MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

Mapping of the fad24

Self-Interaction Domains

Report on Final year Project

KAN, Chun Him (2009 5867)

The Hong Kong University of Science and Technology

Author Note
This Report was prepared for LIFS 4971 & 4981, supervised by Dr Chun Liang

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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1.

Acknowledgements ....................................................................................................... 3

2.

Abstract......................................................................................................................... 3

3.

Introduction .................................................................................................................. 3

4.

5.

6.

7.

3.1.

Background .......................................................................................................... 3

3.2.

The Yeast Two-Hybrid System............................................................................... 4

3.3.

Objective .............................................................................................................. 6

Materials ....................................................................................................................... 7
4.1.

Oligodeoxynucleotides (Primer)............................................................................ 7

4.2.

E. coli strains ........................................................................................................ 7

4.3.

Yeast strains ......................................................................................................... 7

4.4.

Plasmids ............................................................................................................... 7

4.5.

Antibodies ............................................................................................................ 8

4.6.

Media and plates .................................................................................................. 8

4.7.

Buffer and reagent ............................................................................................... 8

4.8.

Equipment and kits ............................................................................................... 9

Methods ...................................................................................................................... 10
5.1.

Polymerase Chain Reaction (PCR) ....................................................................... 10

5.2.

Restriction digestion ........................................................................................... 10

5.3.

Agarose Gel Electrophoresis ............................................................................... 10

5.4.

DNA extraction from agarose gel ........................................................................ 11

5.5.

Determination of DNA concentration ................................................................. 11

5.6.

Ligation .............................................................................................................. 12

5.7.

Transforming E. coli with plasmids ...................................................................... 12

5.8.

Plasmid Extraction form Transformed E. coli ....................................................... 12

5.9.

Transforming budding yeast with plasmids ......................................................... 12

5.10.

Yeast growth, storage and revival ....................................................................... 13

5.11.

Methods for yeast total protein extraction ......................................................... 13

5.12.

SDS-PAGE ........................................................................................................... 14

Results......................................................................................................................... 15
6.1.

Bioinformatics Analysis ....................................................................................... 15

6.2.

Successful Molecular Cloning.............................................................................. 16

6.3.

Yeast Transformants with Correct Inserts Expressed ........................................... 18

6.4.

2 Domains of fad24 Self-Interact ........................................................................ 20

Discussion ................................................................................................................... 21

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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8.

Reference .................................................................................................................... 24

1. Acknowledgements
It is my great pleasure to express my sincere gratitude to Prof. Chun Liang, who
is my supervisor for my final year project. His guidance and support are uttermost
essential in helping me throughout the year.
I would like to express my sincere thanks to all the people working in Prof.
Liangs laboratory. Special thanks have to be given to Dr. Aftab Amin, who taught
and supported me a lot since the first day I joined the laboratory. I am grateful to a
number of postgraduates and technicians who offered great support during my time in
the laboratory
KAN, Chun Him
May 2015

2. Abstract
fad24 (factor for adipocyte differentiation, clone number 24) was found to be a
human homolog of the Noc3p (Nucleolar complex-associated protein). In our study,
fad24 is found to be self-interacting. It possesses two self-interacting motifs which are
also coiled-coils. The first motif is positioned in region from amino acid 1 to 150 while
the second one lies in between amino acid 400 and 599.

3. Introduction
3.1. Background
fad24 encodes a protein consisting of 801 amino acids. The amino acid sequence
of fad24 was found to have a basic leucine zipper motif and a NOC domain

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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(Tominaga, 2004). It is found to localize in the nucleus. Apart from promoting

adipocyte differentiation, fad24 is also believed to be one of the components for
transcription and/or pre-mRNA splicing. (Tominaga, 2004).
However, fad24 was also found to share a significant degree of identity (about
30%) with Noc3p of budding yeast. Therefore, fad24 is believed to be a human
homolog of Noc3p, given that Noc3 is pretty conserved among species of eukaryotes.
Hence, fad24 is speculated to be capable of DNA replication initiation.
Noc3p was identified as a bHLH (basic helix-loop-helix) protein. It is
multifunctional in which it is required for DNA replication initiation and pre-rRNA
processing.
Interaction with many proteins and/or with replicators is required for DNA
replication initiation. For instance, Noc3p interacts with MCM proteins and ORC, as
well as binding to chromatin and replicators throughout the cell cycle. It links between
ORC and other initiation proteins to allow the establishment and maintenance
pre-replication complexes (pre-RCs) (Zhang, 2002).
Given the previous study in our lab, several ORC components and Noc3p were
found to be self-interacting. It is believed that ORC in budding yeast may dimerize to
load the MCM.

