Manipulations of the Drosophila DracI and POSH genes
Ehsan Fadaei, MBB 432, Simon Fraser University 2009
Introduction:
Drosophila melanogaster DracI gene is a member of the Rho family of small guanosine
triphosphatases (GTPases) which include Rho and Cdc42 and are responsible for the temporal and
spatial control of actin filament organization in cytoskeleton that can cause changes in cell shape [2].
The DracI gene is responsible for encoding small-GTP binding proteins of Ras superfamily which
mediate signals from the cell surface receptors into the cell that could affect processes such as
cytoskeletal organization and gene transcription [3]. In Drosophila, Rac and Rho are both involved
in dorsal closure. Thus, through the study of using DracI active and dominant negative mutants,
researchers can study implications of DracI in closure of dorsal epidermis, myoblast fusion, the
establishment of planar cell polarity, and the control of axon growth and guidance [1]. Active
mutants of DracI protein can interfere with the cytoskeletal dynamics which perturbs these
processes. Only through the study of phenotypic loss-of-function mutation in the endogenous Rac
genes, one can determine if the DracI protein is necessary for formation of actomyosin cable and
completion of dorsal closure.
One of the components of DracI signalling is the POSH gene (Plenty of SH3s) that contains
several SH3 domains primarily used for protein-protein interactions. The POSH gene functions as a
scaffold protein for the Jun N-terminal Kinase (JNK) signal transduction pathway, which can lead to
mammalian and Drosophila cell death. The Drosophila homolog of POSH lacks sequence homology
compared to the Rac binding domain of mammalian POSH but their four SH3 domains and RING
finger motif are well conserved [8]. Overexpression of POSH during Drosophila development can
lead to defects such as loss of anterior wing, crossveins, notched wings and disordered hair polarity
[8]. Previous studies have discovered that POSH gene can interact with TAK1, an MLK responsible
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for activation and termination of immunity signalling to further prove that POSH mediates the
activation of the JNK pathway in Drosophila [8].
Researchers often use the GST-fusion protein approach as the initial step for biochemical
studies on the gene of interest such as DracI gene or for raising antibodies against the gene of
interest for further genomic analysis such as RNA in-situ hybridization, Northern blotting and other
experiments. GST fusion proteins are constructed by inserting the Open Reading Frame (ORF) of
DNA of interest (DracI) in frame with the internal sequences encoding GST into the multiple
cloning site of a pGEX vector that contains a tac promoter which is induced by the lactose analog
isopropyl β-D thiogalactosidase (IPTG). pGEX vectors provide tight control over expression of an
inserted gene by the engineered internal lacIq repressor gene that encodes for a protein which binds
to the operator region of the tac promoter to prevent expression until the induction of IPTG. The
GST fusion system allows for the purification of the GST-fusion proteins from other proteins in the
cell via affinity chromatography technique, where GST is bound to its glutathione substrate in the
Glutathione Sepharose 4B beads [3]. The pGEX-2TK vector (appendix I) used in the lab is a
uniquely designed vector since it also contains recognition sequence for the catalytic subunit of
cAMP-dependent protein kinase of heart muscle located between the thrombin recognition site and
the MCS [4]. Expressed proteins in the pGEX-2TK vector can be directly labelled using protein
kinase and [γ-32p] ATP and detected by radiometric or autoradiographic techniques. The thrombin
recognition site in pGEX-2TK is a protease cleavage site for the thrombin cleavage enzyme used to
cleave off the GST tag from the protein of interest. The confirmation of GST-fusion protein
purification from other proteins in the cell can be analyzed on Sodium Dodecyl Sulfate
Polyacrylamide (SDS-PAGE) that separates proteins according to their electrophoretic mobility.
Site-directed mutagenesis is a powerful technique for the purpose of creating an alteration in
the function of a gene of interest by creating a site specific mutation in its DNA sequence. Some of
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the uses for the site-directed mutagenesis include the study of protein structure-function relationships
to define interactions of a protein with other molecules such as ligands, substrates, receptors and
partners in signalling pathways [5]. Reverse genetics approach is based on identifying a gene and
modifying its phenotypic expression to become a mutant gene for further analysis of its function, and
one of the most common techniques used today for reverse genetics is site-directed mutagenesis.
