The Role of Epstein Barr Virsus in Oncogenesis

Abstract:
Epstein- Barr virus is one of the most common human viruses, infecting
approxi matel y 95%of the worl d’s population. EBV viral proteins are able to mani pulate
cellular pat hways essential for mai ntaini ng homeostasis by disrupting nor mal protein
interactions. The field of systems bi ology is ideal for studyi ng the effects of viruses on
human hosts because the reductionist approach tounderstandi ng cellular pathways and
disease pat hogenesis is challenged and disregarded. Ayeast-t wo-hybrid met hod was
utilized to screen 216 EBV proteins agai nst 15, 483 human proteins, producing 188
positive candi dates. The discovery and anal ysis of EBV- human proteininteractions has
the potential toreveal howt he disruption of cellular pat hways can result inthe
devel opment of EBV-associated diseases, includi ng cancer.
I. Introduction:
Syste ms Bi ol ogy and Interactomes
The field of systems bi ology has emerged as a result of the successful completion
of the Human Genome project, which made entire genome sequences for human and
numerous model organisms easily accessible. Before the availability of entire genome
sequences researchers approached compl ex questions regardi ng cellular pathways and
disease pat hways by focusing on indi vi dual components of molecular processes. Systems
bi ol ogy challenges the reductionist approach by proposi ng an integrated approach to
understandi ng biol ogical processesi
. Marc Vi dal explains, “the idea of systems bi ol ogy
presupposes that no life for mcan be i magi ned without compl ex systems for med by
interacting genes and macromol ecules, or cells at a hi gher scalei i
.”

The “system” approach to understandi ng cellular pat hways and disease
devel opment focuses on the mappi ng of compl ete interaction net works, or interact omes.
Inthe field of proteomi cs, interact ome maps are graphical representations of indi vi dual
proteins and the protein-proteininteractions that occur withi n a celli i i
. Once these
interactions are observed and validated, biological processes can be better understood.
More i mportantly, complete interactome maps may reveal howexternal factors, such as
viruses, have the potential to mani pulate cellular processes.
The construction of interact ome maps requires the availability of open-reading
frames ( ORFs), or the protein codi ng sequence of a gene fromthe start codon tothe stop
codon, excl udi ng the 5’ and 3’ untranslated regions ( UTRs)i v
. Earlylarge-scale proteomi c
anal ysis utilized cDNA pools, which were protein-encodi ng genes incl udi ng the 5’ and 3’
UTRs. In many instances, researchers reported hi gh instances of false positives as a result
of hybrid protein expression inthe wrong readi ng frame or fromt he 5’ or 3’ UTRs v
.
ORFs, which excl uded untranslated regi ons, not onl y mi ni mi zed the number of false
positives, but also be easilytransferred into numerous expression vect ors and used by a
variety of protein-interaction screeni ng met hods. The effectiveness of open readi ng
frames has been best observed inthe high throughput yeast t wo-hybrid ( Y2H) screeni ng
met hod.
Y2 H Syste m
Si nce its devel opment in1989 by Fields and Song, the Y2Hsystemhas emerged
as a wi del y accepted method for deter mi ni ng protei ninteractions in vivo. The systemhas
been successfully used in numerous large-scale studies, effectivel yidentifying protein
interactions in humans and several model organisms. It is relativel yinexpensi ve because

eli mi nates the need for antibody production and protein purification prior to screeni ngv i
.
The Y2Hscreeni ng process is flexi ble inthat it relies on readily available genome
sequences, which can be easilytransfor med into a wi de variety of expression vect ors
through recombi national cl oni ng. Lastly, the Y2Hsystemis compatible with Gen Mat e ©,
Aquarius ©and Genzyme© robotic liqui d handling systems, maki ng the screeni ng
process accurate and relatively rapi d.
The general Y2H procedure invol ves splitting a transcription factor, commonl y
GAL 4, intot wo domai ns: the DNA- bi ndi ng domai n ( DB) and the activation domai n
( AD). The DNA- bi ndi ng domai n activates the expression of an adjacent reporter gene by
bi ndi ng tothe upstrea mactivating sequence ( UAS), while the activation domai n is
invol ved in asse mbling transcription factors needed for the initiation of transcription. The
expression of the reporter gene, often HI S3, is dependent on the interaction of AD and
DB, made possible by fusion proteins. ORFs, which code for the proteins of interest, are
expressed as fusion proteins bound tothe AD and DB domai ns. When the protein
encoded by the t wo different ORFs interact, transcription is activated and the reporter
gene is expressedv i i
.
Aut o-activat ors are proteins, fused tothe DB domai n, which do not require
recruit ment of ADt othe promot er sequence toinitiate transcriptionv i i i
. Si mpl y, aut o
activat ors are able toinitiate transcription without interacting with proteins fused tothe
activation domai n. They are a common source for false positives because they appear to
react with fusion proteins on AD, when no interaction is occurring. Aut o activators can
be selected for prior to starting the screen and throughout the phenot ypi ng process.

The Y2Hscreeni ng met hod for proteininteractions utilizes t wo hapl oi d yeast
strains, designated as either AD or DB. ORFs, which are expressed as AD and DBfusion
proteins are transfor med intothe correspondi ng yeast straint hrough a gateway
recombi nation cloni ng process. The t wo yeast strains are mated allowi ng for the
interaction of AD and DB, as well as the expression of the reporter gene. The activation
of the reporter gene, confir mi ng protein-proteininteractions, can be detected by a col or
change or growt h on selective mediai x
. Use of the Y2 H systemhas resulted inthe
construction of interact ome maps for numerous model organis ms, as well as the
identification of virus-host proteininteractions excl usive to Epstein- Barr virus.
There are many advantages to utilizing S. cerevisiae during the Y2Hscreeni ng
process. The first, and possibly most i mportant, is that many yeast genes are homol ogous
wit h human genesx
. This offers an effective way to research and identifythe potential
cause of numerous human diseases, without using human cells, thus avoi ding numerous
et hical dilemmas. S. cerevisiae has a fully sequenced genome, in addition to a fast and
si mpl e life cycle, maki ng it a model organis mfor the Y2Hscreeni ng process.
Gate way Recombi nation Cl oni ng
The Gateway recombi nation cloni ng system, first devel oped by Invitrogen™
company, allows for the transfer of DNAfragments into numerous expression vect ors
wit hout altering the open readi ng frames of the proteins of interest.x i
The devel opment of
the cloni ng technol ogy has compl etely revol utionized the Y2Hscreeni ng by allowi ng
DNA fragments to be easily cloned into compatible vect ors through a t wo-step process.
This process replaces the previ ousl y used enoduclease and ligase- based met hods, which
were not onl ylaborious, but also affected by inappropriately positioned restriction

enzyme sites. The Gateway©systemutilizes site-specific recombi nation that includes
BP and LRreactions.
The gene of interest, the ORF i n many cases, contai ni ng t wo att Bsites on either
side, is transferred into a GAL4-based donor vect ors, through a BP reaction. The donor
vect ors contain ccdB and CmR counter-selectable genes, allowi ng for negative selection
of unwanted by-product plas mi ds after recombi nation occurs.x i i
The t wo att Bsites, att B1
and att B2, on the ORF will interact withthe att Bsites, att P1 and att P2, on the donor
vect or. An enzyme mi xt ure of BP Cl onase will generate entry clones, flanked by aat L
sites, which contain DNA sequences frombot h att Band att P sites.x i i i
See Figure 1.
Fi gure 1: Overviewof BP process perfor med through Gateway recombi national
cl oni ngx i v
.

