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Lab Report
Characterization and tagging of HRT1 in Tetrahymena thermophila
BMS 658
Joshua J Smith
Submitted by: Amruta Ashtekar
Date: 05-06-2011
Abstract
The specificity of ubiquitination is often controlled by ubiquitin-protein ligases (or E3s), which
facilitate the transfer of ubiquitin to appropriate targets. Hrt1, a subunit of SCF, (Skp1,
Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate
diverse processes including cell cycle, immune response, and development. The Hrt1 protein
interacts with many different F box proteins in order to form different ubiquitin ligases. The
RING finger protein, Hrt1, is also required for progression of DNA replication forks through
damaged DNA. The main purpose of this study is to tag and characterize HRT1 in Tetrahymena
thermophila. The HRT1 gene from pENTR vector, an entry vector, was isolated and inserted into
pGTW, an expression vector tagged with either FLAGHis6 or GFP. HRT1 was characterized using
bioinformatics tools and found that homologs in T. thermophila and A. Thaliana are more
similar phylogenetically than in H. sapiens or S. cerevisiae; and also they contain zinc finger
domains indicative of ubiquitin ligases. Expression analysis and western blot analysis of the
protein was performed. HRT1 is likely to be involved in early and late DNA damage responses
by UV light since the expression increased after UV DNA damage. However, Hrt1 protein could
not be expressed in detectable amounts in a western blot. Further study of protein localization
with the help of fluorescent epitope tag will also be done for better understanding the
functional properties of this gene in Tetrahymena.
Introduction
Protein degradation is an important post-transcriptional regulatory process that allows cells to
respond rapidly to intracellular signals and changing environmental conditions by adjusting the
levels of key proteins. One major proteolytic route in eukaryotes involves the ubiquitin (Ub) 26S
proteasome pathway. Proteins that are destined for degradation are modified by the covalent
attachment of multiple Ubiquitins. The ubiquitinated substrates are then recognized by the 26S
proteasome and degraded while the Ub moieties are recycled (Gagne, 2002).
Degradation of a protein via the ubiquitin-proteasome pathway involves two discrete and
successive steps: tagging of the substrate by covalent attachment of multiple ubiquitin
molecules and degradation of the tagged protein by the proteasome complex (Glickman, 2002).
Ubiquitination of proteins is achieved through an enzymatic cascade involving ubiquitin-
activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. Ubiquitination
occurs when an E3 ligase enzyme binds to both substrate and an E2 thioesterified with
ubiquitin (E2<Ub), bringing them in proximity so that the ubiquitin is transferred from the E2 to
the substrate. E3 ligases confer specificity to ubiquitination by recognizing target substrates and
mediating transfer of ubiquitin from an E2 ubiquitin conjugating enzyme to substrate. Based on
their subunit organization and/or mechanism of Ub transfer, currently, five types of E3s have
been identified. In yeast (Saccharomyces cerevisiae), one important E3 type is the SCF complex
which is composed of four primary subunits: cullin1/ Cdc53, Rbx1/ Roc1/ HRT1, Skp1, and an F-
box protein. The activity of most E3s is specified by a RING domain, which binds to an
E2<ubiquitin thioester and activates discharge of its ubiquitin cargo (Deshaies, 2009). Ubiquitin
ligases are classified into two main classes on the basis of structural similarities: the RING-finger
proteins and the HECT-domain proteins. RING-finger type ubiquitin ligases contain a cullin (Cul)
protein subunit (derived from Cullin–RING Ligase or CRLs) and its function is to sort substrates
for degradation. The biological significance of CRLs is manifested by the fact that about 20% of
the proteasome-dependent cellular protein degradation is dependent on CRL activity (Lee,
2010). The RING component of SCF complex consists of 2 family members, RBX1 (RING box
protein 1), also known as ROC1 (regulator of cullins), and RBX2/ROC2 (also known as SAG
[sensitive to apoptosis gene]), both of which are essential for the catalytic activity of SCF (Wei,
2010). The SCF RING subunit is known as either Rbx1, Roc1, or HRT1. SKP1–CUL1–F-box protein
(SCF) complex is one of the six different mammalian CRLs (Frescas, 2008). HRT1, a subunit of
SCF, (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that
regulate diverse processes including cell cycle, immune response, and development (JH, 1999).
A study suggests that HRT1p is a core subunit of multiple SCF complexes (M, 2000). HRT1p
contains a ring-finger domain and interacts with Cdc53p, Cdc34p, and at least three different F-
box proteins (Blondel, 2000). Recent studies in Arabidopsis thaliana indicate that F-box proteins
and SCF complexes play critical roles in various aspects of plant growth and development
(Gagne, 2002). RING finger protein HRT1 is a part of ubiquitin ligase which is required for
progression of DNA replication forks through damaged DNA. Thus, it is likely that the ubiquitin
ligase has a role in dealing with defective rRNA and damaged DNA (Hinnebusch).
This lab will include cloning the gene of interest (HRT1) from entry vector into a
destination vector and transform the E coli cells with the vector having gene of interest. HRT1
can be taken from pENTR vector, obtained from BMS 110 honors class and transfer it to pGTW
destination vector. The vector containing HRT1 will be transformed into the appropriate
organism (Tetrahymena thermophila) and use it either for preservation or expression of the
gene. The gene will express the protein that it encodes in appropriate expression organism. We
will then confirm the presence of the protein and quantify it. The expression of HRT1 can also
be quantified by qRT-PCR. The HRT1 gene was cloned into pGTW, a Tetrahymena expression
vector with any of the four epitopes: GFP, RFP, 2HA and FLAG-His. For tagging of my pGTW
vector, FLAG-His and GFP epitopes were used. The purpose of using epitope tagged pGTW
vector is to be able to immunoprecipitate and study the localization of the protein. FLAG-His is
polypeptide protein tag consisting of amino acid sequence N-DYKDDDDK-C (1012 Da) and a
stretch of histidine residues added to a protein. pGTW vector can be used as destination as well
as expression vector. It is available with different epitope tags (2HA, FLAG, His, GFP, RFP) and
has ampicillin resistance marker. It can be used in LR clonase reaction in Invitrogen Gateway
Cassette technology, in which the gene insert can be swapped between pENTR and pGTW. In
addition, pGTW has Btu1 (pBMFH6-GTW) or rpL29 (pBSMTTGFP-GTW) loci. These loci have
homologus sites in Tetryhymena genome and thus the gene of interest can be inserted in
Tetrahymena genome at homologus sites. pGTW also has cadmium inducible metallothionine
(MTT) promoter to overexpress the gene in Tetrahymena. The recombinant protein can be
pulled down or immunoprecipitated using anti-FLAG antibodies. GFP is a fluorescence tag that
enables visualization of the protein. The Hrt1 protein can be coexpressed with such epitope tag
in Tetrahymena and the presence of the protein can be confirmed by Western blot followed by
Mass-Spec analysis. The expression of HRT1 can be studied using quantitative PCR. The
expression of HTR1 in Tetrahymena can be studied under varying DNA damaging conditions,
such as UV treatment and also log phase, starvation etc. Quantitative PCR can indicate whether
the gene is upregulated, downregulated or unchanged under these various conditions in
Tetrahymena. Gene expression is confirmed the using RT-PCR from cDNA and presence of Hrt1
protein is confirmed using Western blot using anti FLAG antibodies. Finally Tetrahymena strain
transformed with HRT1 gene will be cryopreserved. In addition, comparison of sequences of
HRT1 that we used in cloning experiments to that of published data can confirm whether we
are working with the right gene or not.