QuickChange site-directed mutagenesis kit from Stratagene follows the basic procedure of
synthesizing a short DNA primer that contains the desired base change with the benefits of allowing
site specific mutations in any double stranded plasmid requiring no specialized vectors, unique
restriction sites or multiple transformation that could generate mutants with greater than 80%
efficiency [6]. The desired mutation can be introduced into the target sequence of both strands in a
plasmid (pBluescript II SK +) via complementary reverse and forward DNA primers. The pfuTurbo
DNA polymerase included in the kit replicates both strands in the plasmid with high fidelity without
the displacement of the mutant oligonucleotide primers which are extended during temperature
cycling by the polymerase. The non-mutated parental DNA templates that have been methylated by
dam methylase in E.coli are digested by the DpnI endonuclease that targets methylated and
hemimethylated DNA. To control for the efficiency of mutant plasmid generated by site-directed
mutagenesis, pWhitescript 4.5-kb control plasmid containing a stop codon (TAA) that disrupts the βgalactosidase gene of pBluescript II SK (-) phagemid is used [6]. Formation of blue colonies on LBampicillin agar plates would confirm successful back mutation of the stop codon TAA to CAA
which would generate functional β-galactosidase gene.
The purpose of DracI site-directed mutagenesis experiments was to determine the effects of
the DracI protein when locked in the GTP-bound state (gain-of-function mutant) and when locked in
the GDP-bound state (loss-of-function mutant). The constitutively active state of DracI protein
DracIV12 is created by altering the Glycine at position 12 (GGA) to Valine (GTA), while the
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negative dominant form DracIN17 is generated by changing Serine at position 17 (ACC) to
Asparagine (AAC) [3].
One of the methods used to purify transformed plasmid DNA or RNA in bacterial cells is
through alkaline lysis technique of plasmid purification. The alkaline lysis method uses the SDS
detergent for solubilization of phospholipids and proteins in the cell membrane and sodium
hydroxide (NaOH) for denaturing proteins, genomic and plasmid DNA. Although the base pairing of
the plasmid DNA is also disrupted, and when conditions are neutralized, the native structure of the
plasmid DNA is rapidly restored to its native superhelical form provided that it was not exposed to
alkali conditions for a prolonged period. Qiagen’s QIAprep spin kit based on alkaline lysis used in
the lab is an alternative method to ethanol precipitation that allows rapid purification, separation and
concentration of plasmid DNA from solution via column centrifugation. The kit contains silicacolumn for DNA adsorption in the presence of chaotropic salts such as guanidine hydrochloride (N3
buffer) [7]. The salts dehydrate the phosphodiester backbone of DNA and result in the exposure of
phosphate residues to allow DNA adsorption to the silica. The adsorbed DNA can be washed with
ethanol (>50%), and can be eluted by sterile water which releases the DNA from the silica column.
The QIAprep plasmid purification method was used to purify the plasmid DNA of the mutant DracI
for sequencing via cycle sequencing and checking the plasmid DNA preparation by restriction
digest.
Cycle sequencing is a sequencing method based on the Sanger chain termination approach
but with a difference generating amplified signals due to being similar to a PCR reaction which
contains repeated cycles of thermal denaturation, primer annealing and polymerization. Another
advantage of cycle sequencing over Sanger chain termination is the ability to use smaller amount of
template DNA to obtain good sequence. The BigDye terminator v3.1 cycle sequencing kit used to
sequence the purified DNA plasmid of DracI features four different fluorescently labelled
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dideoxynucleotide triphosphates (ddNTP) that are involved in chain termination once incorporated
to the extending sequence. The four differently labelled ddNTPs are advantageous due to the fact
that it permits sequencing in a single reaction and prevents artifactual bands that are caused by
premature termination [3]. The automated DNA sequencer is used to record and determine the
nucleotide bases from the fluorescent labels in the sequencing reaction. The purpose of sequencing
the purified DracI plasmid DNA is to confirm if the site-directed mutation occurred and determine if
the site-directed mutation is a constitutively active mutation (DracIV12) or a dominant negative
mutation (DracIN17).
Transgenic flies that contain the mutated DracI gene can be created through cloning of the
cDNA of DracI from the site-directed mutagenesis experiment to a special plasmid vector which
contains the upstream activating sequence (UAS), able to facilitate GAL4 yeast transcription factor
to drive the transgene expression of DracI in Drosophila [3]. The DracI cDNA is inserted into the
polylinker with the appropriate restriction enzymes (BglII/ KpnI) which is located next to the Hsp70
(TATA) minimal promoter that can only transcribe the gene of interest when GAL4 bearing flies are
crossed with UAS bearing flies. The vector pUAST features a modified P element transposon that
flanks the gene of interest for transportation of the transgene into the genome. Another plasmid
containing helper P element (pTurbo) is required when pUAST is injected into the posterior of
Drosophila embryo, since it will provide transposase for the P element of pUAST vector.