The LRreaction utilizes the LR cl onase enzyme to transfor mentry clones int o
destination vect ors. Every entry clone will contain aat Lrestriction sites, which will
interact withthe aat Rrestriction sites on the destination vect ors, ADand DB, produci ng
an expression clonex v
. See Figure 2.
Fi gure 2: Overviewof LR process perfor med through Gateway recombi national
cl oni ngx v i
.
Epstei n- Barr virus
Epstein- Barr virus (EBV) was first identifiedin 1964 after virus-like particles
were observed in cells froma Burkitt’s lymphoma bi opsy. After its initial discovery,
EBV beca me the first known human virus directly invol ved inthe devel opment of
mali gnant tumors. Extensive research has confir med the role of EBVi nthe pathogenesis

of Burkitt’s lymphoma, Hodgki n’s disease, non-Hodgki n’s lymphoma, nasopharyngeal
carcinoma and lymphomas, and leiomyosarcomasx v i i
.
The Worl d Health Organization esti mates that 95% of adults worldwi de have
been infected with EBVx v i i i
. Upon initial infection, indi viduals become lifelong carriers
of the virus. The maj ority of infected indi vi duals coexist withthe virus without serious
complications, but a s mall population devel op malignancies as a result.
EBVis trans mitted by saliva and pri marilyinfects the stratified squa mous
epitheliumof the oropharynx. Initial infection may be asympt omatic; however, t wo-thirds
of infected indi vi duals manifest infectious mononucleosisx i x
. Latent infection is thought
to occur when B-lymphocytes and infected oropharynx cells interact inthe oropharyngeal
lymphoi d organs. The virus remai ns in me mory B-cells indefinitely, allowi ng for the
reactivation and devel opment of numerous EBV- associated cancers wherever B-cells
circulatex x
.
EBVis one of six known viruses that constitute the Herpesvirus famil y. All
herpesviruses possess the same struct ural characteristics, but are divided into three
subfa milies ( , , and ) based on genomi c size, content, and organizationx x i
. EBVis a
me mber of the -herpesvirus subfa mil y and possesses a 184-kbp double-stranded DNA
genome that encodes for 89 genes, with onl y 28 bei ng EBV-specific and important in
latent B-lymphocyte infection. Forty-six of the 89 genes are “core” genes, found in all
herpesvirus families, which are essential for successful genome replication, packagi ng,
and deliveryin all cells. Si x of the remai ni ng eighteen “noncore” genes found in bot hthe
-herpesvirus and -herpesvirus subfa milies, and the last t wel ve genes are specific tothe
-herpesvirus subfa mil yx x i i
. Li ke all herpesviruses, EBVis characterized by a toroi d-

shaped protein core, a nucleocapsi d with 162 capsomeres, a proteintegument, and an
outer envel ope with external glycoprotein spi kesx x i i i
.
Thompson and Kurzrock reveal that EBV viral proteins are able to mani pulate
cellular pat hways essential for mai ntaini ng homeostasis by i mitating several growt h
factors, transcription factors, and antiapopt otic factorsx x i v
. Once the virus has control over
essential cellular pathways the potential for EBV- related disorders to develop is vast.
Understandi ng the inter molecular proteininteractions bet ween the viral proteins, as well
as interactions that occur bet ween EBV and a human host, may provi de alternate ways to
prevent and treat EBV-associated diseases.
Si nce the discovery of EBV and its potential to cause a wi de variety of diseases,
extensive research has been focused on known EBV proteins and their role inthe life
cycle of the virus. EBNA-1is a phosphoprotein required for the replication and
mai ntenance of the EBV genome and is consistentlyfound intumors ininfected
indi vi duals. The protein bi nds to DNA sequences on the EBV genome and uses the host
enzymes to mediate the replication process. Because of its abilityto continuousl y
replicate the EBV genome, while activating the expression of other EBV genes, EBNA- 1
is consi dered to play a central role in mai ntaining latent infectionx x v
.
EBNA- 2 is one of the first proteins detected after EBVi nfection and is a known
transcriptional activat or responsi ble for the expression of viral and cellular genes. It is
essential for i mmortalization of human Blymphocytes and increases the expression of c-
myc, a gene known to become an oncogene as a result of over expression. EBNA- 2
increases expression through bi ndi ng with other transcription factors, notablythose
invol ved inthe pat hogenesis of T-cell lymphomax x v i
.

EBNA- LP, often referred to as EBNA- 5, is known tointeract with EBNA shortly
after EBVi nfection. The proteinis known to activate resting Blymphocytes, while
suppressing the tumor repressor protein, retinobl astoma. EBNA- LP, through interaction
wit h EBNA- 2, is also essential tothe i mmortalization of Blymphocytes and one of the
first detectable protei ns after EBVi nfection.x x v i i
EBNA- 3A, EBNA- 3B, and EBNA- 3C are all transcriptional regulators, differing
in specific function. EBNA- 3A and EBNA- 3C are essential for EBVi mmortalization
inside a host cell. EBNA-3Cis also known toinactivate retinobl astoma and increase the
production of LMP- 1, which inhi bits apopt osis of infected cells. The role of EBNA- 3Bis
generally unknown, however, is not essential for EBVi mmortalizationx x v i i i
.
Throughout the years the EBV LMP- 1 protein has been linked to cancer because
of its abilitytoinhi bit apopt osis of cancer cells by raising levels of Bcl-2. The proteinis
able to recruit an array of cellular genes by mi mi cki ng cell growt h signals, increasing the
risk for cancer devel opment. LMP- 1 also up-regulates the expression of B-cell adhesi on
mol ecules, allowi ng for cellular buildup and tumor devel opment.x x i x
LMP- 2 proteins, LMP- 2A AND LMP- 3B, are both essential for EBVlatency in
B-cells. Recent studies have shown the expression of LMP- 2Ai n Hodgki n’s disease and
nasopharyngeal carcinoma, suggesting an unknown role in cancer devel opment. Abetter
understandi ng of the LMP-2 proteins may provi de insight intothe virus’ abilityto
overtake cellular pat hways, causing numerous diseases.x x x
EBV- Encoded RNAs 1 and 2, known as EBERs 1 and 2, are noncodi ng RNAs
expressed is nearly all EBVi nfected host cells and found in all for ms of latency.