Methods/Procedure
1. Plasmid purification from E.coli cells
Cryopreserved E.Coli cells containing the pENTR plasmid with HRT1 gene were obtained from
BMS110 Honor students. In order to confirm the presence of cloned gene in the plasmid pENTR,
the plasmid from the bacteria was isolated using FAST plasmid Mini kit. In this procedure, cells
were lysed using lysis solution and lysozyme. The plasmid was purified using a spin column
assembly and finally the plasmid was collected in a collection tube. (Protocol: Dr. Smith,
BMS558/658 Plasmid Purification and restriction enzyme digest, Spring 2011).
2. Restriction enzyme digest to confirm the presence / orientation of the HRT1
Confirmation of the PCR product (HRT1 gene) in the plasmid was done by restriction digesting
the isolated plasmid with resection endonucleases EcoRV and XhoI. The HRT1 insert does not
contain any restriction sites; hence it could not be cut within the gene. For restriction digest,
3µl of plasmid DNA was incubated at 370C for 1 hour and then run on 2% agarose gel to obtain
the fragments pattern. After confirming that the pENTR contains HRT1 in the correct
orientation, the plasmid was quantified using nanodrop and further treated with Rnase. Gene
construction kit 3.0 was used to set up a mock digest reaction. It gave the predicted sizes and
location of the bands obtained after cutting the DNA with chosen restriction endonucleases.
3. Bioinformatics
HTR1 protein (121 amino acid) from Saccharomyces cerevisiae was considered as starting
protein. The protein sequence of HRT1 was found in Tetrahymena from Tetrahymena genome
database wiki (TTHERM_00313710). Similarly, using NCBI protein database, amino acid
sequence HRT1 was searched among Human, Schizosaccharomyces, Arabidopsis, Drosophila,
Paramecium, and bacteria. TCOFFEE, which computes a multiple sequence alignment was used
to align all four protein sequences from all the species. The phylogenetic tree was constructed
and the sequences of different homologs of HRT1 protein in all four species were graphically
compared with the MEGA 4.0 tool. The phylogenetic tree was constructed and the sequences
of different homologs of HRT1 protein in all four species were graphically compared with the
ClustalW tool. Graphical representation of the HRT1 gene was obtained from TGD wiki. The
gene sequence and structural mapping of HRT1 gene (including Exons, Intron, and EST
sequence) was also obtained from Tetrahymena genome wiki. The genomic sequence was
analyzed for design of the primers for RT-PCR using primer 3 plus program. These primers will
be used in qRT-PCR. (Protocol: Dr. Smith, BMS558/658 Bioinformatics, Spring 2011).
4. Electrocompetent cell preparation:
E.coli cells to be used for transformation by pGTW plasmid containing the gene HRT1 and
tagged with epitope (FLAG-His and GFP) were prepared for receiving the plasmid DNA. E.Coli
DH10B and DB3.1 strains were used. The electro-competent cells were prepared using SOB
media and preserved in 10% glycerol. (Protocol: Dr. Smith, BMS558/658 Production of Electro-
competent E. coli (DH10B & DB3.1), Spring 2011).
5. Bacterial transformation by electroporation and electroporation for competency
determination
The electro-competency of our prepared electro-competent DH10B cells was checked by
electroporation technique (protocol: BMS558/658 Bacterial Transformation by Electroporation,
Spring2011). The electroporation was done using 2 mm gap cell operated at 2.5 kV, 200 Ohms
resistance and 25 µF capacitance. For checking the electrocompetency of DH10B cells, we used
pUC19 (RPD23) (resistant to ampicillin and kanamycin) and pGTW (resistant to ampicillin) and
pENTR (resistant to kanamycin). Transformation efficiency (as CFU/µg DNA) was calculated by
counting the number of colonies grown in the appropriate Kan and Amp plates.
6. LR Clonase Reaction
L-R recombination reaction was used to remove HRT1 from the pENTR-TOPO-D vector (entry
vector) and to replace with the gateway cassette in the new Gateway (GTW) vector (destination
vector). RNase treated purified pENTR with HRT1 insert was used with equal amount of pGTW
vector tagged with either FLAG-His or GFP epitope and incubated with enzyme LR recombinase.
Since E.coli sample #6 showed the best results on gel with concentration = 0.16 µg/mL, it was
used for clonase reaction. For LR Clonase reaction, 400 ng of GTW-GFP and 200 ng of GTW-
FLAG (destination vectors) and 150 ng of pENTR+ HRT1 were used (protocol: Dr. Smith, BMS
558/658 Recombination of Gene into Tetrahymena Gateway Tagging vectors, Spring 2011). The
products of LR recombinase reaction (the recombinant GTW plasmids, pBMFH6-GTW and
pBSMTTGFP-GTW ) were transformed into DH10B electrocompetent E.Coli cells and grown in
LB+Amp plates. The recombinant plasmids in the E.Coli cells were then isolated using boiling
prep method (protocol: Dr. Smith, BMS 558/658 Plasmid Purification & Restriction Enzyme
Digest, Spring 2011).
