was used to inoculate a new culture of 50 mL at a OD600 of 0.38. The

culture was allowed to grow for 4 hours at 30C with shaking. After the 4 hours, the 50 mL

culture was spun down in the microcentrifuge and the resulting cell pellet washed with sterile

water. Then, the cell pellet was resuspended in 1 mL of 100 mM of LiAc. The tubes were

then re-spun in the microcentrifuge at a high speed for 15 seconds. After being re-spun, the

LiAc was taken off the cell pellet and the cells were resuspended to an ending volume of 500

uL using 100 mM LiAc. Then, these suspensions was separated into two tubes; one was the

experimental (T) tube and the other was the negative control tube. The tubes were then

microcentrifuge again using a high speed for 15 seconds. Once done, the supernatant was

removed from the tubes. Next, 240 uL of 50% PEG, 36 uL of 1.0 M LiAc, and 5 uL of

single-stranded DNA was added to the cell pellets in both tubes. Then, the experimental (T)

tube received 70 uL of the DNA fragment and the negative control tube received 70 uL of

sterile water. Both tubes were vortexed for 1 minute or when the cell pellet was fully

dispersed in the tube. The tubes were then put in the incubator for 30 minutes. After

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 incubation, the tubes were heat shocked at 42C for 25 minutes. Once heat shocked, the tubes

were microcentrifuge for 1 minute at 6000 rpm. Following this, the transformation mix was

removed leaving behind the cell pellets. Then, each tube received 200 uL of sterile water so

that the cells could resuspended gently using a pipette. The experimental (T) tube and the

negative control tube were then plated evenly on different -HIS selection plates with the use

of glass beads. The glass beads were discarded after use. The -HIS selection plates were then

placed in an incubator at 30C for a couple of days so that colonies were able to grow. The

professor streaked the colonies on a fresh plate for the next lab. (Burris, 2023)

B. DNA Extractions

To begin, a sterile, wooden stick was used to scrap a pea-sized amount of cells off the plate.

Then, the scraped off cells were placed in 1.5 mL Eppendorf tube that contained 100 uL of

extraction buffer, which was composed of 1 mL of 2% Triton X-100, 2.5 mL of 1% SDS,

0.08 g of 10 mM Tris pH 8, 0.29 g of 100 mM NaCl, and 0.01 g of 1 mM EDTA. The above

procedure was done for cell samples “A” and “B.” Next, one scoop of glass beads were

added to each of the Eppendorf. Then, 100 uL of chloroform was carefully pipetted into each

tube . Both tubes were vortexed for 2 minutes and then microcentrifuge at 13,200 rpm for 5

minutes so that the lipids, proteins, and other cellular debris were pelleted. The DNA was

finally extracted by pipetting 15 uL of the supernatant in the top layer of the tube into a new

500 uL Eppendorf tube. This procedure was done for each sample. (Burris, 2023)

C. Polymerase Chain Reaction (PCR)

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 To start, the two master mixes for the PCRs were made with 206.25 uL of ddH2O, 25 uL of

10x Taq buffer, 5 ul of forward primes, 5 uL of reverse primer (reverse primer #1 was used

for the wild-type PCR and reverse primer #2 was used for the knock-in PCR), 5 uL of

dNTPs, and 1.25 uL of 5 units/uL Taq pol. Once all components for the master mix were

added, the tubes were mixed thoroughly. Then, 49.5 uL of the master mix was pipetted into

each of the tubes for each of the PCR sets. Next, 0.5 uL of the positive control isolated DNA,

0.5 ul of the transformant DNA, and 0.5 uL of sterile water were pipetted into the respective

positive control, experimental, and negative control PCR tubes. Each of the PCR tubes were

vortexed to ensure everything was mixed thoroughly. After being completely mixed, the PCR

tubes were placed in a thermal cycler and the “Taq polymerase” program was begun. This

program is comprised of three stages: denaturation, annealing, and elongation, and each stage

include either an increase or decrease in temperature for a certain amount of time. For

denaturation, the temperature was increased to 95C for 1 minute. After is the annealing

stage, which results in a decrease in temperature for 30 seconds. Finally is elongation, during

this stage the temperature increases to 72C for 7 minutes. The thermal cycler repeated the

cycle of these stages 30-40 times. The number of cycles if dependent on the what the thermal

cycler was set to. (Burris, 2023)

D. Agarose Gel Electrophoresis and Sequencing of DNA

To begin, a 1% agarose gel was made by dissolving 1 g of agarose into 100 mL of 1x TAE in

a 500 mL Erlenmeyer flask. The mixture of agarose and TAE running buffer was heat in a

microwave, and once the buffer began to boil the mixture was gently swirl and then

microwaved again. The process of heating and mixing was repeated until the granules of

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 agarose were dissolved. Next, two combs were added to the casting tray in the casting mold.