I mportantly, bot h EBERs are known to mai ntainthe malignant phenot ype of Burkitt’s
lymphoma cells, suggesting their role in oncogenesis.x x x i
Previ ous Work
In 2007, the first EBV- human interact ome map was constructed by the Center for
Cancer System Bi ol ogy (CCSB) at Dana-Farber Cancer Institute ( DFCI) incollaboration
wit h Mi chael Cal der wood from Bri gha mand Women’s Hospital. The primar y objective
of the screen was toidentifythe interactions of EBV proteins with each ot her and with
human proteins using a stringent Y2Hsystemx x x i i
. The successful construction of
interact ome maps depicting EBV- EBV and EBV- human proteins may be hel pful in
identifying the role of specific proteins inthe virus’ replication and reactivation
processes.
An EBV ORFeome contai ni ng 80 full and 107 partial EBV ORFs, representing 85
of the 89 known EBV proteins, were transferred int o GAL4- DB and GAL4- AD vect ors
by gateway recombi national cloni ng totest EBV-EBV proteininteractions. The
successful fusion of the 187 EBV ORF tothe DB and AD GAL4 domai ns allowed for the
testing of approxi matel y 35, 000 EBV proteininteractions via the Y2Hsystem. The
resulting fusions were transfor med intot wo different hapl oi d yeast strains, then mated
and anal yzed on selective media. Potential interactors were deter mi ned through
Pol ymerase Chai n Reaction (PCR) amplification and sequenci ng. The 43 EBV- EBV
proteininteractions, involving 44 EBV proteins, identifiedinthe Cal der wood screen
were merged with published EBVi nteractions toconstruct the current EBV- EBV
interact ome net work (Fi g.1.)x x x i i i

Fi g 1. “EBV–EBVi nteract ome net work resulting fromt he mergi ng of interactions identified inthe
Cal der wood et al. study with published interactions. Previ ousl y identified published interactions are shown
as purple lines and interactions identified inthe Cal der wood et al. study are shown as red lines. Core
herpesviruses are shown as yellowcircles and noncore proteins are shown as green circles. Hi gh
confidence interactions are shown as solidlines and lowconfidence interactions are shown as dashed lines.
The EBV- EBVi nteract ome consists of 52 proteins invol ved in 60 interactionsx x x i v
”.
EBV- human proteininteractions were then screened using si milar testing
techni ques. Acompl ete human spleen cDNAlibrary was fused tothe Gal 4-AD domai n
and 113 EBV ORFs, representing 85 EBV proteins, were fused tothe Gal 4- DB domai n.
The resulting fusions were then transfor med intotwo different yeast strains, allowi ng the
screeni ng of 85 known EBV proteins agai nst 100,000 to 1, 000, 000 human proteins. The
t wo yeast strains were mated and potential activators were anal yzed by PCR and
sequenci ng. The resulting EBV- human interactome net work incl uded 173 different EBV-

human proteininteractions bet ween 40 different EBV proteins and 112 human proteins.
(Fi g. 2.)x x x v
Fi g 2. “The EBV- human interact ome resulting fromt he Calder wood et al. Y2Hscreen. Core herpesvirus
proteins are shown as yellowcircles and noncore proteins are shown as green circles. Human proteins are
shown as blue squares. The interactions are shown as solid red lines. The EBV- human interactome
net work represents 40 EBV proteins and 112 human proteins connected by 173 interactionsx x x v i
.
In 2009, the CCSB at DFCI perfor med a second EBV- human screen in hopes of
expandi ng the previ ousl y constructed EBV- human interact ome net work. The maj or
difference bet ween the pri mary EBV- human screen ( CCSB wit h Cal der wood) and the
2009 screen ( CCSB wit hout Cal der wood) was the use of the human ORFeome v. 3. 1
(hORFeome v3. 1), instead of the human spleen cDNAlibraryx x x v i i
. During the screen,
hORFeome v3. 1 was the largest publicly available library of human open readi ng frames,
containi ng 12, 212 human ORFs, representing 10, 214 human genesx x x v i i i
.

The EBV ORFs, containing 85 full-lengt h genes and 127 fragments, were
expressed as fusion proteins tothe Gal 4- DB domain. The hORFeome v3. 1library was
expressed as fusion proteins tothe GAL 4- AD domai n, allowi ng the screeni ng of 216
EBV proteins agai nst approxi mately 12, 000 human proteins in a stringent Y2 H system.
Pot ential activat ors were anal yzed using PCR a mplification and sequenci ng and the
resulting interactions were merged withthe pri mary screen result. The CCSB screen
revealed 241 interactions bet ween 38 EBV proteins and 124 human proteins. (Fig. 3)x x x i x
Fi g 3. CCSB EBV- human interactome net work constructed in 2009.

Redundant interactions bet ween the Cal der wood and CCSB EBV- human screen
were anal yzed and dis missed in order to confir m68 additional interactions fromthe
secondary screen. The mergi ng of bot h screens identified 381 interactions bet ween 49
EBV proteins and 219 human proteins, resulting in the construction and expansi on of an
updated EBV- human interactome map. (Fi g. 4.)x l
Fi g. 4. EBV- human interact ome net work constructed fromthe consolidation of the Cal der wood and CCSB
Y2 H systems. EBV proteins are expressed as red circles and the blue circles showt he interacting human
protein. The EBVi nteractome net work represents 49 EBV proteins and 219 human proteins connected by
381 interactionsx l i