7. Restriction enzyme digest to confirm the presence of the HRT1 in GTW plasmid.
The colonies were selected for each epitope tag after transformation and the recombinant
plasmid was isolated by boiling prep method. Restriction digest was performed using BamH1
enzyme for cutting both FLAG and GFP tagged recombinant GTW plasmids. The restriction
digested samples were loaded in 1% agarose gel to confirm the LR recombination. (Protocol: Dr.
Smith, BMS 558/658 Plasmid Purification & Restriction Enzyme Digest, Spring 2011).
8. Biolistic transformation of Tetrahymena with recombinant gene.
The recombinant pGTW was isolated from E. Coli cells by Midi prep boiling method. HRT-1 gene
with GFP or FLAG-His tag was further digested and linearized for better transformation. The
linearized pGTW containing HRT-1 gene fragment + epitope tag was coated on gold beads for
transformation. The Tetrahymena cells (Cu522 strain for FLAG tagged plasmid and wild type
strain for GFP tagged plasmid) were starved to bring them to G0 stage. After starving, the cells
were transformed using gene gun; by bombarding the cells with gold particles coated with DNA,
so that they take up the DNA. The cells were then grown in recovery media for four hours, so
that they replicated the recombinant DNA along with their genomic DNA. Then the cells were
grown in media containing appropriate drug (Paclitaxel for FLAG tagged plasmid / Cu522 strain
and cyclohexamide for GFP tagged plasmid / wild type strain). The transformed cells were
plated in 96 well plates and allowed to grow for 4-5 days. The transformed cells are alive with
glistening appearance, whereas the cells that have not taken up the plasmid die in presence of
the drug. Thus, the live cells were subcultured into fresh media and used for protein isolation.
The culture was kept going, passaging whenever required in the presence of appropriate drug
and finally, it was frozen after growing in 2mL stock tubes. (Protocol: Dr. Smith, BMS 558/658,
Biolistic Transformation of Tetrahymena, Spring 2011).
9. PCR
a) GoTaq RT-PCR
The primers were designed to amplify HRT-1 gene and GoTaq RT-PCR was performed on
genomic as well as cDNA to check the efficiency of primers. Gotaq PCR uses a reverse
transcriptase to convert mRNA to cDNA and then a Taq polymerase does the regular PCR
reaction. Forward primer 5’ TATTAAGTGCTTGGCATGTCTTGT 3’ and reverse primer 5’
AATGTGATGCCAACTAAAACTCAA 3’ were used. The average melting temperature for both
forward and reverse primers was calculated as 65.50 C. Four melting temperatures were used:
52.70 C, 54.60 C, 57.40 C, 60.80 C. (Protocol: GoTaq® PCR Core Systems www.promega.com). The
PRC products were run on a 2% agarose gel to estimate the product size and compare between
gDNA and cDNA, if the gene had any introns.
b) qRT-PCR protocol (SsoFast Protocol)
After confirming which melting temperature works the best for PCR, quantitative PCR was
performed using MiniOpticon RT-PCR system. The cDNA was obtained from Tetrahymena cells
under following conditions: Starved, Log phase, no UV, 0 hour UV treatment, 1 hour post UV, 2
hours post UV, 3 hours post UV and 4 hours post UV. Also, 3 standards were used with genomic
DNA at 3 concentrations: STD1 = 0.1 µg/µL, STD2 = 0.01 µg/µL and STD3 = 0.001 µg/µL. PCR
reaction was set using HRT-1 and HHP-1 forward and backward primers. The PCR products were
loaded on a gel and amplification as well as melting curves were obtained. HHP-1 is involved in
stress sensitivity and used as an indicator for stress. HHP-1 expression values are used for
normalization of HRT-1 expression. HRT-1 expression values are made relative to that of starved
cells and a graph is obtained. (Protocol: http://www.bio-
rad.com/webroot/web/pdf/lsr/literature/Bulletin_10014647.pdf)
10. Tetrahymena Protein Isolation and Immunoprecipitation
Tetrahymena cells transformed with HRT-1 + FLAG-His tag were taken from wells E7 and E12
and grown for protein isolation. Cells were induced with CdCl2 for 2 hours. The recombinant
HRT-1 gene can be overexpressed under the influence of Cd inducible MTT promoter and
hence protein can be overexpressed. (Protocol: Dr. Smith, BMS 558/658, Tetrahymena
Protein Isolation and immunoprecipitation, Spring 2011). The protein was isolated and
diluted. The standard curve was prepared using Bradford reagents and protein
concentration was calculated as 2.5 µg/µL and 3.3 µg/µL for sample E7 and E12,
respectively. The protein was loaded on 8% SDS gel and Western blot was performed using
Anti FLAG anitibodies. X ray film was developed at low and high exposure.
11. HRT1 sequencing
The HRT1 insert that we cloned was amplified using M13 forward and reverse primers
and sent for sequencing. The sequence was compared using CodonCode Aligner to
published sequence from gene construction kit.
12. Cryopreservation of the E.coli cells with the pGTW and HRT1 gene
The E.coli clone that we used to extract pGTW plasmid containing HRT1 gene was
cryopreserved in 50% glycerol. (Protocol: Dr. Smith, BMS 558/658, Cryopreservation of
E.Coli containing plasmid with gene, Spring 2011).
Results
The pENTR plasmid containing HRT1 gene was isolated using FAST plasmid mini Kit from
E.Coli cells. Gene construction kit was used to predict the correct orientation and band pattern
(Figure 1). After cutting the plasmid with EcoRI and XhoI, two bands of estimated sizes 2452bp
and 600bp would be obtained if the gene was present in the right orientation. The restriction
digestion of pENTR-HRT1 confirmed that the gene was present in the correct orientation in
samples #3, 4 and 6 (Figure 2). Digest of sample #3 and #4 show three bands each; of
approximate sizes 3000bp (blue arrow), 2400bp (red arrow) and 600bp (yellow arrow). The
additional band could be due to relaxed form of the plasmid. Sample # 6 showed only two
bands, of approximate sizes 2400bp and 600bp. Since E.coli strain # 6 agreed the most with
predicted band sizes, it was chosen for future use. After we confirmed the presence of HRT1 in
the entry vector, isolated plasmid was quantified using a nanodrop (Table 1).