It would be interesting to know if the same scenario happen to

human as well. Therefore, the fad24 was targeted in our study to search for
self-interacting domain(s).
3.2. The Yeast Two-Hybrid System
In our study, the yeast two-hybrid (Y2H) was used.

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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The yeast two-hybrid can be done using mating or co-transformation. Regarding

mating, yeast strain a (like AH109) & (like Y187) were transformed with one
plasmid each (pGADT7 or pGBKT7). They are then mated to form diploid cells with
both pGADT7 and pGBKT7. Regarding co-transformation, AH109 can be transformed
with pGADT7 and pGBKT7 together. Co-transformation was used in our study due to
the easiness in transformation procedures.
After co-transformation, transformants containing both pGADT7 and pGBKT7
can survive on SCM-L-T plate (synthetic complete medium minus Leucine and
Tryptophan).
Originally, the activation of gene transcription in yeast require transcription factor
to bind on the upstream activation sequence (UAS) using the DNA binding domain
(BD) as well as the activation domain (AD) to activate transcription. The physical
distance does not affect the transcription. In the yeast two-hybrid system, the AD and
BD are separated and fused with the desired protein to check for protein interaction.

Fig. 1: Diagram Demonstrating How the Y2H Works (Clontech, 1999)

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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If the desired protein pair interacts, the AD and BD will be brought together to
recruit other transcription factors and RNA polymerase II to kick-start expression of
the reporter genes (fig 1). Interaction between the bait (BD-fused protein) and the prey
(AD-fused protein) allows the transformant to express HIS3. Hence, the yeast can
synthesis Histidine and thus capable of growing in SCM-L-T-H plate (SCM minus
leucine, tryptophan and histidine). Strong interaction between the bait and the prey
allows the transformant to express HIS3 and ADE2. Hence, the yeast can synthesis
Histidine and Adenine. Consequently, it is now capable of growing in SCM-L-T-H-A
plate (SCM minus leucine, tryptophan, histidine and adenine). It is important to assume
that the inserted protein can neither be an AD or a BD for the system to work.
3.3. Objective
In our study is to investigate the possibility of self-interaction in fad24. If
self-interaction is possible, we would like to map out any self-interacting motif.
Therefore, we divide the fad24 into the 10 fragments (1-10 as in figure) according to
the coiled-coil analysis (by MULTICOIL) and multiple sequence alignment analysis
(by Clustal W2). We have designed overlapping sequence for double-checking.

Fig. 2: Planned Dissection of fad24 for the experiment

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4. Materials
4.1. Oligodeoxynucleotides (Primer)
Name

Sequence
F hNoc3 0 - 150

GAATCCCATATGATGAAGGCGAGAAGAAA

R hNoc3 0 - 150

GAATCCCTTAAGCTAAATCAGTTCCTTCTCTGGT

F hNoc3 151 270

GAATCCCATATGGCACCAGAGAAGGAACTGATT

R hNoc3 151 - 270

GAATCCCTTAAGCTACATCAGAGAAACAATTACC

F hNoc3 271 399

GAATCCCATATGCCTTCATATAAAATCCGGCCCC

R hNoc3 271 399

GAATCCCTTAAGCTAAAGAGAAGCCTGGCCTAA

F hNoc3 400 599

GAATCCCATATGGTGATTTCTGGTTTTGTGAAGGG

R hNoc3 400 599

GAATCCCTTAAGCTAACCTTCATTGGTAGCACC

F hNoc3 600 801

GAATCCCATATGGAGATTGTACTCCAGTG

R hNoc3 600 801

GAATCCCTTAAGCTAGTGTAGTGATGT

4.2. E. coli strains

DH5 (from our lab stock)
4.3. Yeast strains
Strain
AH109

Genotype
MATa,

trp1-901,

leu2-3,

112,

ura3-52,

his3-200,

gal4 ,

gal80 ,

LYS2::GAL1UAS-GALATATA-HIS3,GAL2UAS-GAL2TATA-ADE2,
URA::mel1UAS-MEL1TATA-lacZ, MEL1 (from Clontech, MountainView, CA)