The EBV diseasome was compl eted shortly after the 2009 CCSB EBV- human
screen by Natali Gul bahce, Han Yan, and Laszl o Barabasi. The diseasome interact ome
map was used to depict the interactions EBV- human proteins and their likelihood inthe
pat hogenesis of various EBV associated diseases. The diseases were characterized as
either first or second degree based on results fromthe Online Mendelian Inheritance in
Man ®( OMI M) database, which contains infor mation on all known genetic diseases and
12, 000 genes. First-degree diseases are those potentially caused by the EBV virus and
second-degree diseases are those that potentially devel op as nei ghboring human proteins
are affected by viral intrusion. See Fi g 5.
Fi g. 5. The EBV Diseasome. The green squares represent the diseases, the yellowcircles represent human
proteins, the red diamonds represent viral proteins, and the bl ue circles represent human genes.
Gul bahce utilized the resulting EBV- human interact ome maps fromt he
Cal der wood and CCSB screens to hel pidentify positive disease links. 49 EBV proteins

were found tointeract with 257 human proteins, de monstrating 436 EBV- human disease
links. The diseasome map is effective in highlighting how EBV may be i mplicated inthe
devel opment of numerous diseases, includi ng cancer. It provi des additional confir mation
of interact ors identifiedin bot h EBV- human screens and numerous medical journals,
while summarizing the devastation that may result from EBVi nfection.
II. Mat erials and Met hods:
Objective:
The goal of this study is to further expand the EBV- human interact ome networ k
by using a stringent Y2H system. The Y2H approach will utilize human and EBV ORFs
expressed as fusion proteins int wo S. cerevisiae strains toidentify positive interact ors.
An expanded EBV- human interact ome will be helpful inidentifying the role of
proteininteractions inthe virus’ abilityto replicate and persist. Abetter understandi ng of
the virus will be useful in treating and preventing EBV-associated disorders.
BP cl oni ng reaction:
1. The BP reaction was assembl ed based on the followi ng chart.
5 μl 10 μl
PCR cl one 5 μl 5 μl
pDONR vect or 75 ng 150 ng
5x BP Buffer 1 μl 2 μl
TE pH 8. 0 To 4 μl To 8 μl
BP cl onase 1 μl 2 μl
Table 1: Constructing the BP Gateway reactionx l i i
.

1. The resulting entry clones were incubated at 25° overni ght.
2. 2 μl of Proteinase Ksol ution was added to each reaction. The constructs were
incubated for 10 mi nutes at 37°C.
3. The constructs were transfor med into competent cells and plated ont o solid media
containi ng Spectinomycin.
LR cl oni ng reaction
1. The LRreaction was asse mbl ed based on the followi ng chart.
5 μl 10 μl
Entry clone (50-150 ng) 5 μl 5 μl
Destination vect or 7 ng 150 ng
5X LR Buffer 1 μl 2 μl
TE pH 8. 0 To 4 μl To 8 μl
LR Cl onase 0. 5 μl 1 μl
Table 2: Constructing the Gateway LRreactionx l i i i
.
2. The resulting expression cl ones were incubated at 25° C overni ght.
3. The expression clones were transfor med into competent cells and plated onto
solid media containi ng Spectinomyci n.
EBV ORF Li brary:
The EBV constructs were generated in Eric Johansson’s laborat ory and were
provi ded to Cal der wood and CCSB as EBV constructs harbored in E. Coli. PCR anal ysis
was perfor med on the sampl es to confir mthe presence of the EBV constructs, to
deter mi ne the exact sizes of the constructs, and toisolate single EBV cl ones.

1. The PCR reaction was perfor med based on the followi ng chart:
Mat erials μl needed.
10X buffer 30
10 mMdNTPSs 6
MgSO4 12
Pri mer 1 0. 3
Pri mer 2 0. 3
EBVtempl ate 1-2
HI- Fi Taq pol ymerase 1. 2
Di stilled water 240
Table 3: Constructing the PCRreaction to confir m proper vect or presence in generated
EBV constructsx l i v
.
2. EBVi nserts containing the proper vect or presence were selected fromt he
origi nal plates. The inserts were then organized into fragments, containi ng a full-
lengt h EBV ORF, and placed into 96 round bottom Costar ©cell culture plates.
3. The EBV DNAfragments were first transfor med int o E. coli cells then purified
usi ng the QI Aprep Spi n Mi ni prep Kit ©. Fragments were transfor med into the
Y8930 yeast strain and glycerol stocks were made of the resulting EBVlibrary,
whi ch were frozen for future use.
The gl yercol stocks, of the EBVlibrary, originally made by Cal der wood and CCSB,
were the starting poi nt for this study. The EBV ORFs were transfor med into the DB
domai n through Gateway recombi national cloni ng.

hORFeo me v. 5. 1
The long-ter mgoal of the Ma mmalian Gene Collection ( MGC) is toidentify and
cl one all human cDNA clones, containing a functional ORF. The collection is readily
available tothe research community and served as a starting poi nt for hORFeome v5. 1
construction. Human ORFs were isolated from MGC cDNA cl ones and were amplified
usi ng PCR machi nery. The amplified ORF were cloned and transfor med into E. coli
vect ors by a recombi national cloni ng reaction. PCR was used to verify and sequence
present ORFs. Present ORFs were identified by BLAST anal ysis and hORFeome v. 5. 1
was successfully compl eted. Gl ycerol stocks of the library were made and frozen for
future use. The hORFeome v5. 1libraryis currentlythe largest publicly available human
open readi ng frame library, includi ng 15, 483 human ORFs, representing 12, 794 human
genesx l v
. The libraryincl udes all previ ous ORFeome v1. 1 and v3. 1 products and the
addition of 3, 272 newl y identified ORFs from MGC templ ates. The expansion of the
human ORFeome has increased the effectiveness and potential of Y2Hscreeni ng
met hods by provi di ng a larger library of potential interact ors. The hORFeome v. 51
library was transfor med int o the AD domai n through Gateway recombi national cloni ng.
S. cerevisiae strai ns
Two Y S. cerevisiae strains, Y8800 and Y8930, were utilized throughout this
study and served as the expression vect ors. Bot h strains were generated by Xi aofeng Xi n
in Charles Boone’s laborat ory. S. cerevisiae strains Y8800 ( MATa) and Y8930 ( MATα)
containthree GAL4p induci ble reporter genes, proxi mal to an upstrea mactivation
sequence, which provi des four indicat ors for positive interact ors. The HIS3 reporter gene
identifies interact ors by allowi ng growt h on media lacki ng histidine. The ADE2 and LacZ
reporter genes bot h utilize col ori metric detection of Gal 4p activity, which varies