Bioinformatics tools were used to characterize the HRT1 gene. Nucleotide and amino
acid sequence of HRT1 was obtained using Gene prediction program (Figure 3). It shows start
and possible stop codons in the sequence. Expasy Prosite was used to predict protein domains
in Hrt1 in Human, S. cerevisiae and Tetrahymena thermophila homologs (Figure 4). Homo
sapiens homolog has a helix loop helix domain; S. cerevisiae and T. thermophila homologs have
zinc finger domain. It is interesting to note that ExPasy did not find any predicted domain for a
holmolog in Arabidopsis. The size of human homolog is almost 2-fold larger than others. The
phylogenetic tree was constructed and the sequences of different homologs of HTR1 were
graphically compared using MEGA 4.0 tool (Figure 5). The scaled lines show relative genetic
distance between different homologs. The phylogenetic relationship shows that Arabidopsis
and Tetrahymena homologs are more closely related, than its yeast or human homologs. Homo
sapiens homolog seems to be most diverted and distantly related. Tetrahymena genome
database (TGD) wiki was used to characterize the sequence of HRT1 in Tetrahymena (Figure 7).
The entire HRT1 gene has no introns (indicated by black color coded nucleotides) and many
expressed sequence tags or ESTs (underlined sequences). In fact, the entire gene sequence is
identified as EST. Primer 3.0 was used to design primers for RT-PCR, using the genomic
sequence of HTR1 (Figure 8). The sequence highlighted in yellow shows a forward primer and
sequence highlighted in blue shows a reverse primer. The size of product is 195 bp.
After bioinformatics analysis, E.Coli cells were prepared for transformation with pENTR-
HRT1. The purpose of this was to isolate the entry vector in large amount. The E.Coli cells were
grown for 3-4 hours (O.D. 550 = 0.8) in log phase and prepared to be electocompetent. pGTW
vector has ampicillin resistance marker and pENTR has kanamycin resistance marker. DH10B
cells transformed with GTW plasmid did not grow on plates. DB 3.1 cells transformed with GTW
plasmid did not grow on Kanamycin or Ampicillin plates. (However, DB 3.1 cells should have
grown on amp plates as they contain mutation for ampicillin resistance). In addition, pGTW has
ccdB gene that is lethal to E.coli cells. Unless ccdB gene is knocked out and replaced by HRT1
insert in pGTW, DH10b cells die. The average transformation efficiency of DH10B cells on AMP
plate was 0.81*104 CFU/µg, after growing at 370C for 30 minutes. The average transformation
efficiency of DH10B cells on KAN plate was found to be 2.23*103 CFU/µg, after growing at 370C
for 30 minutes. (Note: Average efficiency is calculated for both DH10B#1 and DH10b#2 cells).
Since the transformation efficiency of the cells was OK, they were used in transformation. To
prepare the DNA for transformation, LR clonase reaction was performed. The product of LR
recombinase reaction, pGTW-HRT1- epitope was transformed into E.coli DH10b cells and grown
on LB+amp plates at 370C for 24 hours. There were 8 colonies on plate containing cells
transformed with pGTW-GFP and 650 colonies observed on plate containing cells transformed
with pGTW-FLAG. Control plates were also set for RFP, GFP, FLAG-His and 2HA (vectors only).
The LB+amp plates showed RFP= 0 colonies, GFP = 0 colonies, FLAG-His = 1 colonies and 2HA =
2 colonies. The last two were probably due to contamination. In addition, E.Coli cells
transformed with no DNA were used as additional control and the plates did not show any
growth. Four colonies each for pGTW-GFP containing HRT1 and pGTW-FLAG containing HTR1
plates were used to start 2 mL cultures and these cells were used to check if the cells contained
pGTW-epitope- HRT1 insert correctly. After transformation of E.Coli cells with epitope tagged
(FLAG or GFP) pGTW containing HRT-1 insert, a restriction digest was performed using BamHI
enzyme. After isolating recombinant plasmid by boiling prep method from electro-competent
DH10B cells, followed by restriction digest, it was confirmed that plasmid pBM-GTW contained
HRT1 gene insert in the right orientation.
GFP tagged plasmid, pBSMTTGFP_gtw with HRT1 insert has size of 9418 bp (Figure 9). It
has rpI29 locus and an MTT promoter. Similarly, FLAG-His tagged plasmid, pBMF6_gtw with
HRT1 insert is of 6976 bp size (Figure 10). It has Btu1 locus and an MTT promoter upstream of
the gene. 1% agarose gel was run to confirm which E.Coli clones contained HRT1 gene (figure
11). All four transformed clones of pGTW-GFP contained HRT1 insert, as 2 bands are seen in
lanes with pGTW-GFP-HRT1 from clones whereas 3 bands are seen in pGTW-GFP without any
gene. Similarly, all four clones transformed with pGTW-FLAG-HRT1 contained HRT1 insert as
only one band is seen in those lanes, whereas 3 bands are seen in pGTW-FLAG lane. Hence, any
clone can be used for further use. Since the HRT1 gene does not have any restriction sites
within it, the pGTW plasmid with and without the HRT1 insert was digested using BamH1
enzyme and the digest was compared. Gene construction kit was used to get a mock digest.
After confirmation of the correct recombination product containing the epitope tag and
gene (HRT-1), biolistic transformation of Tetrahymena was performed. The Tetrahymena
(Cu522) cells were transformed with linearized pGTW-HRT1-FLAG plasmid. Four transformants
were found and they were subcultured into fresh media. For Tetrahymena (wild type) cells that
were transformed with linearized pGTW-HRT1-GFP plasmid, high transformation efficiency was
observed with 17 transformant colonies. After transformed Tetrahymena clones were obtained,
quantitative PCR was performed to look at expression of HRT1 gene from the cells. Forward
primer (sequence 5’ TATTAAGTGCTTGGCATGTCYTGT 3’) and reverse primer (5’
TTGAGTTTTAGTTGGCATCACATT 3’) for HRT1 amplification were designed and stocks were
made. Average melting temperature was calculated as 56.5o C. GoTaq PCR was performed on
genomic and cDNA from Tetrahymena cells using four temperatures as melting temperatures
(Tm) to check the efficiency of primers and see if the PCR was working. The products of GoTaq
PCR were loaded on a 2% agarose gel (Figure 12). However, PCR reaction at low (52.7o C) and
high (60.8o C) melting temperatures did not work very well, middle temperatures 54.6o C and
57.4 o C as Tm showed low amplification. To make sure if the primers were working correctly,
PCR reaction was set using SsoFastEvaGreen supermix, a hot start PCR. cDNA and genomic DNA
was amplified in miniOpticon thermocycler. The products were loaded on a 2% agarose gel, and
amplification (figure 13) and melting curves (figure 14) were obtained. qRT-PCR was done to get
expression profile of HRT1 in tetrahymena (figures 15, 16). The HRT1 expression was
normalized using values for HHP1 (figures 17 & 18) and also the HRT1 expression values were
made relative to value for starved cells. There is positive trend between UV treatment and
HRT1 expression (Figure 19). Expression profile of HRT1 was also obtained from TGD wiki for
comparison (Figure 20). The gene expression is upregulated in growing cells and starvation, but
downregulated in conjugating cells.