4.4. Plasmids
Name

Description

Abbrevation

pGADT7

2u, LEU2, Amp, GAL4-AD (Clontech)

AD

pGBKT7

2u, TRP1, Kan, GAL4-BD (Clontech)

BD

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4.5. Antibodies
Description

Name
Anti-Myc (Mouse)

Roche

Anti-HA (Mouse)

Roche

HRP-conjugated anti-mouse IgG, light chain

Jackson ImmunoResearch Inc. West Grove

secondary antibody

4.6. Media and plates

Name

Description

LB medium

Bacterial tryptone 10 g, Yeast extract 10 g, NaCl 10 g, add water to 1L.

LB + Amp.

Add 100 g/ml ampicillin to LB medium

LB + Kan.

Add 30 g/ml kanamycin to LB medium

SOC

Bacterial tryptone 10 g, yeast extract 5 g, NaCl 10 g, 2 M KCl 1.25 ml, 1 M

MgCl2 10 ml, 1 M MgSO4 10 ml, glucose 36 g, add water to 1 L

YPD medium
SCM Mix

Bacterial peptone 20 g, yeast extract 10 g, glucose 20 g, add water to 1 L

Yeast nitrogen base 500 g, L-Alanine 7.5 g, L-Arginine 7.5 g, L-Asparagine 7.5
g, L-Aspartic acid 7.5 g, L-Cysteine 7.5 g,L-Glutamic acid 7.5 g, L-Glycine 7.5
g, L-Isoleucine 7.5 g, L-Lysine 7.5 g, L-Phenoalanine 7.5 g, L-Proline, 7.5 g,
L-Serine 7.5 g, L-Thronine 7.5 g, L-Tryosine 7.5 g, L-Valine, 7.5 g, Inosital 2.5 g

Agar plate

20 g of bacterialogical agar were added to medium before autoclaving

4.7. Buffer and reagent

Name
SDS solution

Description
1 M Tris-HCl, pH 9.0 10 ml, 20% SDS 10 ml, 500 mM EDTA, pH 8.0 2 ml,
add H2O to 100 ml

Transfer buffer

NaHCO3 1.05g, Na2CO3 0.901g, Mehnanol 300 ml, 10 N NaOH 0.9 ml,
add H2O to 2L

TBST buffer

1 M Tris-HCl, pH 7.4 40 ml, NaCl 8 g, KCl 0.2 g, 20% Tween 20 5 ml, add
H2O to 1 L

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Tris base 242 g, Acetic acid 57.1 ml, 500 mM EDTA pH 8.0 100 ml, add

50x TAE

H2O to 1 L
2x Laemmlis buffer

60% Glycerol 37 ml, 20% SDS 33.3 ml, 1 M Tris-HCl, pH 6.9 13.9 ml, add
H2O to 100 ml

6x DNA loading buffer

Bromophenol blue 0.25 g, xylene cyanol FF 0.25 g, 60% Glycerol 10 ml, add
H2O to 100 ml

4.8. Equipment and kits

DNA cloning and manipulation kits:

1.

QIAEX II Gel extraction kit (QIAGEN, Valencia CA)

2.

DP103-TIANprep Mini Plasmid Kit (Tiagen biotect (beijing) co Ltd.)

Molecular Cloning Enzymes:

1.

KOD DNA polymerase (Novagen)

2.

T4 DNA ligase (New England Biolabs, Beverly, MA)

Restriction Enzymes (New England Biolabs):

1.

DNA markers:
1.

EcoR1 & Nde1

1 Kb ladder marker (Fermantas Inc. Glen Burnie, MA)

Western Blotting:
1.

Vertical Mini-Gel System (C.B.S Inc. Del Mar, CA) EBU-204

2.