dependi ng on the strength of expression. ADE2, in addition to provi di ng a notable col or
change, allows for growth on media lacking adeninex l v i
. Although LacZis present in both
strains, it was not utilized inthis study.
Y8800 and Y8930 were genetically engi neered to contain deletions of the GAL4
and GAL80 genes, which code for GAL4p and GAL80p, t wo regulatory GAL-transcription
genes. The deletion of GAL80p, a repressor gene, prevents the inhi bition of GAL4
transcription throughout the phenot ypi ng process. Two auxotrophic markers, leu2 and
trp1, were utilized to select for yeast cells harboring bot h AD and DB domains. Use of
auxotrophic markers allows for selection of yeast cells that were successfully
transfor med. Bot h strains are cycl ohexi mi de resistant, aiding in plas mi d shuffling and
identification of aut o activat orsx l v i i
.
Inthis study, Y8800 was the AD yeast strain and Y8930 was thus the DB strain.
The Y8800 strain woul d thus harbor the GAL4- AD domai n, which contained the
hORFeome v5. 1library expressed as fusion proteins. The auxotrophic marker for Y8800
was leu2 (-trp1), indicating that trypt ophan coul d be used to confir mthe successful
transfor mation of the AD plas mi d intothe yeast strain. Y8930 was designated for the
GAL4- DB domai n, which contained the EBV ORFeome expressed as fusion proteins.
The auxotrophic marker for the Y8930 strain was trp1 (-leu2), allowi ng for confir mation
of successful transfor mation of the DB plas mi d and EBV ORFs when grown inleuci ne
liqui d media.

Yeast Strain Harbors ORF Auxotrophic
Mar ker
Gr ow on
Y8800 GAL4- AD
domai n
Hu man Leu2 -Trp media
Y8930 GAL4- DB
domai n
EBV Trp1 -Leu media
Y8800 +
Y8930
GAL4- DB
domai n and
GAL4- AD
domai n
Hu man and
EBV
N/ A -leu, -trp –his,
+1mM3-a mi no
triazole (3AT)
Table 4: Summary of Y8800 and Y8930 strains, their contents, and the selective media
required through the Y2H screeni ng process.
Procedure:
1. The the Genmat e ©robotic liqui d handi ng systemwas used toinoculate 10μl of
AD cl ones, the Y8800 strai n was transfor med with the GAL4- AD domai n and
human ORFs, into 80μl of 1XSC- Trp liqui d media. Inoculation was performed in
96 round bottom well Costar ©cell culture plates. Synt hetic Compl ete (SC) drop
out media contains essential ami no aci ds and vitami ns needed for yeast growt h,
while still selecting for auxotrophies. The cultures were grown at 30°Cfor t wo
days.
2. Si milarly, 10μl of DB clones, containing the GAL4- DB domai n with fused EBV
ORFS, were inoculated int o 80μl of 1XSC-leu liqui d media using the Genmat e ©
robot. The cultures were grown at 30º Cfor t wo days.
3. To test for the presence of aut o activat ors prior tostarting pri mary and secondary
phenot ypi ng processes, 5μl of the DB cl ones were pipetted ont o –leu and –his
plates. Growt h was observed after 24 hours. If the DB cl ones survi ved on
-histidine plates, without the presence of the AD clones, they were aut o-activat ors

and removed fromthe screen. Additional steps were taken throughout the screen
process toidentify aut o activat ors that were not detected prior to screeni ng.
4. The AD and DB cl ones that grewinthe 1XSC-trp and 1XSC-leu and were not
dee med aut o activat ors were mated using the Genmat e ©robot. The AD and DB
cl ones were spotted directly on top of one anot her allowi ng for the hapl oi d cells to
become di pl oid. Di pl oi d yeast cells then harbored bot h the GAL4- AD domain and
GAL4- DB domai n, containing bot h human and EBV ORFs, allowi ng for potential
proteininteractions to occur. 5μl of each yeast culture were spotted on YEPD
plates, which provi de essential nutrients for yeast growt h. Si x controls were added
tothe bottomof the YEPD plates, which were then grown for 24 hours at 30º
The Si x Controls
As wit h any scientific study, controls were used and monitored to detect any
abnor mal growt h patterns or lack of growt h all toget her. Six controls, specific to Y8800
and Y8930, were utilized to ensure effective screeni ng techni ques. Any deviations from
expected growt h patterns were consi dered before continui ng the screen.

Control DB cl one AD cl one Expected Growt h
1 pPC97: An e mpt y
DB expression
vect or.
pPC86: An e mpt y
AD expression
vect or.
No growt h is
expected because
onl y empt y vect ors
are present.
2 Rb: Weak
Positive
E2F1: Weak Positive Little or no growt h
3 Fos HLH: Strong
Positive
Jun HLH: Strong
Positive
Fai nt growt h
4 GAL4 AD full protein Maxi mu m growt h
5 DP1: E2F-1: Strong growt h
6 DP1 E2F-1 with
cycl ohexi mi de
Equal to control 5
Table 5: The six controls and their expected growth patterns.x l v i i i
.
5. Aft er 24 hours of incubation, pri mary phenot ypi ng began with replica plating.
Yeast col onies were replicated ont o selective media to assess their abilityto grow
in an interaction -dependent manner. Replica plating ont o selective media
confir ms that the yeast vect ors are transfor med with bot h AD and DB, and
potential interactions can occur. This process requires the availability of replica
plating bl ocks and vel vet squares, bot h of which must be sterile prior to use. The
replica blocks and vel vet squares were aut oclaved to prevent the probabl y of
outside contami nation.
6. Yeast cells were transferred ont othe sterile vel vets by evenl y pressing the bottom

of the plate. The cells fromt he vel vets were then transferred to a newplate
containi ng selective media through the same even pushi ng techni que. Yeast cells
were replica plate fromthe YEPD plate ont o
1. –His, +Ade, +3AT plates.
2. Cycl ohexi mi de plates.
Gr owt h on –His and +Ade indicated the interaction of AD and DB and the
expression of bot h reporter genes. 1mMof 3-ami no triazole (3AT), which inhi bits the
HI S3 gene product, was used to decrease the background expression associated with
HI 3x l i x
. The specific concentration of 3AT ensuredthat weak interactions are not lost, but
the probability of false positives was eli mi nated. The cycl ohexi de plates were used to
identify aut o activat ors.
7. Aft er 24 hours, the –His, +Ade, +3AT and cycl ohexi mi de were cleaned. Cleani ng
the plates required the use of replica plating blocks, sterile velvets, and a roller. A
sterile vel vet was placed on the replica plating bl ock then the plate was firml y
placed ont othe vel vet. Aroller was used to carefully and evenl y roll across the
back of the plate until the col onies were no longer visible. In some cases, t wo
vel vets were necessary. Cl eani ng ensured that there were an equal and
comparable number of cells in each spot, by removi ng background growt hl
. Plates
were grown at 30°Cfor 5 days.
8. Positive col onies fromt he - His, +Ade, +3AT plates were picked using a sterile
toot hpick into 80μl of 2XSC- Leu- Trp liqui d media. The toot hpicks were
aut oclaved before use and discarded after picki ng each col ony. Candi dates were
grown at 30º C overni ght.