The positive tranformants of Tetrahymena for GFP tagged HRT1 were used for protein
isolation. The protein concentration of the transformant samples were calculated (figure 21). A
western blot did not confirm HRT1 protein (Figure 22). The protein was probably not
expressed.
Finally, the sequence of HRT1 gene that we cloned was compared to published
sequence of HRT1 gene and it was found to be exactly same. The E.coli strain that was
transformed with pGTW-HRT1-GFP and pGTW-HRT1-FLAG-His clone #2 and used for plasmid
isolation was cryopreserved.
Figures:
Figure 1: Plasmid map of pENTR-HRT1 insert obtained using gene construction kit. HRT1
sequence in green and all of the restriction enzymes marked around the edges. The pink is the
antibiotic resistance (Kanr), brown is the recombination sites and the yellow is the origin of
replication.
Figure 2: 2% agarose gel showing restriction digest of pENTR +HTR1 plasmid using restriction
endonucleases EcoRV and XhoI. Lane 1 contains 1kb DNA ladder; Lane 2 contains Ecoli sample
#3; Lane 3 contains E.coli sample #4; Lane 4 contains E.coli sample #6. The band sizes and
location indicate that the HRT1 insert has been inserted in the correct orientation.
Sample #3 Sample #4 Sample #6
260/280 1.97 1.92 1.39
Conc (µg/mL) 0.21 0.14 0.16
Table 1: quantification of isolated pENTR +HTR1 using nanodrop.
1 2 3 4
3kb
2 kb
500 bp
Bioinformatics
Figure 3: Nucleotide and amino acid sequence given by Gene prediction program. Predicted
amino acid, coding, and genomic sequences of the HRT1 of Tetrahymena thermophila
(TTHERM_00313710) obtained from TGD wiki.
Homo sapiens: H-S-HRT1 (304 aa)
PS50888 HLH Myc-type, 'helix-loop-helix' domain profile :
S. cerevisiae: S-C-HTR1 (121 aa)
PS50089 ZF_RING_2 Zinc finger RING-type profile :
Tetrahymena thermophila: T-T-HRT1 (152 aa)
PS50089 ZF_RING_2 Zinc finger RING-type profile :
>TTHERM_00313710(coding) HRT1 ATGGAAACAGAAAAAAGATTCGAAGTCAAAAAATGGAACGCTGTTGCTCTTTGGCAATGG GATATTGAAGTTGAAAATTGCGCCATTTGCAAGAAGTAAGTTTAATCATCTCACAACCTT TTTTTATTAAGTGCTTGGCATGTCTTGTTTTACATTAATTAGATTATTTTCAAAGATTTC GTATAAAAATTTTAAAAGCCAAATAGGTTTTATAAGCACATAAAATAAAATGTTTAACTA TATTTGTAACCATATTTAATTTAAATAATTAGCAACATTATGGAACCTTGTATTGAATGT GATGCCAACTAAAACTCAAATAGTGCTTCCGAATGTGATGTTGCTTGGGGTACTTGCAAT CACGCATTCCATTTCCATTGTATTTCCAGATGGTTAAAAACTCGTAATACTTGTCCTCTT GATTCTCGTCAATGGGATTATCAAAGATATGGTCGTTGA
>T.t.HRT1 METEKRFEVKKWNAVALWQWDIEVENCAICKKQVQSSHNLFLLSAWHVLFYINQIIFKDFVQKFQKPNRFYKHIKQNVQLYLQPYLIQIISNIMEPCIECDANQNSNSASECDVAWGTCNHAFHFHCISRWLKTRNTCPLDSRQWDYQRYGR
Figure 4: Graphical representation of the HRT1 gene obtained from the ExPasy prosite in
organisms Homo sapiens, S. cerevisiae and Tetrahymena thermophila, however no domains are
found in Arabidopsis thaliana.
Figure 5. The phylogenetic tree was constructed by two different methods as mentioned in the
figures and the sequences of different homologs of HTR1 in Tetrahymena thermophila (T.t.),
Arabidopsis thaliana(A.t), Saccharomyces cerevisiae (S.c.) and Homo sapiens (H.s.) were
graphically compared using MEGA 4.0 tool.
Figure 6: Graphical representation of Tetrahymena gene HRT1 homolog (TTHERM_00313710)
using TGD wiki. Alignment P-value (BLAT) or E-value (TBLASTN) color codes: black < 1e-50; dark
grey > 1e-60; purple > 1e-80; blue > 1e-100; green > 1e-130; pink > 1e-160; yellow > 1e-
200; red > 1e-350
ATGGAAACAGAAAAAAGATTCGAAGTCAAAAAATGGAACGCTGTTGCTCTTTGGCAATGGGATATTGAAGTTGAA
AATTGCGCCATTTGCAAGAAGTAAGTTTAATCATCTCACAACCTTTTTTTATAAGTGCTTGGCATGTCTTGTTTTACA
TTAATTAGATTATTTTCAAAGATTTCGTATAAAAATTTTAAAAGCCAAATAGGTTTTATAAGCACATAAAATAAAAT
GTTTAACTATATTTGTAACCATATTAATTTAAATAATTAGCAACATTATGGAACCTTGTATTGAATGTGATGCCAACT
AAAACTCAAATAGTGCTTCCGAATGTGATGTTGCTTGGGGTACTTGCAATCACGCATTCCATTTCCATTGTATTCCA
GATGGTTAAAAACTCGTAATACTTGTCCTCTTGATTCTCGTCAATGGGATTATCAAAGATATGGTCGTTGA
Figure 7: The genomic sequence of Tetrahymena HRT1 gene including protein coding regions
and ESTs. The entire gene is no introns and many expressed sequence tags.