Another feature of the pUAST vector is the white reporter gene that encodes for the wild type
eye color of Drosophia used as an indicator to determine if the insertion of P element occurred in the
endogenous genome and if the progeny is fully transgenic. The main advantage of using GAL4-UAS
system to create transgenic flies is the ability of being able to express the gene of interest in specific
tissues and creating stable transgenic progenies of many generation to observe phenotypic
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characteristics and study the gene function of mutant genes that maybe lethal to the species during
early development.
To determine the binding partners for POSH gene, the yeast two-hybrid screening that
utilizes the interaction trap system was used in the lab. This system is based on the interaction
between the binding domain (BD) fused to a bait protein and the activation domain (AD) fused to
the prey proteins that would express reporter genes during interaction. In the yeast-two hybrid
interaction system used in the lab, the bait protein (POSH) was fused to the binding domain of the
bacterial repressor (LexA) in a yeast- E.coli shuttle vector (pEG202) that contains the Histidine
(HIS3) selection marker for growth in His- media. While the library proteins (prey) were fused to an
activation domain of B42 protein contained in the yeast- E.coli shuttle vector (pJG-45) plasmid that
also features the Tryptophan selection marker (TRP1) for growth in Trp- media and Simian
Vacuolating Virus 40 (SV40) T nuclear localization signal used as a transcriptional enhancer to
increase rate of synthesis [3].
The third plasmid in the interaction system is a lacZ reporter plasmid (pSH18-34) that
contains the lacZ reporter gene under lexA operator that produces β-galactosidase when the bait and
the prey interact, and as a result, form blue product in the presence of X-Gal organic compound.
Another reporter gene for positive selection incorporated in the genome of the host yeast strain
(EGY48) is Leucine (LEU2) which is under control of lexA operator and is also expressed when the
functional transcription factor of the BD-AD fusion product binds to the lexA operator to allow
growth of yeast cells in Leu- media. Galactose is the sugar source that is used for the expression of
the library-AD fusion protein, while glucose used as a counter selection, inhibits its expression, thus
inhibiting the interaction between the bait and prey proteins.
The method used in the lab to detect the DracI transgene expression was RNA in-situ
hybridization that aids in the study of transcript distribution of DracI in the Drosophila embryo. The
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UAS-GAL4 system can be used in combination with the RNA in-situ hybridization to drive gene
expression in specific tissues such as embryonic tissue (amnioserosa) in Drosophila by AS-GAL4 or
segmentally repeated stripes (patch gene) of embryo by ptc-GAL4. RNA in-situ hybridization
method is based on the hybridization of radioactively, chemically or enzymatically (DIG) labelled
antisense probes (riboprobes) that are complementary to the mRNA of gene of interest (DracI)
which can be detected by autoradiography (radioactive label), fluorescence microscopy
(fluorochrome) or colorless substrate (DIG label).
Roche DIG RNA labelling and detection system incorporates digoxigenin (DIG) labelled
RNA nucleotide triphosphate (DIG-UTP) into riboprobe during in-vitro transcription reaction by
cellular machinery (T7 RNA polymerase). Digoxigenin is a steroid produced only in flowers and
leaves of plants Digitalis purpurea and Digitalis lanata which can prove valuable in raising specific
antibodies against it, thus minimizing background reactions. To detect the DIG labelled DracI
riboprobes, anti-DIG antibodies targeting DIG product can be conjugated to alkaline phosphatase
enzyme that are exposed to two colorless chemical compounds, 5-Bromo-4-chloro-3-indolyl
phosphate (BCIP)/x-phosphate and Nitro Blue Tetrazolium chloride (NBT) for a colorimetric
reaction. BCIP binds to the alkaline phosphatase active site as the alkaline phosphatase substrate and
can be released from the active site by reacting to NBT that serves as an oxidant, to form blue
coloured precipitate.
Utilizing Green Fluorescent Protein (GFP) to visualize distribution of proteins of interest
such as cadherin that is affected by DracI in live cells of Drosophila can aid in studies of signal
transduction, tissue morphogenesis and building of 3D structural views of protein in cells. GFP,
isolated from jellyfish Aequorea victoria, is composed of 238 amino acids (26.9 kDa) and fluoresces
green under blue light which can be detected by fluorescent microscopy [9]. In cases where
morphological distinction is made by fusing GFP to different structures, the product of GFP is
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spliced into the genome of the organism in the DNA coding region of the target protein and is
expressed by the same promoter. For visualization of cadherin, a cell adhesion protein, the construct
of GFP-cadherin is made by ligating the 3’ end of shotgun gene encoding cadherin to the GFP
coding sequence and expressing the product in every embryo cells of Drosophila by an ubiquitous
promoter [3]. Drosophila embryos are investigated through confocal and epifluorescence microscopy
to visualize the cells that express the fused GFP-cadherin products.