One comb was placed at the top of the tray and the other comb was placed half-way down the

casting tray to ensure there was enough wells for the PCR samples. After, the dissolved

agarose mixture was poured into a casting tray that was in the sideways positions in the

casting mold.

Once the agarose gel solidified, the gel was submerged in TAE running buffer in an

electrophoresis apparatus. The agarose gel was positioned so that that the wells were toward

the negative end of the electrophoresis apparatus. Before the PCR samples could be placed in

the wells, 43 uL of loading buffer was pipetted on a piece of parafilm and 5 uL were

aliquoted to each PCR tube. Then, 5 uL of DNA ladder was pipetted on the parafilm and 1

uL of loading buffer was mixed into the DNA ladder. Finally, all the samples were loaded

into the gel using the following gel map (Figure 2.). Once the samples were all loaded, the

lid was placed on the gel chamber and the electrodes that were attached to the power supply

were placed in the designated positions. The gel was ran for 25 minutes at 110 volts. After

the 25 minutes were up, the gel was placed under the UV illuminator so that the bands on the

gel could be visualized.

After the bands on the gel were visualized, one band was chosen to be prepared for DNA

sequencing. To prepare the selected the band for sequencing , the band was cut out of the gel

using a razor blade and placed in a 1.5 mL microcentrifuge tube with 500 of DF buffer. Next,

the microcentrifuge tube was placed in a 55C water bath for roughly 10 minutes. During the

10 minutes in the water bath, the tube was inverted every 3-4 minutes. Once the agarose gel

was fully dissolve, the microcentrifuge tube was left to cool to room temperature. After the

sample was cooled, the contents were transferred into a DF column and then placed in a

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 microcentrifuge for 30 seconds at 13,200 rpm. The flow through was discarded and 600 uL

of wash buffer was added into the DF column. The column was left to sit for 1 minute and

then microcentrifuge for 30 seconds at 13,200 rpm. The flow through was discarded and the

column was microcentrifuge for 3 minutes at 13,200 rpm, and then left to dry. Once dried,

the column was placed in 1.5 mL microcentrifuge tube and 30 uL of elution buffer was added

to the center of the matrix of the column and the column was left to sit for 2 minutes. Finally,

the column after the 2 minutes was microcentrifuge for 2 minutes at 13,200 rpm so that the

newly purified DNA fragments could be collected.

To set up for sequencing the Nanodrop was blanked with elution buffer. Next, 1 uL of the

purified DNA fragments were placed on the pedestal of the Nanodrop. Both the

concentrations and A260/A280 ratio were recorded. The DNA sequencing company,

Genewiz, required that the sent off PCR tube had 27 ng of DNA. The concentration from the

Nanodrop was used to calculate the amount of uL of purified DNA that was needed for the

PCR tube. The final PCR tube that was sent off to the sequencing company contained 1.79

uL of purified DNA fragment, 10.7 ul of sterile water, and 2.5 uL of forward primer. (Burris,

2023)

E. Sequencing Analysis and Fluorescent Microscopy

To begin, the “N”’s on the chromatogram that was received from Genewiz were assigned

either an A, C, T, or G nucleotide identity based on the color of the peak at the “N”’s position

on the chromatogram. Then, the sample sequence was referenced with the S288C strain of

Saccharomyces Cerevisiae using the NCBI “Blast Genome” website. As a result, the identity

of what sequence, chromosome gene, and protein that the sample sequence closely matched

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 with was given. Next, the gene name that resulted was put into the SGD and all significant

information about the gene was recorded.

To prepared the transformed yeast cells for fluorescent microscopy, the cells were

streaked two days prior of the lab so actively growing cells in log phase could be collected

for the microscopy. On the day of the lab, two 5 uL drops of water were pipetted on a slide

separately for each group’s sample. Then, a pipette tip was used to take a small amount of

yeast cells off the plate from the colony that was sent off for sequencing, either colony “A”

or “B.” Next, the yeast cells on the pipette tip were mixed in with one of the water droplets.