ATGGAAACAG AAAAAAGATT CGAAGTCAAA AAATGGAACG CTGTTGCTCT 51
TTGGCAATGG GATATTGAAG TTGAAAATTG CGCCATTTGC AAGAAGTAAG 101
TTTAATCATC TCACAACCTT TTTTTATTAA GTGCTTGGCA TGTCTTGTTT 151
TACATTAATT AGATTATTTT CAAAGATTTC GTATAAAAAT TTTAAAAGCC 201
AAATAGGTTT TATAAGCACA TAAAATAAAA TGTTTAACTA TATTTGTAAC 251
CATATTTAAT TTAAATAATT AGCAACATTA TGGAACCTTG TATTGAATGT 301
GATGCCAACT AAAACTCAAA TAGTGCTTCC GAATGTGATG TTGCTTGGGG 351
TACTTGCAAT CACGCATTCC ATTTCCATTG TATTTCCAGA TGGTTAAAAA 401
CTCGTAATAC TTGTCCTCTT GATTCTCGTC AATGGGATTA TCAAAGATAT 451
GGTCGTTGA
Figure 8: RT-PCR primer designed with the Primer 3 Plus Program using the genomic sequence
of HRT1 gene. The sequence highlighted in yellow shows a forward primer and sequence
highlighted in blue shows a reverse primer. The size of product is 195 bp.
Figure 9. Plasmid map pBSMTTGFP-GTW with HRT1 insert (green). Location of restriction sites is
shown. The green arrow indicates location and direction of HRT-1 gene and pink color shows
antibiotic resistance gene.
Figure 10. Plasmid map pBMFH6-GTW with HRT1 insert (green arrow). The plasmid contains
Btu1 locus. The arrow indicates the direction of the gene. Location of restriction sites is shown
on the plasmid. The pink is the antibiotic resistance. It also has MTT promoter upstream of the
gene.
Figure 11. 1% Agarose gel picture of restriction digested GTW recombinant (with HRT1) and
without HRT1 plasmid, with BamH1 enzyme. Lane 1 contains 1kb ladder; Lane 2 contains
pGTW-GFP without HRT1 insert; Lane 3 contains recombinant pGTW-GFP-HRT1 from clone #1;
Lane 4 contains recombinant pGTW-GFP-HRT1 insert from clone #2; Lane 5 contains
recombinant pGTW-GFP-HRT1 insert from clone #3; Lane 6 contains recombinant pGTW-GFP-
HRT1 insert from clone #4; Lane 8 contains pGTW-FLAG without HRT1 insert; Lane 9 contains
recombinant pGTW-FLAG-HRT1 insert from clone #1; Lane 10 contains recombinant pGTW-
FLAG-HRT1 insert from clone #2; Lane 11 contains recombinant pGTW-FLAG- HRT1 insert from
clone #3; Lane 13 contains recombinant pGTW-FLAG-HRT1 insert from clone #4. Size of 1kb
DNA ladder bands is shown on the left. The digest confirms that all four clone of each pGTW-
GFP and pGTW-FLAG contain HRT-1 insert.
1 2 3 4 5 6 7 8 9 10 11 12 13
6kb
3kb
2kb
1.5kb
500 bp
Figure 12. 2% agarose gel showing products of GoTaq PCR reaction. Lane 1 contains 100kb DNA
ladder; lane 2 contains genomic (gDNA) sample amplification at Tm=52.70 C; lane 3 contains
cDNA sample amplification at Tm=52.70 C; lane 4 contains gDNA sample amplification at
Tm=54.60 C; lane 5 contains cDNA sample amplification at Tm=54.60 C; lane 6 contains gDNA
sample amplification at Tm=57.40 C; lane 7 contains cDNA sample amplification at Tm=57.40 C;
lane 8 contains gDNA sample amplification at Tm=60.80 C; lane 9 contains cDNA sample
amplification at Tm=60.80 C. The low band intensity and absence of bands in lanes 2,3 and 9
indicate inefficient PCR. The red arrow indicates position of bands in lanes 5,6,7,8.
1 2 3 4 5 6 7 8 9
500 bp
400 bp
300 bp
200 bp
100 bp
Figure 13. Test qRT-PCR to confirm primers. Amplification curve is obtained for cDNA and gDNA
samples using GoTaq PCR. Forward and reverse primers designed for HRT-1 are used. STD1
(gDNA 0.1ug/uL) and STD2 (gDNA 0.01ug/uL) are RED, Negative control (-TEMPLATE) is BLACK,
and cDNA is GREEN. The cDNA shows amplification above threshold. However, STD1 (gDNA
0.1ug/uL) shows very low amplification, probably due to very high concentration of DNA.
Negative control also shows amplification, indicating contamination. However, the curves
confirm that primers are working.
Figure 14. Test qRT-PCR to confirm specificity of primers and purity of the PRC product. Melting
curve is obtained for cDNA and gDNA samples using GoTaq PCR. STD1 (gDNA 0.1ug/uL) and
STD2 (gDNA 0.01ug/uL) are RED, Negative control (-TEMPLATE) is BLACK, and cDNA is GREEN.
The sharp and single peaks confirm that only one product is formed, which has melting
temperature at around 740 C. The primers are specific.
Figure 15. qRT-PCR to show expression profile of HRT-1 in tetrahymena. Amplification curve is
obtained for cDNA and gDNA samples using SsoFastEvaGreen supermix PCR protocol. Forward
and reverse primers designed for HRT-1 are used. STD1 (RED), STD2 (YELLOW), STD3 (BRIGHT
BLUE), Log growing cells (DARK GREEN), Starved cells (TEAL), No UV damage (BRIGHT GREEN),
0hr after UV (ORANGE), 1hr after UV (GREY), 2hr after UV (PINK), 3hr after UV (DARK BLUE), and
4hr after UV (PURPLE). There is positive trend between HRT-1 expression and UV treatment.
HRT-1 expression increases with increasing UV exposure time, but it is low in case of log
growing, starved and No UV samples.
Figure 16. Melting curve is obtained in qRT-PCR expression profile of HRT-1 in tetrahymena.
Melting curve is obtained for cDNA and gDNA samples using SsoFastEvaGreen supermix PCR
protocol. STD1 (RED), STD2 (YELLOW), STD3 (BRIGHT BLUE), Log growing cells (DARK GREEN),
Starved cells (TEAL), No UV damage (BRIGHT GREEN), 0hr after UV (ORANGE), 1hr after UV
(GREY), 2hr after UV (PINK), 3hr after UV (DARK BLUE), and 4hr after UV (PURPLE). The sharp
and single peaks confirm that only one product is formed, which has melting temperature at
around 740 C. The primers are specific.