9. Gl ycerol stocks of potential candi dates fromt he –Hi s, +Ade, +3AT plates were
made and stored in 40%gl ycerol. They were frozen to allowfor reuse during
future studies.
10. The positive dipl oi d cells fromt he liqui d media were spotted ont o –leu, -trp
plates. Col onies were grown for t wo days at 30º C. This process began
phenot ypi ng t wo, where positive interactions were then picked and sequenced
usi ng PCR anal ysis.
11. The –leu, -trp plates were replica plated ont othe flowi ng plates, inthe given
order, after the t wo days.
1. –His, +3AT plates
2. +3AT, +cycl ohexi mi de plates
12. The plates were cleaned i mmediately after completing the replication process to
remove any background growt h. The plates were grown for five days at 30º C.
13. The secondary plates were placed side by side for comparison. Each indi vidual
spot was compared bet ween the –his, +3AT and +3AT, +cycl ohexi mi de plates to
deter mi ne the presence of aut o activat ors before PCR anal ysis and sequencing.
Any growt h observed on the +3AT, +cycl ohexi mide plates were noted on the -
Hi s, +3AT plates and deemed aut o activat ors. Colonies that grewon the +cycl o
plates were not picked because they were aut oactivat ors. Col onies that showed
growt h on –His, +3AT and not +3AT, +cycl oheximi de were candi dates, which
were thus picked and analyzed.
14. A sterile toot h was used to pick positive candi dates fromt he –His, +3AT plates.
Candi dates were then picked ont o –Leu, -Trp plates, which were grown for 24

hours at 30º C.
15. Fi ve milliliters of Z-buffer, which contained 1. 0 milligrams of zymol yase per 1. 0
milliliter of lysis buffer, was made. The zymol yase enzyme lysed the yeast cells
and released the DNAi nto sol ution.
16. 15μl of Z-buffer was distributed intothe each 96 round bottom well on the
Costar ©cell culture plates. As mall amount of each candi date was picked and
placed into a round-bottom well. Anewsterile toothpick was used to pick each
col ony.
17. The followi ng zymol yase/lysis progra m was run, inthe given order, in a ther mal
cycler.
1) Incubation- 37º Cfor 15 mi nutes.
2) Activate zymol yase enzyme- 95º Cfor 5 mi nutes.
3) 10º Cfor t wo hours.
18. After the zymol yase/lysis progra m was compl eted and the DNA was released
into sol ution, 100μl of distilled water was added to each Costar © well. The
Costar ©plates were centrifuged for 5 mi nutes at 2000 rpm. This process diluted
the template and was necessary for further PCR anal ysis.
19. Additional PCR anal ysis was run to amplify segments of the AD and DB
segments. The followi ng progra m was run inthe given order.
1) 94º Cfor 2 mi nutes
2) 94º Cfor 30 seconds
3) 58º Cfor 30 seconds
5) 94º Cfor 30 seconds, 30 ti mes

20. The resulting AD and DB PCR plates were store at 4º Cfor five days before they
were sent to Agencourt Bioscience Corporation inBeverly, MA for sequencing.
III. Results
The EBV- human screen allowed for the screeni ng of 216 EBV proteins against
15, 483 human proteins and was successfully completed three ti mes. The three screens
yielded 188 potential candi dates, which were analyzed using PCR machi nerythen sent to
the Agencourt Bi oscience Corporation for sequenci ng.
St udy Co mplications
Nu merous complications arose throughout the duration of this study. Many were a
result of human error, however systematic errors also negativel y affected the EBV- human
screens.
Intotal, eight EBV- human screens were attempted, but onl ythree were
successfully compl eted. The first t wo screens were disregarded after pri mary
phenot ypi ng was compl eted. Upon exa mi nation, the plates showed faint growt h in half of
the expected spots at best. It was deter mi ned that the replica plating or cleani ng processes
were not compl eted correctly. It is possible that during the replicating process, the
“ mot her” plates were not pressed fir ml y ont othe vel vets, thus causing the insufficient
transfer of col onies ont o the selective media. It is also possible that during cleani ng the
roller was pushed too firml y ont othe vel vets, for too long of a ti me period, causing the
col onies totransfer solely ont othe cleani ng vel vets.
The next three screens were also disregarded after pri mary phenot ypi ng because

the same growt h pattern was observed on every plate after the t wo yeast strai ns were
mat ed. Adifferent growth patternis expected on each plate throughout a screen because
each human gene can interact differently. Through collaboration with other me mbers in
the CCSB depart ment, it was concl uded that intermi xi ng of the wells caused an aut o
activat or to spill into adjacent wells. Initially 120μl of 1XSC-trp and 1XSC-leu liqui d
medi a were used toinoculate the AD and DB cl ones, in stept wo of the procedure.
Before the clones were spotted ont othe –leu and –his plates, the Costar ©cell culture
plates needed to be spun. It was later deter mi ned that the wells shoul d hol d 80μl instead
of 120μl. Too much liquid media was used the wells were inter mi xi ng when spun,
causi ng the same growt h patternto be present on every plate.
There were also numerous instances when contami nation rui ned plates used
during the screens. Contami nation was likely a result of i mproper techni ques and air
pollutants.
Co mplications continued to arise even after the successful compl etion of the three
screens. Three plates containing 188 candi dates were placed inthe col d room and stored
at 4º C until PCR coul d be perfor med. During this ti me the candi dates were disposed of,
leavi ng one plate left for anal ysis.
I V. Discussion
Proteins are possiblythe most i mportant class of macromol ecules inthe human
body, servi ng a variety of functions. Each indi vi dual protein has a specific role in
ensuring that vital life processes are perfor med correctly and safely. Many proteins do so
by interacting with other proteins, catalyzi ng bi oche mi cal reactions, servi ng as antibodies
agai nst antigens, acting as che mi cal messengers, or carrying i mportant molecules from