Mini-Blotting System (C.B.S Inc)

3.

Bio Trace pure nitrocellulose membrane (Pall Corporation,

Washington, NY)

4.

SuperSignal west Pico Chemiluminesent Substrate (Thermo Fisher

Inc.)

5.

Prestained protein marker

6.

Protein marker

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

5. Methods

10

5.1. Polymerase Chain Reaction (PCR)

KOD was used instead of the Taq as it has higher fidelity.
The PCR reaction mixture contains 33.2 ul of distilled watr, 5 ul of buffer, 2 ul of
MgSO4, 5 ul of dNTP, 1 ul of DNA template, 1.4 ul of forward primer, 1.4 ul of
reverse primer and 1 ul of KOD.
The parameters were set up as follows: 95 oC for 5 min for 1 cycle; denature
DNA at 95 oC for 15 sec, anneal DNA at 50 oC for 30 seconds, extend DNA at 68 oC
for 2 minutes, in total 35 cycles; 68 oC for 10 minutes, 1 cycle.
5.2. Restriction digestion
Digestion reaction mixture for the inserts (PCR products) and vectors (plasmids)
include 10 ul of DNA sample (insert or vector), 7 ul of sterile water, 2 ul of buffer 4, 0.5
ul EcoR1 and 0.5 ul Nde1. The inserts were digested at 30oC for 2 hours. Meanwhile,
after 1 hour of incubation at 30oC, 1 ul of CIP was added to the reaction mixture that
contained the plasmid. After that, 1 more hour was needed for total digestion of the
plasmid.
5.3. Agarose Gel Electrophoresis
Agarose gel electrophoresis separates DNA fragments according to their
molecular weights. Therefore, the DNA fragments can be found as the samples were
run together with a DNA ladder.
To run a gel, the DNA samples were mixed with 6x DNA loading buffer and
loaded into 1.0% (w/v) agarose gel. Horizontal gel electrophoresis apparatus and 1x
buffer TAE (Tris-acetate-EDTA) were used. DNA was then stained with EB

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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(ethidium bromide) for 10 minutes and de-stained with distilled eater for 10 seconds.
UV at 254 nm was used to visualize the bands by employing the UV transilluminator
(Bio-Rad ChemiDoc XRS Gel Photo Documentation System).
5.4. DNA extraction from agarose gel
Target DNA fragments of digested inserts and plasmid were separated by gel
electrophoresis. After staining, the desired band was cut and purified with QIAEX
Gel Extraction Kit.
To one volume of gel slice, three volumes of Buffer QX I and 20 ul QIAEX II
were added, and the mixture was incubated at 55oC for 10 min during which it was
gently vortexed every 2 minutes. (If solution turned orange or purple, Sodium acetate
(5 M) can be used to modify the pH until the color is yellow.) After spinning at 13,000
rpm for 30 seconds (same setting for all other centrifugation in gel extraction),
supernatant was removed and re-suspended with 500 ul of QX1. This procedure was
repeated twice using PE buffer instead. The pellet was then air-dried for 10~15 minutes.
After that, it was re-suspended with 20 ul of sterile water and incubated at room
temperature for 5 minutes. After spinning, supernatant was removed and stored at
-20oC
5.5. Determination of DNA concentration
UV absorbance at 260 nm for each digestion product (50 fold dilution by sterile
water) was checked. The concentration of sample would be (absorbance*50*50)
ng/ul.