Figure 17. qRT-PCR to show expression profile of HHP-1 in Tetrahymena, used for normalizing
the expression of HRT-1. Amplification curve is obtained for cDNA and gDNA samples using
SsoFastEvaGreen supermix PCR protocol. STD1 (RED), STD2 (YELLOW), STD3 (BRIGHT BLUE), Log
growing cells (DARK GREEN), Starved cells (TEAL), No UV damage (BRIGHT GREEN), 0hr after UV
(ORANGE), 1hr after UV (GREY), 2hr after UV (PINK), 3hr after UV (DARK BLUE), and 4hr after UV
(PURPLE). HHP-1 expression increases with increasing UV exposure as predicted and thus we
know that PCR reaction works as predicted.
Figure 18. Melting curve of HHP-1 expression in tetrahymena. Melting curve is obtained for
cDNA and gDNA samples using SsoFastEvaGreen supermix PCR protocol. STD1 (RED), STD2
(YELLOW), STD3 (BRIGHT BLUE), Log growing cells (DARK GREEN), Starved cells (TEAL), No UV
damage (BRIGHT GREEN), 0hr after UV (ORANGE), 1hr after UV (GREY), 2hr after UV (PINK), 3hr
after UV (DARK BLUE), and 4hr after UV (PURPLE). The sharp and single peaks confirm that only
one product is formed, which has melting temperature at around 750 C. The primers are
specific.
Figure 19. Relative expression profile of HRT1 in Tetrahymena under different conditions as
indicated in the figure legend. There is positive correlation between UV treatment time and
HRT1 expression. The cDNA was obtained from Tetrahymena cells under following conditions:
Starved, Log phase, no UV, 0 hour UV treatment, 1 hour post UV, 2 hours post UV, 3 hours post
UV and 4 hours post UV.
Figure 20. Expression Profile of HRT1 from TGD wiki. Growing cells are denoted by L, starvation
by S and conjugation by C. The gene expression is upregulated in growing cells and starvation,
but downregulated in conjugating cells.
0123456789
LOG STV No UV UV 0hr UV 1hr UV 2hr UV 3hr UV 4hr
Nor
mal
ized
Rel
ativ
e ex
pres
sion
Expreession of HRT-1
Figure 21. Standard curve using BSA Protein Standard. Standard curve was used to calculate
protein concentration of unknown samples, E37 and E12.
y = 0.0242x + 0.4284R² = 0.9421
00.10.20.30.40.50.60.7
0 2 4 6 8 10micrograms Protein
1 2 3 4 5
1 2 3 4 5
A
B
Figure 22. Western blot of protein samples isoalted from Tetrahymena tranfomants using anti
FLAG antibody. Lane 4 contains E7 sample; Lane 5 contains E12 sample. Band size (kD) and
position of the rainbow marker is shown on left. Panel A shows X ray image after 20 minutes of
exposure. Lanes 4 and 5 indicate some bands; however they are not specific to those lanes and
simple could be background or non-specific binding. Pane B shows X ray image after 5 seconds
of exposure. Lanes 4 and 5 show absence of bands. The figure indicates no HRT-1 protein was
expressed.
Discussion/Conclusion
The aim of this experiment was to clone HRT1 gene into Tetrahymena and express the protein
in order to study its function and localization. HRT1 gene was characterized using
Bioinformatics tools and the functional domains and phylogenetic relationship of the HRT1
homologs was predicted. The primers were designed for quantification of the cloned HRT1 gene
using qRT-PCR for HRT1 genomic sequence. Expression profile of HRT1 was obtained in
Tetrahymena under different conditions. Expression analysis, immunoprecipitaion of the
protein was performed with the help of epitope tag to better understand the functional
properties of this gene.
In order to achieve this, the pENTR plasmid was isolated from the E.coli cells provided by
BMS110 honors class and presence of HRT1 gene insert in the correct orientation was
determined, with the help of restriction digestion. The plasmid was RNase treated and
quantified in order to be used in LR clonase reaction. Using Invitrogen Gateway cloning
cassette, HRT1 gene from pENTR was swapped into a destination vector GTW with either FLAG-
HIS or GFP tag. The epitope tagging of the gene enables immunoprecipitation of the protein. To
confirm that epitope tagged GTW vector contained HRT1 gene after the successful recombinase
reaction, the recombinant plasmids was digested with restriction endonucleases BamH1. After
confirmation of the insert in the GTW vector, E.coli DH10b cells were transformed with
recombinant pGTW-FLAG and recombinant pGTW-GFP plasmids. After growing the cells,
recombinant pGTW-FLAG-HIS and recombinant pGTW-GFP vector from DH10b cells was
isolated. Again, presence of HRT1 gene in GTW plasmid was confirmed before proceeding for
biolistic transformation. It is important to confirm if our plasmid has correct gene and epitope
tag at every step in the tranformation experiment.
Bioinformatics results showed that the protein sequence of HRT1 gene is found in
Tetrahymena (TTHERM_00313710) with e value 3.3e-28. Hence, it is the closest homolog to S.
cerevisiae HRT1 protein. There is only on homolog of HRT1 in Tetrahymena
(TTHERM_00313710), as found in TGD wiki. The HRT1 protein has several homologs across
different species like humans, Tetrahymena, Arabidopsis thaliana (a plant) and S. cerevisiae, but
it is not found in bacteria or paramecium. Homologs are found in eukaryotes, but not in more
ancient species like bacteria. S. cerevisiae, so HRT1 must have come at a later point on
evolutionary time scale. HRT1 protein in S. cerevisiae has 121 amino acids, so it’s a fairly small
protein. Moreover, there are not many predicted domains that are conserved across the
homologs of different species. Because HRT1 does not seem to be much conserved across
species, it may not be very clear which particular protein domain is functionally important. It
was found that homologs in S. cerevisiae and T. thermophila contain zinc finger domains while
H. sapiens have a helix loop helix, a different domain. A. thaliana does not have predicted
domains. Either the ExPasy program could not identify the domains in A. thaliana HRT1 protein,
or the domains are not important in function of A. thaliana homolog and thus are not
conserved. As not many conserved domains are found in homologs in spite of their sequence
similarity, the function of the protein probably depends on modification of few amino acids.