different sites withinthe body.
I mportant questions are posed when external factors can disrupt the nor mal
function of these indi vi dual proteins and the functions they serve. What happens when
proteins stop interacting wit h proteins that hel pthe mcarry out i mportant life processes?
What is the effect when proteins start interacting wit h different proteins? Ne wresearch
has shown that the development of numerous human diseases is attributed to
malfunctions in nor mal proteininteraction. Often ti mes, the host’s i mmune systemis
compromised, allowi ng for an external factor to hijack and disrupt i mportant cellular
processes.
Viruses, such as EBV, have the abilityto enter into a host undetected through
respiratory air ways. They are essentially genes, protected by a protein coat, which require
a host cell to survi ve. Viruses use the host’s enzymes and ot her cellular machi nery to
express their proteins, synt hesize proteins, and replicate. The cells used for viral
replication rarely survi ve, releasing numerous newlyfor med viruses, which attack
nei ghboring host cellsl i
. As the host cells are infected and lysed, nor mal protei n
interaction and cellular function may be effected.
As nor mal cellular processes are affected, host cells may not de monstrate nor mal
cell growt h patterns. Healthy functioni ng cells shoul d show bot h growt h stimul at ory
signals and inhi bitory signals. Basically, cells will continue to divi de as long as there is
enough roomand they shoul d stop divi di ng once they come in contact with one anot her.
Ho wever, virallyinfected cells may not have excessive growt h sti mulat ory signals or too
fewinhi bitory signals as a result of abnor mal protei ninteractions, resulting intumor
for mationl i i
.

EBVis one of the most common human viruses, esti mated toinfected 95%of the
worl d’s population. The virus has been i mplicated in numerous diseases, incl udi ng
Burkitt’s lymphoma, Hodgki n’s disease, non- Hodgki n’s lymphoma, and nasopharyneal
carcinoma. Unlike most viruses, EBVis able to mai ntainits viral genome wit hout
da magi ng or marki ng host cells for destruction. The virus’ abilityto establish latency has
proposed numerous questions regardi ng the effect on bot h viral and human proteins and
the devel opment of cancer.
Ne wresearch has identified the role of indi vi dual EBV proteins, while suggesting
that proteins that react with numerous ot her proteins may somehow be involved in
disease devel opment. However, research focused on the EBV virus still remai ns
inadequate. Wit h such a large percentage of the world’s population affected by the virus,
more attention shoul d be focused on preventing and treating the virus before the disease
devel ops.
Inthe 2007 EBV- human interact ome project, CCSB and Cal der wood defined
EBV “hubs” as proteins wit h relatively short pat hways to human proteins or those with a
large number of proteininteractions. They found that “hubs” inthe EBVinteract ome
where significantly more essential to yeast survi val than proteins with a s mall number of
interactions. More specifically, they found cancer devel opment is likely a result of “hub”
interaction. However, even with significant evi dence for the pat hogenesis of disease as a
result of protein “hubs” the necessary research is not bei ng funded and perfor med.
Si nce EBV-associated diseases are not as common as other infectious diseases,
many argue that research is not a priority. While onl y a s mall population devel op

mali gnancies as a result, the potential for more EBV- associated disease cases is vast. As
our environment becomes more polluted and less resourceful, it is inevitable that humans
will be exposed to har mful external factors that may reactivate the virus ininfected
indi vi duals. Very little is known about how or why the virus reactivates, however, it is
obvi ous that dangerous toxi ns or changes have the potential to do so.
Ne w controversial research has also suggested the role of EBVi nthe
devel opment of breast cancer. Some studies have shown traces of EBV viral material in
51 %of the breast cancer tissue sampl ed, while others failedto detect EBVin any tissue
sa mpl es. The variance instudy results may be a result of different screeni ng techni ques,
the EBV proteins and RNAs studied, or in breast cancer itself. However, wit h an
esti mated 192, 370 new breast cancer cases in 2009 alone, it is i mportant to understand
the potential role of EBV and the pat hogenesis of this disease as well. The linkage
bet ween EBV and breast cancer see ms probable after anal yzi ng the staggering number of
breast cancer cases and the large population that serve as hosts for the virus.
Understandi ng if the virus is invol ved, and the role it plays if so, in oncogenesis woul d be
beneficial for all patients and healthcare professionals.
Treat ment of EBV- associ ated malignancies
Si nce the discovery of EBV-associated malignancies, treat ment options have
proven relatively unsuccessful. Antiviral agents, immune-based therapies, and specific
monocl onal antibody options are currently bei ng expl ored and have shown promisi ng
results. Antiherpesvirus and anticyt omegal ovirus agents, such as ganciclovir,
famcycl ovir, acycl ovir, valaciclovir, foscarnet, and cidofovir, have been applied in a
clinical setting, but all varyintheir effectiveness. Many antiviral treat ment options are

used toinhi bit EBVreplication, initiate the apopt osis of EBV- positive lymphoma cells,
or block EBV antigen activation. Regardless of the encouragi ng clinical results, antiviral
agents are unable totreat the i mmunodeficiency issues that allowfor the devel opment of
EBV-associated malignanciesl i i i
.
I mmunot herapy procedures, utilizing EBV-specific cyt ot oxic Tlymphocytes
( CTLs), have also been successful intreating patients with EBV-related tumors, while
addressing i mmunodeficiency issues. The treat ment option has been most successful in
patients with Hodgki n’s disease, helpi ng reestablish i mmunocompetence. EBV-specific
CTLs can be taken froma seropositive donor and directlyinfused into a patient, or
expanded further in vivo then infused intothe patientl i v
. While the success rate for the
i mmunot herapy procedures remai ns mi ni mal, the abilityfor the procedure to fight
i mmunodeficiency may be successful in combating the cellular pat hway disruption
caused by EBV.
The useful ness of EBV-specific CTLs is bei ng used inthe efforts to devel op and
EBV vacci nationl v
. Vacci nes efforts are under way to hel p protect agai nst initial EBV
infection. If proven successful, the vacci ne woul d have the pot ential to eradicate
numerous EBV-associated diseases. Wit hthe majority of the worl d’s population infected
wit h the virus, a vacci ne woul d undoubtedl y be beneficial. Therefore, it is vital that
clinical studies are funded and supported to ensure that efforts are continued and
resources are available. Lastly, the vacci ne, once devel oped and approved, must be
available in all countries, regardless of the cost. It woul d not be effective if onl y certain
countries had access because EBV-associate diseases affect indi vi duals worldwi de.
While options for EBVtreat ment are available, the success rate of antiviral drugs

and i mmunot herapy remai ns inadequate when looki ng at the number of cases of EBV-
associated diseases and mali gnancies. In order to successfullytreat disease, a better
understandi ng of the virus and its abilityto disrupt nor mal protein function is required. A
number of studies have focused on the interactions bet ween EBV and human proteins and
the possible result on cells and the human body. However, the findi ngs of the Y2H
systemhave not been further anal yzed as a result of insufficient fundi ng for further
research. Indi vi dual protei ns have been indentified and their roles in EBV infection have
been theorized, however, much is still vague or unknown. More research needs to be
perfor med to not onl yidentify addition EBV- human interact ors, but alsoto further
anal yze known interactors role in EBVi nfection, latency, and reactivation.
Understandi ng the ability of EBVt o disrupt proteins that hel p carry out vital cellular
function may provi de insight intothe devel opment of numerous diseases. Once the virus
is better understood, researchers can focus on the devel opment of a vacci ne that woul d
protect indi vi duals agai nst initial infection and other treat ment options for indi vi duals
already infected.
Fut ure Di rections:
This study ended at DFCI inthe early anal ysis stage. Once the candi dates are
sequenced and sent back to CCSB at DFCI they need to be anal yzed by bi oinfor matics to
deter mi ne which interactions were detected inthe screen. Any newinteractions shoul d be
noted and redundant interactions shoul d not be disregarded. If the same interactions are
observed in numerous screens they very well may be “hub” proteins or essential to EBV
function. Special attention shoul d be focus on redundant interactions, especiallythose
observed all three EBV Y2 H screens.