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5.6. Ligation
20 ul of Ligation mixture contain insert, vector, 1ul of T4 DNA Ligase, 2ul of
T4 DNA Ligase buffer and water. The mole ratio of vector to insert is 1:3. After
mixing, the reaction mixture was stored at 4oC for overnight.
5.7. Transforming E. coli with plasmids
Some 10 ul of plasmids were mixed with 100 ul of E. coli competent cells for one
transformation, and the mixture was incubated on ice for 30 min. The mixture was then
heat shocked at 42oC for 1.5 min, and some 250 ul of SOC were added to the cells. The
mixture was incubated at 37oC for 1 hr for the cells to recover. Finally, the cells were
plated on LB selective plates and incubated at 37oC for overnight. (pGADT7
transformed E. coli required Ampicillin while pGBKT7 transformed E. coli required
Kanamycin).
5.8. Plasmid Extraction form Transformed E. coli
TIANprep Mini Plasmid Kit was used and procedure followed the manufacturer
manual.
5.9. Transforming budding yeast with plasmids
Starter culture was generated by picking a single colony of yeast cells from the plate
and put into 5 ml of medium and growing the culture overnight in 25oC. The starter was
then diluted in 50 ml YPD to O.D 0.2 (at 600 nm) and cultured at 30oC until the O.D
reached 1. The cells were then harvested into a 50 ml falcon tube (sufficient for 10 full
transformations). Cells were washed with 20 ml of sterile H2O and re-suspended with 1
ml of sterile water. After being transferred into a new Eppendorf tube, the cells were
spun down again, and 500 l of 0.1 M LiAc were added to the tube. The mixture was
then shaken at room temperature for 15 min.

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For each transformation, some 50 ul of cells were transferred to separate

Eppendorf tube. After that, they were spun down and the LiAc was removed. A
transformation mixture (240 ul of 50% PEG (w/v), 36 ul of 1 M LiAc, 20 ul of 10x TE
buffer containing DNA or plasmids to be transformed, 50 ul of 1mg/ml ssDNA (boil for
10 minutes before use and then let cool)) was added to the cell pellet. The mixture was
re-suspended and then shaken at room temperature for 30 minutes. The cells were then
put to heat shock in a 42oC water bath for 20 minutes and then spun down at top speed
for 15 sec. Some 60 ul of sterile water were added to the cell pellet, and the cells were
then plated on SCM-L-T (40 ul) and SCM-L-T-H (20 ul). Plates were then incubated at
30oC for 3~5 days.
5.10. Yeast growth, storage and revival
For incubation in liquid medium, wild type S. cerevisiae cells were cultured in
YPD in a shaker at around 200 rpm, and the temperature was 25oC or 30oC depending
on the need. Normally wild-type cells double in number every 3 hrs at 25oC and every
1.5-2 hrs at 30oC in a rich medium. Selective media were used for cells transformed
with plasmids according to the selectable markers in the cells.
For storage of budding yeast cells, 0.5 ml of cells in log-phase was transferred to a
screw-capped tube and 0.5 ml of 30% glycerol was added. The mixture was then stored
at -80oC.
5.11. Methods for yeast total protein extraction
Alkaline Extraction method was employed to extract total protein from yeast for
expression checking. The starter culture was diluted to OD 0.1 with fresh medium.
After the O.D reached 1.5-2, cells were harvested, washed with ice-chilled water twice,
resuspended in 500 l of 0.1 N NaOH, and then put into a shaker to incubate at room

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temperature for 15 min. The mixture was then spun down, and the cell pellet was added
with Laemmlis buffer (with 2-mercaptoethanol) and boiled for 5 minutes. Samples
were stored at -20oC and then used for SDS-PAGE later.
5.12. SDS-PAGE
Yeast protein samples were separated by SDS-PAGE. The stacking gel was 5%
while the resolving gel was 12.5%. Separation was performed on a Slab Gel system.
Proteins were first stacked in the stacking gel under electric field of 60-80 V and then
separated in the resolving gel at 120-160 V.
When the tracking dye reached the desired position, the proteins were transferred
onto a nitrocellulose membrane (pore size 0.2 or 0.45 m) in the transfer buffer using
the Electrophoretic Blotting Systems. Protein transfer was carried out at constant
current of 500 mA for 1.5 hours.
The membrane was stained with Ponceau S working solution (0.5% Ponceau S in
1% acetic acid). The protein marker was marked by pencil after rinsing. The
membrane was then de-staining with TBST buffer. After that, it was blocked with 5%
non-fat milk in TBST for 30 minutes. Next, it was incubated with the primary
antibody for overnight at 4oC with agitation. The membrane was washed with TBST
for three times with agitation for 10 minutes each and then incubated with the
secondary antibody conjugated with HRP (horseradish peroxidase) for 2 hours at
room temperature followed by three washes by TBST as aforementioned.
The membrane was then mixed with SuperSignal chemiluminescence substrate
for 5 minutes. Excess substrate was drained off and exposed to light-sensitive X-ray
films (Fuji) immediately. Finally, the films were developed in a film-processing
developer (Kodak) in a dark room.