Finally, a coverslip was placed on the water droplet and the yeast cells were visualized with

the fluorescent microscope. (Burris, 2023)

IV. Results

After the process of transformation,

the negative control (-) tube and

experimental (T) tube were placed on

-HIS selection plates. The absence of

grow on the negative (-) control -HIS

selection plate was recorded. For the

experimental (T) -HIS selection plate,

a colony forming unit count (CFU)

was conducted due to growth on the plate. The CFU results were 5 small, white colonies

and these results were recorded (Figure 1.).

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 At the conclusion of the DNA extraction process, samples “A” and “B” were

visualized in the respective tubes and two distinctive layers were seen. The top layer was

clear and the bottom layer was cloudy. Both samples did have a thin, white scum portion

on the top of the clear layers. The DNA, which was located in the top, clear layer was

extracted using a pipette. This extraction process was done carefully so that the bottom,

cloudy layer, and white scum portion was not disturbed in both samples.

Following the PCR, an agarose gel electrophoresis was run with the wild-type PCR

sets, knock-in PCR sets, and the DNA ladder. Before the electrophoresis was ran, all the

PCR sample were loaded correctly in the wells in the agarose gel. A UV illuminator was

used to visualized the results of the electrophoresis and a total of four bands were seen.

The wild-type positive control in lane 2 had a band at 500 bp. The wild-type “B” sample

in lane 4 had a band at 3,000 bp. Also, both knock-in PCR samples “A” and “B" had

bands at 1,500 bp. These results were recorded and can be seen in (Figure 2.).

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 After visualization of the gel, the knock-in sample “B” was cut out of the gel and

purified using a DF column. Both the concentration and A260/A280 ratio were recorded

using the newly purified knock-in sample “B” DNA. The concentration was 15.1 ng/uL

and the A260/A280 ratio was 2.72. Finally, the sample was sent to the DNA sequencing

company, Genewiz, for sequencing.

A sample was taken from knock-in

sample “B” for visualization and analysis

under the fluorescent microscope. On the

fluorescent microscope, both the

brightfield and GFP setting were used on

the cells to compared structure of the yeast

cell and probable location of the GFP tagged protein. The location of the fluorescence on

the GFP setting image was recorded (Figure 3.).

Following the arrival of the

knock-in sample “B” sequencing

results from Genewiz, the resulting

sequence was copy and paste into the

NCBI “Blast Genome” program for

sequence analysis (Figure 4.). The

“Blast” resulted in two matches that

matched the knock-in sample “B”

that was put in. The first match, chromosome XVI, had a query coverage of 82% and a

percent identity of 100%, and was chosen for further analysis (Figure 5.). The protein

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 sequence that best matched the knock-in sample “B” sequence in the database for

chromosome XVI was PRE2, which is a protein for the proteasome core particle subunit

 5. Proteasome core particle subunit  5, produced by the gene PRE2, works in a multi-

protein complex. This protein is responsible for the chymotryptic activity of the

proteasome and localizes to the peroxisome in oleate-growing cells. The proteasome core

particle subunit  5 is found in the animalia and fungi kingdoms and functions to enable

peptidase or endopeptidase activity. This protein is located in the nucleus, cytosol, ER

membrane, and the proteasome storage granule. PRE2 has a gene length of 864 bp and is

287 amino acids long. Found just upstream of PRE2 is LEC1 and just downstream is

FHIL1. The molecular weight of proteasome core particle subunit  5 is 31631.9 Da and

there is 10384 ( 2615) molecules of this protein per cell. Lastly, proteasome core

particle subunit  5 has a total of 970 physical interaction for 721 unique genes.

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 V. Discussion/Conclusion

After the process of transformation, a colony forming unit count (CFU) was

conducted on both the experimental (T) -HIS selection plate and the negative control

(-) -HIS selection plate. The experimental (T) -HIS selection plate had a total of 5

colonies, which meant that the transformation on the wild-type yeast cells was a

success. This successful result was because the transformant DNA that was given to

the experimental (T) yeast cells contained a selection cassette, HIS3, that allowed for

the cells that had undergone the process of transformation to produce histamine, an

essential amino acid, on the histidine-lacking selection media. Therefore, any cells

that did not receive or take up the transformant DNA would not have grown on the -

HIS selection plates. This statement aligns with the negative control (-) -HIS selection

plates because when the CFU was conducted for this plate there were 0 colonies or no

growth. Since the transformant DNA was not given to the negative control (-) cells,

but instead sterile water, there should not have been any growth on the selection plate,

making this result valid (Figure 1.).