Materials and Methods:
The methods used for all the experiments relating to DracI and POSH genes were followed
according to the standard procedures noted in the MBB 432 lab manual [3] except for some of the
procedural steps in the following experiments:
Purification of DracI GST fusion protein: step 11(week 3), addition of 50µl of 2U DNAseI (stock).
Step 5 (week 4), for the SDS-PAGE gel staining, SYPRO orange protocol was used.
Spyro orange protocol: Add 10µl of SYPRO orange to 50 ml of 7.5% acetic acid, mix components
and transfer to staining dish. Add gel to staining solution and incubate at room temperature for 30
minutes with gentle agitation (cover container to protect against light). Remove gel from staining
solution and rinse with fresh 7.5% acetic acid at room temperature for 30 seconds and photograph
gel.
Site-directed mutagenesis of DracI: step 2 (week 4), 2µl β-ME mix not added since it was not
necessary.
Subcloning DracI coding sequences into pUAST: step 6 (week 7), for the heat shock transformation
procedure; addition of 0.5 ml of SOC broth (pre-warmed) to heat pulsed sample.
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Yeast two-hybrid screening using the interaction trap system: step 11 (week 8), addition of 25 µl
library (400ng/µl) and 0.1mg salmon sperm DNA (ssDNA) to 15 ml round-bottomed falcon tube to
obtain more positive colonies. Steps 17-20 of protocol skipped for the optimization of procedure.
Step 21, centrifugation at room temperature for 1 minute at max speed. Step 23, plate 1/3 of 0.5ml
Tris resuspended cells on 3 CM plates. Additional “Lazy Bones” yeast transformation protocol
followed to compare transformation efficiency to the standard yeast transformation in the MBB 432
lab manual.
Lazy Bones yeast transformation protocol: Add 500 µl of PLATE solution to a 1.5 ml eppendorf
tube. Boil carrier DNA (ssDNA) for 5 minutes, chill on ice immediately after. Add 10 µl of boiled
ssDNA to the tube (ssDNA at 10 mg/ml). Add transforming DNA (10-25 µl). Pick a large colony
from yeast plate using the flat end of the toothpick and add to tube and re-suspend in solution. Mix
well by vortexing or shaking and incubate at 30ºC overnight. Pellet cells for 10-20 seconds in
microfuge and remove supernatant. Resuspend cells in 1 ml sterile TE and plate (200µl/plate) onto
five CM plates (his-, ura-, trp-, leu+) and incubate at 30ºC for at least 4 days. Note: Plate solution
contains 40% PEG, 0.1M lithium acetate, 10mM Tris-HCl (pH 7.5) and 1mM EDTA (pH8.0).
Step 4 (week 9), centrifugation at max speed in clinical centrifuge for 10 minutes at room
temperature to get rid of agar. Step 10, preparation of 1ml serial dilutions of 1:100, 1:1,000,
1:10,000, 1:1,000,000 and 1:10,000,000 from library using sterile TE, to titre the library.
Steps 1-5 (week 10) modified; calculation of volume of library needed to get 30,000 colonies to
plate on 3 plates (10,000/plate). If volume of library needed is between 10 µl-100 µl, add 1 ml of
Gal/Raff/CM leu+ liquid media to it and incubate for 3.5 hours at 30ºC with shaking at 200 rpm. If
volume of library needed is less than 10 µl, dilute with 1X TE such that the volume needed is 100 µl
then add add 1 ml of Gal/Raff/CM leu+ liquid media to it and incubate for 3.5 hours at 30ºC with
shaking at 200 rpm. If volume of library needed is more than 100 µl, then add 10 X volume of
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Gal/Raff/CM leu+ liquid media to it and incubate for 3.5 hours at 30ºC with shaking at 200 rpm.
Step 6, centrifuge at max speed for 1 minute and discard supernatant. Protocol repeated again except
10 ml of inoculated yeast library in 1ml of Gal/Raff/CM leu+ liquid media used instead of serial
dilutions.
Additional step 3 (week 12), patch out 10 colonies of undiluted library plate onto a Gal/Raff/XGal/leu+ plate and omit patching onto CM plate.