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Monoclonal antibody (mAb) used in the study includes anti-HA antibody (12CA5)
from Roche at 1:20,000 dilution and anti-Myc antibody (9E10) from Roche at 1:
5,000 dilution. Second antibody used includes HRP-coupled anti-mouse (1:20,000 in
TBST containing 5% non-fat milk).

6. Results
6.1. Bioinformatics Analysis
Using peptide sequence from NCBI and multiple sequence alignment tool Clustal
W 2.1, substantial homology was found among many existing species as shown in
figure 3.

Fig. 3: Matrix showing the percentage identity among species (from Clustal W2)
P.S. : (Same order as following figures)
1: Homo Sapien
2: Pan troglodytes
3: Pongo abelii
4: Mus musculus
5: Rattus
6: Gallus gallus
5: Danio rerio
8: Drosophila
9: Saccharomyces cerevisiae

From the previous study on Noc3p in our lab, a particular sequence was found to
be conserved among species especially Homo Sapien and Saccharomyces cerevisiae.
Using the MULTICOIL analysis, the sequence was found to be a coiled-coil (fig 4)
(CC2 accroding the previous study in the lab (Wu, 2011)). It was found to be one of
the self-interacting motif of Noc3p of budding yeast.

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Fig.4: Multiple Sequence Alignment of a Conserved Sequence among species (from Clustal W2)

Fig. 5: Assembly of the Screenshot of Multicoil result from Species (from Multicoil)

Other than this sequence, one other coiled coil was found to be shared among
species. It is called CC1 (from amino acid 1 to 169) for Noc3p (Wu, 2011) and found
to be self-interacting motif as well. Although little similarity was found between the
exact amino sequences of that coiled coil of fad24 (of Homo sapien) and Noc3p (of
Saccharomyces cerevisiae), it is also intriguing to investigate the possibility of
self-interaction of the coiled-coil of fad24.
6.2. Successful Molecular Cloning
All of the vectors used were successfully inserted into competent E. coli. The
plasmids were extracted by miniprep. After that, the extracted DNA was under
restriction digestion by Nde1 and EcoR1. The digested DNA were separated by
agarose gel electrophoresis and then visualized. It was found that the fragment fitted
into the corresponding size of the inserts and the plasmid. It preliminarily shows that

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the inserts are positioned properly into the vectors. In order to confirm the sequence
of insertion, some volume of each transformed E. coli were sampled and sent for
sequencing (by BGI). Sequences of the insert fit in our desired sequence. Hence, the
plasmid extracted from the E. coli can be used for subsequent transformation of yeast
for the yeast two-hybrid system.

Fig.6: Double Restriction Digestion of the Plasmids Extracted from E. coli Transformed with AD
ligated with various inserts (Accompanied with a 1 kb DNA ladder)
P.S. :
(The Lanes of the above Figure follow the following order & M represents for 1 kb DNA ladder)
1:
AD-Full Length
2:
AD-Fragment 1
3:
AD-Fragment 2
4:
AD-Fragment 3
5:
AD-Fragment 4
6:
AD-Fragment 5
5:
AD-Fragment 6
8:
AD-Fragment 7
9:
AD-Fragment 8
10: AD-Fragment 9
11: AD-Fragment 10

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Fig.7: Double Restriction Digestion of the Plasmids Extracted from E. coli Transformed with BD
ligated with various inserts (Accompanied with a 1 kb DNA ladder)
P.S. :
(The Lanes of the above Figure follow the following order & M represents for 1 kb DNA ladder)
1:
BD-Full Length
2:
BD-Fragment 1
3:
BD-Fragment 2
4:
BD-Fragment 3
5:
BD-Fragment 4
6:
BD-Fragment 5
5:
BD-Fragment 6
8:
BD-Fragment 7
9:
BD-Fragment 8
10: BD-Fragment 9
11: BD-Fragment 10