The following lab was DNA extraction, which was performed so that the newly

transformed DNA from the experimental (T) plates could be prepared for PCR.

During the DNA extraction process, samples “A” and “B” were visualized in the

respective tubes and two distinctive layers were seen. When the samples were spun in

the microcentrifuge, the nonpolar and polar layers of the sample separated resulting in

the two layers that were seen. The dense, nonpolar bottom layer contained cell

membranes, lipids, cell walls, and proteins. The less dense, polar layer contained the

DNA that was wanted for the extraction. Also, there was a thin, white scum layer that

13
 was on the top of the polar layer, which most likely contained other cellular debris

that had not yet settled to the bottom layer. When extracting the DNA to different

tubes, caution was used so that the bottom and white scum layer were not bothered.

The resulting tubes that contained just the DNA from samples “A” and “B” were

clear, indicating that no cellular debris was in the final samples.

Several bands were expected to be seen on the agarose gel under the UV

illuminator. For the wild-type PCR set, we expected to see a band on the wild-type

positive control (+) at 500 bp because the forward and reverse primers that were

received in this sample were complementary to the untransformed, wild-type version

of the gene. Also, for the wild-type PCR, a band at 3,000 bp should be present for

both samples “A” and “B,” because the forward and reverse primers were

complementary to untransformed, wild-type version of the gene. The reason this band

is longer than the wild-type positive control (+) is because the transformant DNA did

not delete the wild-type sequence, but rather annealed downstream of the original

sequence, meaning that during the PCR process both the wild-type and transformant

sequence were transcribed together. For the knock-in PCR, a 1,500 bp band should be

seen for both samples “A” and “B” because samples received forward and reverse

primers that were complementary to the transformant DNA. The bands at samples

“A” and “B” should be the only bands seen for the knock-in PCR.

Following the completion of the gel electrophoresis, the agarose gel was

visualized under the UV illuminator and a total of 4 bands were seen. The wild-type

positive control was seen at 500 bp as expected (Figure 2, Lane 2). Also, wild-type

sample “B” was seen at the expected 3,000 bp, which meant that the transformant had

14
 integrated at the right location of just downstream of the wild-type sequence (Figure

2, Lane 3) . The wild-type sample “A” band was not seen as expected, but this

unexpected result was most likely due to errors by Taq polymerase since Taq

polymerase is not a perfect tool and makes frequent mistakes. For the knock-in PCR,

both knock-in samples “A” and “B” had a band at 1,500 bp, which meant that

transformation was successful and that the transformant integrated at the correct

location on the gene (Figure 2, Lanes 7 and 8). Both the wild-type and knock-in

PCR’s negative controls did not produce a band, which was a valid result as it showed

that there was no contamination in the master mixes of the PCR (Figure 2, Lanes 5

and 9). If contamination was present in the PCR master mixes, then a band in the

negative controls of both the wild-type and knock-in PCR would have been present.

Also, no band was apparent for the knock-in positive control (+) and this is a valid

result (Figure 2, Lane 6). If a band was present for the knock-in positive control (+),

then that would have meant that the wild-type sequence was still present in the

transformant sample and that the transformant DNA had integrated somewhere else.

After visualizing the gel under the UV illuminator, the knock-in sample “B” band

was chosen to be sent off to the DNA sequencing company, Genewiz, for sequencing.

In order to be sent off, knock-in sample “B” had to be purified with the use of a DF

column. Once purified, the A260/A280 ratio and concentration was recorded. The

A260/A280 ratio was 2.27. Since the A260/A280 ratio was above 1.8, this meant that

the DNA sample was relatively pure and ideal for sequencing. The concentration of

the DNA sample was 15.1 ng/uL.

15
 When knock-in sample “B” DNA sequence was sent back from Genewiz, the

sequence was put into NCIB “Blast Genome” program so that knock-in sample “B’s”

sequence could be compared to other sequences in the database. The “Blast” resulted

in two matches that matched the knock-in sample “B” that was put in. The first

match, chromosome XVI, had a query coverage of 82% and a percent identity of

100% . The second match, chromosome VIII, had a query coverage of 9% and a

percent identity of 100%. Due to the higher query coverage of the first match,

chromosome XVI was chosen. After selecting the first match, two possible ranges

were given and the first range was selected for further analysis due to continuity of

the provided sequence. By comparing knock-in sample “B’s” sequence to sequences

in the database, the unknown gene was identified to be PRE2.