Detection of DracI transgene expression by RNA in-situ hybridization: Step 2 (week 11), before the
first wash, rinse embryos with 1 ml of pre-warmed wash buffer. Step 4, wash buffer changed four
times at 2-3 hour intervals, with the last wash incubated overnight at 52ºC and 75 rpm.
Results and Discussion:
The initial step for biochemical studies, reverse genetics studies and genetic modification of
the DracI gene is by obtaining the wild type protein of DracI to raise antibodies against it for
applications such as Northern blot, RNA in-situ hybridization, comparison with the mutated DracI
protein among other experiments. Purification of DracI protein by GST protein purification method
allowed successful expression of DracI protein in E.Coli cells and isolation of the GST tagged DracI
from other endogenous soluble and insoluble proteins of E.Coli. The results of the purified DracI
protein were run in SDS-PAGE gel to confirm the size of DracI protein and check for contamination
in the solution (fig. 1).
In the gel image shown, the 1st lane contains the protein marker (appendix II) while the 2nd
lane contains the pellet sample of all proteins including cell membrane proteins and 3rd lane contains
supernatant of sample of all soluble proteins including GST-DracI fusion protein. The GST-DracI
purified sample which was separated from other soluble proteins via affinity chromatography, was
ran in the 4th lane which contains four bands of varying sizes. The sizes of the four bands were
calculated by standard calibration curve with Log Molecular weight =-1.1022(Relative mobility RF)
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+ 2.214 and R2=0.9236. Three bands identified in lane 4 correspond to DracI protein (21 Kd), GST
protein (27 Kd) and DracI-GST fusion protein (~48 Kd). The GST and DracI protein band should
not be expected to appear in lane 4 of gel, but the problem may have arisen due to protein
degradation in the freeze/thaw cycle of cell lysis step. The other reason for this problem could be due
to bacterial pellet containing some colonies with empty pGEX-2TK vector (GST protein) and other
colonies containing DracI-GST plasmid. The unknown fourth band (13 Kd) in lane 4 of the gel
could be an endogenous soluble E.Coli protein that was transferred from miniscule volume of
discarded supernatant accidentally with the pelleted DracI-GST sample.
Fig 1. DracI-GST purification (SDS-PAGE): 1st lane contains 10µl of protein marker with its band sizes shown. 2 nd lane
contains 20µl of pellet sample of all un-soluble proteins after cell lysis. 3rd lane contains 20µl supernatant sample of all
soluble proteins including DracI-GST fusion protein. 4th lane contains the purified DracI-GST fusion products. There are
4 bands in the 4th lane that correspond to DracI-GST fusion protein (48kd), GST protein (27kd), DracI protein (21kd)
and an unknown protein (13kd). Note: the 5th lane contains 10µl of protein marker loaded to balance the dye front.
To genetically modify the DracI gene, site-directed mutagenesis method was used for reverse
genetic study of DracI by introducing site-specific mutation to create either dominant negative
protein (DracIN17) or dominant positive protein (DracIV12). In the experiment, DracI cDNA
cloned into the pBluescript SK+ (3.0 kb) was used as a template for mutagenesis and pWhitescript
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4.5-kb control plasmid was used to confirm successful site-directed mutagenesis [3]. After
successful transformation of mutated plasmids into XL1-blue competent cells and plating, the results
confirmed successful site-directed mutagenesis of DracI due to the fact that the control plate
(pWhitescript) showed blue colonies. Unsuccessful site-directed mutagenesis of pWhitescript would
show white colonies since the lacZ gene is natively disrupted by a stop codon (TAA).
Four white colonies suspected of being positive for mutagenized plasmid DNA was cultured
and purified using alkaline lysis technique by QIAprep spin miniprep kit. The purification of
plasmid DNA allows for isolating the mutagenized plasmid DNA for sequencing via BigDye
Terminator v3.1 cycle sequencing. To check for plasmid DNA preparation, in order to determine the
DNA concentration, and the right clone, the plasmid DNA was digested by BamHI and EcoRI
restriction enzymes. The BamHI/EcoRI digest is expected to liberate ~600bp DracI coding sequence
from the pBluescript SK+ vector and yield two bands on Agarose gel. Agarose gel electrophoresis
method after restriction digest was used to determine the size of the insert in the pBluescript SK+
plasmid.
The results of Agarose gel electrophoresis for the BamHI/EcoRI digest of mutagenized
plasmid DNA of DracI in pBluescript SK+ is shown in the gel image of figure 2. The gel image
shows that there are two bands in lanes 2-5 corresponding to BamHI/EcoRI digested DNA clones of
samples 8A1-8A4 respectively. The two bands shown on the gel (lanes 2-5) are spotted at about
2826 bp and 676 bp that can correspond to pBluescript SK+ vector and liberated DracI coding
sequence respectively. The results from the gel electrophoresis confirms that all four clones of the
mutagenized DracI plasmid DNA are the right clones since the two band sizes are close to the
expected band sizes of ~600 bp for DracI and 3.0 kb for pBluescript SK+.