6.3. Yeast Transformants with Correct Inserts Expressed

The vectors used were checked for presence of the inserted DNA sequence by
double restriction digestion (Nde1 & EcoR1). Furthermore, E. coli containing the
plasmids (AD-FL & BD-FL) were sent for sequencing, which allow verification of the
sequence. However, we need to confirm if the yeast was transformed with proper
vector and insert so that the yeast can expressed the proper AD- or BD- fused protein.
In our study, the transformants were lysed to check for expression using western blot.
The AD-fused protein contain the HA tag (approximately 1.1 kDa) while the
BD-fused protein contain the myc tag (approximately 1.202 kDa). Therefore,

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monoclonal antibodies anti-HA and anti-Myc can be used to probe for the AD-fused
protein and BD-fused protein respectively. We have only checked the expression of
particular group of the transformants as stated below:
Transformants with Expression Checked
AD vector/ BD vector
AD FL/ BD vector
AD F1/ BD vector
AD F3/ BD vector
AD F4/ BD vector
AD F5/ BD vector
AD F7/ BD vector
AD F8/ BD vector
AD F9/ BD vector
AD vector / BD FL
AD vector / BD F1
AD vector / BD F3
AD vector / BD F4
AD vector / BD F5
AD vector / BD F7
AD vector / BD F8
AD vector / BD F9
Table 1: Transformants with Expression Checked

AD F2/ BD vector
AD F6/ BD vector
AD F10/ BD vector
AD vector / BD F2
AD vector / BD F6
AD vector / BD F10

The transformants have found to contain the protein of desired molecular weight
(fig 8 &9).

Fig 8: Result of the Expression Check (For AD-fused protein: Probed with anti-HA/ anti-mouse)

Fig 9: Result of the Expression Check (For BD-fused protein: Probed with anti-myc/ anti-mouse)

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

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Lane Human Approximate size (kDa)
1
FL
92.5
2
1
18
3
2
29.98
4
3
24
5
4
24.2
6
5
47.8
7
6
48.2
8
7
71.8
9
8
78.12
10
9
14.4
11
10
15.48
12
Null
Null (empty vector)
Table 2: Table showing the identity of the lane in fig 8 & 9
P.S.
Size of tag not included here.
Proteins in Fig 8 & 9 are tagged with HA (1.1kDa) & c-myc (1.202 kDa) respectively.

6.4. 2 Domains of fad24 Self-Interact

After co-transformation of yeast strain AH109 with different combinations of
AD and BD vectors, each of the different combination of yeast (144 combinations in
total) was streaked onto SCM double dropout plate (SCM-L-T) and triple dropout
plate (SCM-L-T-H).
All the transformant can grow on double dropout plates, proving that all of them
contain AD vector and BD vector (i.e. successful transformation if regardless of the
insert).
It is found that the fad24 (FL for full length) can self-interact, which
allowed the corresponding transformant to survive on triple dropout plate. fad24 (FL)
can also interact with fragment 1 (F1), 2 (F2), 3 (F3), 4 (F4), 5 (F5), 7 (F7) and 9 (F9)
in two ways (except fragment 9). In other word, either AD- or BD-fused fragments 1,
2, 3, 4, 5 and 7 can interact with full length fad24 counterpart, except that only BD-F9
interact with AD-FL.
As in figures 10, 11& 12, interactions among fragments were found. F7 can
interact with F1, F3, F5 and F9 in two ways. F8 can only interact with F3 in one way

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

21

(AD-F3 interact with BD-F8, but not the other way). Most importantly, F1 and F3 can
self-interact.