Phylogenetic tree shows that HRT1 in A. thaliana and Tetrahymena thermophila are more
closely related, than in S. cerevisiae or humans. Multiple sequence alignment using TCOFFEE
shows that there are some amino acids sequences that are conserved across all or most the
homologs. Amino acids in such regions could be functionally important for the role of HRT1
protein. TGG wiki shows sequence and graphical representation of the gene and the striking
feature is the entire gene has no introns and many expressed sequence tags (ESTs). A gene
without any introns may mean that many isoforms of the protein are not present. ESTs
represent short sub-sequence of a cDNA, regions of the genomes that are have been used to
identify gene transcripts. A small size of gene could be another reason why all of the sequence
has been identified as EST. It also means HRT-1 is always expressed in form of mRNA and may
have an important role in housekeeping functions of the cell.
Biolostic tranformation of Tetrahymena cells was done using linearized plasmid pGTW+
HRT1 gene. Tetrahymena Cu522 strain has a mutation in Btu1 locus which makes them
sensitive to the drug paclitaxel, a microtubule stabilizer. Tranformation of these cells with
linearized pBSMF6_GTW + HRT1 disrupt the gene for sensitivity for Pac and thus the
tranformants become resistant towards the drug. Wild type Tetrahymena receive resistance
gene for cycloheximide along with pBSMTTGFP_GTW +HRT-1. Hence, both type of tranformants
can be selected using specific drugs in the medium.
Tetrahymena cells were treated with increasing does of UV light and cDNA was
obtained. Quantitative PCR was performed on cDNAs from cells under different conditions,
such as UV treated, log growing or starved. Expression profile of HRT-1 under different
conditions of UV treatment showed a definite positive relation between HRT-1 expression and
increasing UV exposure time. It is likely that HRT-1 is involved in both early and late DNA
damage responses. Since HRT-1 is a part of ubiquitin ligases (E3), it is not affirmative to find the
positive correlation between HRT-1 expression and DNA damage by UV. It is also possible that
HRT-1 is involved in apoptosis and not DNA repair. Hence, to predict the exact function of HRT-
1 in UV response, more experimentation is required. No significant expression of HRT-1 is seen
in log growing, starved or untreated cells. When this was compared to the expression profile of
HRT-1 obtained published on TGD wiki, the expression was upregulated in case of log growing
and starved cells, but down-regulated in conjugating cells. However, no published information
was available about UV response of the cells in terms of HRT-1 expression and thus this data
could not be compared.
Moreover, protein extraction of transformed cells followed by immunoprecipitation by
FLAG antibody showed no presence of protein. It is possible that HRT-1 was not expressed in
enough quantity or it was being degraded. We may want to induce cells with Cadmium for
longer time and see the expression of protein. We do not have enough information on what
post-transcriptional modification are required to make a stable and functional protein. It might
be informative to see mRNA and protein expression of HRT-1 at the same time. It also might be
interesting to study HRT1 knockout phenotype. It would have been better if we could see
expression of pHRT1 with GFP tag. That ways we would know if there was problem with
expression altogether, or detection of the protein with immunoblot.
It was also interesting to see that the sequence of HRT1 that we cloned into pGTW
vector was identical to the published sequence. It was confirmed that we cloned the correct
gene into E.Coli and Tetrahymena and thus our results should be comparable to the published
data. Cryopreservation the strains of E.coli that were used in transformation enables us to
preserve exact clones of cells containing pGTW+HTR1, without the chance of introducing
mutation for future use. Investigators who wish to study this gene can start from preserved
cultures.
In summary, this lab was very informative. We learned many procedures including
isolation of plasmid using different methods, LR clonase reaction, tranformation of organisms,
and immunoprecipitation. It was interesting to know that HRT1 has some role in UV damage. It
might be interesting to see effect of other DNA damaging conditions, environmental stresses or
naturally dying cells. We can also make point or multiple mutations in HRT1 to see which amino
acids play important role in function of the protein. Further study is required to pin point the
exact function of the protein.
Work cited
Blondel, M. Galan JM, Peter M. (2000). Isolation and Characterization of HRT1 Using a Genetic
Screen for Mutants Unable to Degrade Gic2p in Saccharomyces cerevisiae. Genetics , 1033–
1044.
Deshaies, R. J. Claudio A.P. Joazeiro (2009). RING Domain E3 Ubiquitin Ligases. Annu. Rev.
Biochem , 399–434.
Frescas, D. Michele Pagano (2008). Deregulated proteolysis by the F-box proteins SKP2 and β-
TrCP: Nat Rev Cancer , 438–449.
Jennifer M. Gagne, Brian P. Downes, Shin-Han Shiu, Adam M. Durski, Richard D. Vierstra
(2002). The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in
Arabidopsis. PNAS , 99, 11519–11524.
Glickman, M. H., Aaron Ciechanover (2002). The Ubiquitin-Proteasome Proteolytic Pathway:
Destruction for the Sake of Construction. Physiol Rev , 373-428 .
Hinnebusch, A. G. (n.d.). Active destruction of defective ribosomes by a ubiquitin ligase involved
in DNA repair. GENES & DEVELOPMENT , 891–895.Seol JH, Feldman RM, Zachariae W,
Shevchenko A, Correll CC, Lyapina S, Chi Y, Galova M, Claypool J, Sandmeyer S, Nasmyth K,
Deshaies RJ, Shevchenko A, Deshaies RJ. (1999). Cdc53/cullin and the essential HRT1 RING-H2
subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. Genes Dev ,
1614-26.
J. Eugene Lee, Michael J. Sweredoski, Robert L. J. Graham, Natalie J. Kolawa, Geoffrey T. Smith,
Sonja Hess and Raymond J. Deshaies (2010). The Steady-State Repertoire of Human SCF
Ubiquitin Ligase Complexes Does Not Require Ongoing Nedd8 Conjugation. Molecular and
Cellular Proteomics .
Blondel M, Galan JM, Peter M. (2000). Isolation and characterization of HRT1 using a genetic
screen for mutants unable to degrade Gic2p in saccharomyces cerevisiae. Genetics , 1033-44.
Wei, D., Yi Sun (2010). Small RING Finger Proteins RBX1 and RBX2 of SCF E3 Ubiquitin Ligases.
Genes & Cancer , 700-707 .