Aft er the interactions are deter mi ned, retesting needs to be perfor med to ensure
that those interactions were actually observed. Once the retesting sequences are anal yzed,
interactions that were observed inthe first screen and agai ninthe retesting shoul d be
added tothe current EBV interact ome map. An expanded interact ome map woul d be
hel pful in understandi ng the virus’ effect on host cells and infected indi vi duals.
Next, efforts need to be made toidentify and incorporate the four unknown EBV
proteins intothe current EBV ORFeome. The readily available EBV ORFeome contains
85 of 89 known EBV genes. It is possible that the four unknown EBV protei ns may be
the most i mportant inthe virus’ abilityto establish latency and cause the devel opment of
EBV-associated diseases. In expandi ng the current EBV ORFeome toincl ude all possible
interact ors, more thorough and effective screens coul d be perfor med.
In addition, efforts to expand hORFeome v5. 1library shoul d be consi dered to
ensure that the maxi mu m number of interactions is screened, tested, and analyzed. By
expandi ng the human gene librarytoincl ude numerous ORFs additions, a wi der range of
potentially vital interactions may be identified.
Once the EBV ORFeome and hORFeome libraries are expanded, additional
screens must be perfor med. Expanded ORFeome libraries woul d allowfor the most
effective, accurate, and groundbreaki ng screens to be perfor med. These screens woul d
have the potential to provide insight into unknown interactions occurring between EBV
and human proteins, EBV- EBV proteins, and human-human proteins, which may be
detri mental to disease devel opment.

V. Concl usi on
In concl usion, the Y2H hybrid systemis an effective means for understanding
proteininteractions and their role in disease development. The field of syste ms bi ol ogy
uses the Y2Hsystemto devel op interactome maps, which depict all known interactions
occurring withi n a cellular net work. Because protei ns play vital roles inlife processes, it
is i mportant to understand howexternal factors can disrupt nor mal interactions in
indi vi duals, havi ng fatal consequences.
Al ong-ter mgoal of the CCSB at DFCI is understand the effect of EBV on a
human host and the virus’ role in oncogenesis. Two screens have been successfully
compl eted, identifying 381 interactions bet ween 49 EBV proteins and 219 human
proteins. This study has been the first screeni ng of EBV- human proteins since the
expansi on of the human ORFeome library. The results of this screen have the potential to
expand the current EBV-human interact ome, provide insight intothe virus’ abilityto
hijack cellular net works, and identify vital unknown proteininteractions in infected
indi vi duals.
It is i mportant that additional screens are perfor med and efforts are continued to
compl ete the EBV ORFeome and expand the current human ORFeome. In doi ng so,
infected indi vi duals would have safer and more effective treat ment options, and ot hers
may be fortunate to be protected agai nst initial EBVi nfection.

VI. Acknowl edge ments
It is with great pleasure that I thank the many people inthe Center for Cancer
System Bi ol ogy Depart ment at Dana Farber Cancer Institute for welcomi ng me i nt ot heir
teamand showi ng me the i mportance of hard work and dedication. Their endless hel p
and understandi ng made my experience surely unforgettable. I woul d like to especially
thank Davi d Hill for welcomi ng me intothe Marc Vi dal Laborat ory and maki ng sure I
was comf ortable withthe concepts and me mbers of the team. I woul d alsolike tothank
Dr. Jennifer Roecklein- Canfield for allowi ng me to be a part of her research and hel pi ng
me realize the i mportance of EBV-associated diseases. It is with most i mportance that I
thank Lila Gha msari for bei ng the first person to wel come me intothe lab, havi ng the
patience toteach me i mportant concepts, and allowi ng me totake part in her personal
research.
I woul d alsolike tothank my professor, ment or, and friend, Marl ene Sa muelson
for her support throughout the years. Thank you for blessing me withthis a mazi ng
experience, challengi ng me t hroughout the years, and encouragi ng me to fulfill my
drea ms.
To all the me mbers of the Curry College Bi ol ogy Depart ment I woul d like to
thank you for provi di ng me wit h an amazi ng support systemthroughout the years. Thank
you for bei ng more than just professors, but dedicatedindi vi duals who believe intheir
students. I amforever grateful for everyt hi ng each one of you has done for me and I will
mi ss you dearly.
To my closest friends, Jai me Callanan and Amanda Leger, I woul d like tothank
you for maki ng my years at Curry College more than me morable. Thanks for the laughs,

love, and support.
Lastly, and most i mportantly, I woul d like tothank the me mbers of my famil y. To
my mot her and father I thank you for lovi ng and supporting me unconditionally. I
appreciate every sacrifice you bot h had to make toensure that I was gi ven the means to
make my drea ms come true. To my brot her, Matt, I thank you for al ways being there for
me t hrough the good and the bad. To my grandmother, I thank you for bei ng like a
second mot her to me throughout my life. Thank you for your selflessness, endless love,
and support. To all other me mbers of my famil y, I woul d like tothank you for some of
the best ti mes in my life. I love you all.

VII. Appendi x:
Yeast Medi a:
1. YEPD
*per liter
20g of pept one
10g yeast extract or 20g for solid plates
50 ml of 40%gl ucose
.15 ml of adeni ne
2. Synt hetic Co mpl ete dropout medi a
*per liter
1. 3g of ami no aci d powder, containi ng adeni ne
1. 7g of yeast nitrogen base
5g of ammoni um Sulfate
500 ml of distilled water
10 MNa OH was used toreach a pHof 5. 9
Selection Medi a
1. 10X TE Buffer
*per liter
100 ml 1 MTris- HCl buffer
20 ml 500 mMEDTA
880 ml distilled water

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