Fig .10& 11: Patching Result of the Y2H

P.S.: Growth on this SCM-3 plates indicates interaction among fragments
pGADT7
V
FL
F1
F2
F3
F4
F5
F6
F7
F8
F9
F10
V
FL
+
+
+
+
+
+
+
F1
+
+
+
F2
+
F3
+
+
+
F4
pGBKT7
F5
+
+
F6
F7
+
+
F8
F9
F10
Fig 12: Combined Result of the yeast two-hybrid result
P.S.: + refers to growth on SCM-L-T-H plates (-3) & all Transformants can grow on SCM-L-T (-2)

7. Discussion
From the aforementioned result, the following interpretation was given.
Regarding the yeast two-hybrid result, full length fad24 was found to be
self-interacting. The possibility of false positive due to possibility of fad24 or its
fragment being either a DNA binding domain or activation domain was refuted as any

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

22

transformant with AD- or BD- fused protein alone failed to survive. Besides, we have
tested the interacting partners in reverse way and introduce multiple overlapping
fragments.
Furthermore, there was direct evidence proving the presence of the
self-interacting motifs. To begin with, fragments 1 and 3 interact with themselves but
not with each other. That means that fragment 1 and 3 alone are independent
self-interact motifs.
Indirect evidence was found to prove the presence of the two motifs as shown in
Fig 13 and 14. Fragment 5 contains the F1 region (first coiled-coil) and hence it can
interact with fragment 7 and full length fad24. Fragment 7 contains both coiled-coils
and thus interacting with fragment 1, 3, 5 and full length fad24.
One point of note is that some additional interaction pairs were found. First,
fragment 9 interacts with full length fad24 and fragment 7 but not to itself. Second,
fragment 2 interacts with full length fad24 but does not self-interact. One speculation
is that the self-interacting motif exactly extends slightly into fragment 9. Hence,
fragments containing fragment 9 can weakly interact. Other speculation is false
positive aroused from the yeast two-hybrid system as false positives are known to
happen using the system. However, this is not likely as interacting pairs between these
fragments worked in both ways.
From the yeast two-hybrid result, some strange result is found. F6 and F8 both
contain F3 but fail to interact with itself or other. One possibility is that the additional
parts on F6 and F8 hinder the CC2 in interacting.

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

23

Fig.13: Diagram showing fad24 dissection and position of F1 & F3 in each fragments

Fig. 14: Diagram showing How the Insert Interact

Nevertheless, some more study may be needed to further verify the result. IP/coIP
is highly recommended for all those transformants but the effort in investigating all
144 combinations would be huge. We can also try using the approach used in the
previous study about Noc3p self-interaction (Wu, 2011) like using versions of fad24
with deletion of the coiled-coils in the yeast two-hybrid system. GST pull down, ICT
and FRET are some possible choices as well.
There are some possible field for future study. It would be ideal if we can explore
further like how, when and where the fad24 self-interacts. Moreover, we can also try
some functional study of fad24. It is because Noc3p has separate functions like DNA
replication initiation, RNA biogenesis and even transcription factor. Hence, it is

MAPPING OF THE FAD24 SELF-INTERACTION DOMAINS

24

possible for fad24 to have additional functions. Adipocyte differentiation is an

instance of function of fad24 being discovered.
Al in all, fad24, human homolog of the yeast Noc3p can self-interact and it
possesses two coiled-coils as the two self-interacting motifs. Such result is similar to
the yeast counterpart (Noc3p). Such phenomenon may suggest fad24 may dimerize
and initiate DNA replication like Noc3p does as in budding yeast. In additional,
counterparts in other higher eukaryotes may be similar in self-interaction as many also
possess the coiled-coil 1 and -2 (Fig.5).

8. Reference
CLONTECH (1999, June 22) MATCHMAKER GAL4 Two-Hybrid System 3
& Libraries User Manual pp1-38
Nacalai USA, Inc. (n.d.) DNA Ladder Retrieved 23 April 2015 from
https://www.nacalaiusa.com/product.php?id=90
Tominaga.K, Johmura.Y, Nishizuka.M and Imagawa. M (2004, September 16)Fad24,
a mammalian homolog of Noc3p, is a positive regulator in adipocyte
differentiation, Journal of Cell Science 117, pp 6217-6226,
doi:10.1242/jcs.01546
WU.R.T (2011, July) Studies of DNA Replication-Initiation Proteins in the Budding
Yeast Saccharomyces cerevisiae, HKUST, pp1-286
Zhang. Y.X. , Yu. Z.L., Fu. X.R. and Liang. C., (2002, June 28) Noc3p, a bHLH Protein,
Plays an Integral Role in the Initiation of DNA Replication in Budding Yeast,
Cell, Vol. 109, 849860