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Fig 2. BamHI/EcoRI digest of mutagenized DracI in pBluescript SK+ plasmid ( 1% TBE Agarose gel): 1st lane contains
10µl of 1kb DNA ladder with its bands sizes shown. 2nd – 5th lane contains 12µl of BamHI/EcoRI digested DNA clones
of 8A1-8A4 respectively. There are 2 bands in 2nd – 5th lanes that are 2826 bp (A) and 676 bp (B) in size which
corresponds to pBluescript SK+ vector (3.0kb) and DracI coding sequence (~600 bp). The gel was ran at 110V for ~40
minutes and visualized by Blue light transilluminator.
The size of the two bands for lanes 2-5 on the Agarose gel image of figure 2 was calculated
based on the standard calibration curve graph, log molecular weight vs. Relative mobility RF (fig.
3). The calibration curve was constructed by comparing the known band sizes of 1kb ladder with the
relative mobility calculated in the equation: RF = (distance migrated by protein)/ (distance migrated
by dye), and measuring the fragment’s migration distance (cm) relative to the distance traveled by
the dye front. The properties of the graph are Log Molecular weight = -1.7304 (Relative mobility
RF) + 4.2942 and R2=0.9848 which shows high correlation between a fragment’s log molecular
weight and its relative mobility.
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Calibration Curve
4.00
1 Kb ladder
Linear (1 Kb ladder)
log molecular weight
3.50
3.00
2.50
2.00
1.50
1.00
y = -1.7304x + 4.2942
R2 = 0.9848
0.50
0.00
0
0.2
0.4
0.6
0.8
1
RF (cm)
Fig 3. Calibration curve for mini-prep: The graph shows comparison between each band’s log molecular weight and their
relative mobility RF (cm). To determine the fragment size of an unknown band, the equation RF=distance migrated by
fragment/ distance migrated by dye is used. The properties of the graph are Log MW=-1.7304 (RF) + 4.2942 and
R2=0.9848 which suggests strong correlation between the log molecular weight and RF.
To determine the sequence of each clone and investigate the unknown forward and reverse
primers used in the site-directed mutagenesis, three positive colonies (clone 8A2, 8A3 and 8A4) of
the purified plasmid DNA of DracI in pBluescript SK+ were picked for cycle sequencing via
BigDye Terminator v3.1. Cycle sequencing is based on the Sanger chain termination method, but
yields a stronger signal due to the PCR amplification of the template DNA and also uses four
different dyes for the four ddNTPs [3]. In the cycle sequencing reaction, T3 primer is used since the
DracI coding sequence in the pBluescript SK+ vector has its 5’ end next to the BamHI site at the
side of the T3 primer binding site where the sequencing should occur (reads 5’ to 3’).
The change in the DNA sequences of each clone was analyzed by NCBI Basic Local
Alignment Search Tool (BLAST) application, where the sequence of each clone was scanned against
the BLASTP database to compare the amino acids of each mutated DracI clone with the wildtype
DracI (fig. 4). The query nucleotide sequences of each mutated DracI clone was also scanned
against the BLASTN database to determine the single base changes in the DNA sequences of each
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clone. The results for site-directed mutagenesis is shown in figure 4, and the unknown forward and
reverse primers used in the site-directed mutagenesis reaction was determined to contain DracIV12
mutation, since it targeted an amino acid change in Glysine (GGA) of position 12 to Valine (GTA)
in the sequence of sample 8A3 DracI mutated DNA clone (Appendix II).
Sample 8A2
Sample 8A4:
Sample 8A3:
Fig 4. Comparison of mutated DracI sequences vs. wildtype via BLAST application: The amino acid sequences for the
three mutated clones (8A2, 8A3, 8A4) scanned against the BLASTP database using NCBI BLAST (Basic Local
Alignment Search Tool) application. The results show that the amino acid query for the clone 8A3 has a mismatched
amino acid compared to its wildtype counterpart as shown by the arrow. The query of clones 8A2 and 8A4 show no
mismatched amino acids compared to their wildtype counterpart as shown by the arrow, thus clones 8A2 and 8A4 were
not mutated.