The PRE2 gene produces a protein for the proteasome core particle subunit  5.

Proteasome core particle subunit  5 is part a complex that is comprised of many

proteins. This protein is responsible for the chymotryptic activity of the proteasome

and localizes to the peroxisome in oleate-growing cells. The PRE2 gene is not only

found in yeast, but is also found in Homo sapiens, Mus musculus, Rattus norvegicus,

Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans. In Homo

sapiens, the proteasome core particle subunit  5 enables peptidase activity and acts

upstream of or within proteolysis. For Mus musculus, this protein enables

endopeptidase activity, is involved in differentiation of fat cells, and acts upstream of

or within antigen processing and presentation. In Drosophila melanogaster, the

proteasome core particle subunit  5 functions is predicted to enable endopeptidase

activity, is involved in the centrosome cycle, and involved in proteasome-mediated

16
 ubiquitin-dependent protein catabolic process. The locations of proteasome core

particle subunit  5 is the nucleus, cytosol, ER membrane, and the proteasome storage

granule.

The PRE2 gene makes a total of 970 total physical interaction for 721 unique

genes. Two specific interactions of PRE2 gene is phenotypic enhancement and

synthetic growth defect. A phenotypic enhancement interaction is inferred when

mutation or overexpression of genes results in enhancement of any phenotype

associated with mutation or over-expression of another gene. The synthetic growth

defect interaction is inferred when mutations in separate genes, each of which alone

causes a minimal phenotype and results in a significant growth defect under a certain

condition when combined in the same cell.

A sample of knock-in “B” was prepared on a slide for visualization under the

fluorescent microscope with both brightfield and GFP settings (Figure 3.). When the

slide was viewed under the GFP setting, fluorescence was seen in the nucleus and

cytoplasm. This result is consistent to the possible locations of the proteasome core

particle subunit  5 that were provided by the SGD website. The cells under both the

brightfield and GFP settings were normal in appearance.

Further analysis of the PRE2 could be conducted by adding mutations to specific

regions on the gene. By adding mutations the effects on the PRE2 and the products of

the gene can be observed. These observations could further the understanding of the

possible functions of PRE2. Previous studies have been done on mutated forms of

PRE2. It was found that two specific missense mutations on the PRE2 create a

homolog to RING10, which is a gene that is part of the class II major

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 histocompatibility complexes in humans (Heinemeyer, et al, 1993). By performing

similar studies, further knowledge on the function and other potential purposes of

PRE2 can be acquired.

VI. Literature Citation

Burris, A. (2023) Genetics Lab Protocols 1-5. BIO305, Missouri Southern State University.

Heinemeyer, W., Gruhler, A., Möhrle, V., Mahé, Y., and Wolf, D.H., (1993) PRE2, Highly

Homologous to the Human Major Histocompatibility Complex-linked RING10 Gene, Codes

For a Yeast Proteasome Subunit Necessary For Chrymotryptic Activity and Degradation of

Ubiquitinated Proteins. Journals of Biological Chemistry. 268, 5515-5120

Kaishima, Misato., Ishii, Jun., Matsuno, Toshihide., Fukuda, Nobuo., and Kondo, Akihiko.,

(2016) Expression of Varied GFPs in Saccharomyces Cerevisiae: Codon Optimization Yields

Stronger Than Expected Expression and Fluorescence Intensity. Scientific Reports. 6, 1-15

Kremers, Gert-Jan., Gilbert, G. Sarah., Cranfill, J. Paula., Davidson, W. Michael., and Piston,

W. Davis., (2011) Fluorescent Proteins At a Glance. Journals of Cell Science. 124, 1-5

Kung, H. Stephanie., Retchless, C. Adam., Kwan, Y. Jessica., and Almeida, P. P. Rodrigo.,

(2013) Effects of DNA Size on Transformation and Recombination Efficiencies in Xylella

Fastidiosa. Applied and Environmental Microbiology. 79, 1712-1717

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 Tullio, Vivian. (2022) Yeast Genomics and Its Applications in Biotechnological Processes:

What Is Our Present and Near Future? Journal of Fungi. 8, 1-16

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