The DNA samples of clones 8A2 and 8A4 showed no mismatched amino acid sequence
when scanned against the wild type DracI amino acid sequence via BLASTP. Thus, the site-directed
mutagenesis were unsuccessful for clones 8A2 and 8A4 which could be caused by inhibitors of the
reverse and forward primers in the PCR reaction from dirty prep of DracI plasmid DNA. The
DracIV12 mutation is a gain-of-function mutation which locks the DracI protein in GTP bound
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state. Studies of these active mutants can aid in analyzing how DracI affects the cytoskeleton
dynamics and its signalling component, POSH gene.
One of the uses for a mutated DracI gene via site-directed mutation is the creation of
transgenic flies that bear the mutated DracI genes. To observe the affects of the altered DracI
protein in-vivo, the mutated DracI coding sequence could be inserted into the pUAST expression
vector that is inducible by GAL4. The DracI coding sequence is first digested out of the pBluescript
vector via BamHI and KpnI restriction enzymes that are compatible with the restriction sites of
pUAST vector BglII and kpnI. The BamHI/KpnI digest of DracI gene is ran on a 1% TBE Agarose
gel for the confirmation of the liberated DracI which gave fragments of 2.9kb and 0.6kb in size
matching the expected size for both bands. The gel purification method via QIAgen QIAquick
column can be used to purify the isolated 0.6kb DracI insert for ligation into the pUAST vector.
Since BamHI cut site of the DracI insert is compatible with the BglII cut site of pUAST, the
BamHI/KpnI digested DracI can be ligated into BglII/ kpnI cut pUAST DNA via T4 DNA ligase.
The DracI-pUAST ligated product are then transformed to DH5α competent cells via heat shock
procedure and plated. The five suspected positive colonies containing the DracI-pUAST plasmid
picked from the plate are then cultured and its DracI-pUAST plasmid DNA can be prepared via
QIAprep column purification.
Restriction digest by EcoRI for analyzing of the right clone containing the appropriate DracIpUAST plasmid was done and all five digests was run in 1% TBE agarose gel electrophoresis. The
expected results should yield two band fragments in the agarose gel, with fragment sizes of 9050 bp
(pUAST vector) and ~600 bp (DracI coding sequence). The gel image shown in figure 5, shows that
lanes 2, 3, 5 and 6 have two fragments of sizes of 3940 bp (pUAST vector) and 784 bp (DracI
coding sequence) which indicates the pUAST vector has the correct insert and full digest occurred.
Although the band sizes measured based on the calibration curve with the equation; logMW =
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-1.7107 (RF) + 4.1538 and R2=0.9954, shows a big discrepancy in the calculated size compared to
the expected fragment size. The reason for the huge discrepancy in the expected pUAST vector size
and the calculated band size of pUAST is due to the dye front not migrating far enough down the gel
to allow for better separation between the bands which can give a more accurate calculation of band
size. The calculated DracI fragment size (784bp) is slightly larger than the expected DracI insert
size (~600bp) due to the high DNA load (~8 µl) which can cause slow migration of the DracI
fragment through the agarose gel. In lane 4 of the gel, only one band of size 2310bp is seen that
could be attributed to incomplete digestion of pUAST plasmid DNA. Incomplete digestion yields
circular plasmid DNA, linearized DNA and supercoiled DNA which could be caused by absents of
restriction digest in the reaction.
Fig 5. EcoRI digest of mutagenized DracI in pUAST plasmid ( 1% TBE Agarose gel): 1st lane contains 10µl of 1kb
DNA ladder with its bands sizes shown. 2nd – 5th lane contains 8µl of EcoRI digested DNA clones. There are two bands
in the 2nd, 3rd,5th and 6th lane of the gel spotted at 3940bp(A) and 784bp(C) which corresponds to pUAST vector
(9050bp) and DracI coding sequence (~600bp) respectively. An undigested fragment at size of 2310bp is shown in the
4th lane. The gel was ran at 110V for ~35 minutes and visualized by Blue light transilluminator.
The method of transporting the DracI-pUAST transgene into a Drosophila embryo to create
transgenic species is through injection via glass needle at the posterior end of the embryo. The
17
procedure of inserting the needle into the embryo is by driving the slide which contains the embryo
on the microscope stage towards the stationary glass needle and observations can be made through a
monitor connected to the microscope. The DNA injection mix includes 40µg of DracI-pUAST DNA
and 8µg of pTurbo herlper DNA that helps transposes the mutated DracI gene in the Drosophila
genome via P elements of pUAST vector.
Screening for the binding partners that interact with the signalling components of DracI such
as the POSH gene, can be beneficial for studying the properties of DracI signal transduction
pathway and how DracI affects other proteins in the cell. One of the ways to determine the
interacting proteins of POSH gene is through yeast two-hybrid screening which utilizes the
interaction trap system as done in the lab. The POSH gene is called the bait in the yeast two-hybrid
screening and is fused in frame to the binding domain (BD) of LexA in pEG202 vector that contains
constitutive LexA promoter and selectable marker gene (HIS3) which allows growth in his- media.
The cDNA library of Drosophia called the prey is fused to the activation domain (AD) of B42
protein in pJG4-5 vector that contains the yeast GAL1 promoter and selectable marker gene (TRP1)
which allows growth in trp- media. The two reporter genes used as indicators of binding between
bait and prey are the lacZ gene in the pSH18-34 reporter plasmid which contains LexA operator and
selectable marker gene (URA3) which allows growth in ura- media. The other reporter gene is
leucine (LEU2) incorporated in the genome of the host yeast strain EGY48, to allow growth in leumedia is under control of the LexA operator.
The yeast two-hybrid procedure allows time for the expression of the POSH-BD fusion
protein, cDNA-AD fusion proteins and reporter gene plasmid in the EGY48 yeast cell through the
use of CM media and CM plates that contain tryptophan that permits cell growth. Only the positive
yeast cells with the appropriate expressions selected from the leu- media are plated onto the four
plates Gal/Raff/leu-, Glu/leu-, Glu/X-Gal and Gal/Raff/X-Gal plates for the screening of binding
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partners of POSH protein. The results are shown in figure 6 which displays that POSH protein has
interaction with other proteins from the cDNA library of Drosophia. The Gal/Raff/leu- plate (B)
used for selection of positive binding partners that allow expression of leucine indicated positive
colonies in quadrants 1,2,4,5,6,7,9 and 10, while Glu/leu- plate (A) used to select against selfactivation of the cDNA-AD fusion protein showed no colonies grown on the plate. The Leu+ Glu/XGal (C) plate also used as a control to select against self activation of the cDNA-AD fusion protein
showed white colony growth in quadrants 1, 4, 5, 6, 7, 8, 9 and 10 while no colony growth occurred
in quadrants of 2 and 3. The growth of only white colonies instead of blue colonies due to no
expression of lacZ gene in the Leu+ Glu/X-Gal plate and no colony growth in Glu/leu- plate
confirms that no self-activation of the cDNA-AD has occurred, since glucose used in both plates as a
sugar carbon source should inhibit expression of cDNA-AD fusion protein.
To finally determine the protein binding partners of POSH protein, the Leu+ Gal/Raff+ XGal plate is used for positive selection of the interaction, since interacting proteins would allow for
expression of lacZ and leucine reporter genes when using galactose as a sugar source, which results
in expressing a functional transcription factor of lexA. The results of the interacting partners are
indicated to be positive in quadrants 1, 4, 7 and 10 of Leu+ Gal/Raff+ X-Gal plate (D) from the
diluted Drosophia cDNA library. Similarly, the positive results of interacting partners for POSH
protein are indicated in all the quadrants of Leu+ Gal/Raff+ X-Gal plate (E) from the undiluted
Drosophia cDNA library. Lack of growth in quadrant 3 of plates (A-D) indicates that a satellite
colony from the positive plate was picked to re-streak the four plates.
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Fig 6. Selection of positive binding partners of POSH protein in Yeast two-hybrid screen: The screen is used to determine
binding partner of POSH protein by fusing POSH gene to BD of lexA and fusing Drosophila cDNA library to AD of B42
protein. The interactions are selected by utilizing the leucine and lacZ reporter genes. The plates are shown in panels (A-E)
Glu/leu- plate (A), Gal/Raff/leu- plate (B), Leu+ Glu/X-Gal plate (C), Leu+ Gal/Raff+ X-Gal plate from diluted library (D) and
Leu+ Gal/Raff+ X-Gal plate from undiluted library(E). Note: (A-D) was plated from the positive plate of diluted yeast cDNA
library contributed by the lab technician while (E) is plated from the positive plate of undiluted yeast cDNA library of group
8A.
Some of the problems encountered during the yeast-two hybrid screening experiment, was
the absence of colony growth in Gal/Raff/CM leu- plates that would select for yeast with functional
lexA transcriptional factor that have binding partners . The problem may have arisen from the
preparation of the leu+ media that allows for the activation of leucine in positive yeast cells. To
solve this problem, undiluted Drosophila cDNA library (E) was used instead of serial dilution of the
library to inoculate fresh leu+ media and longer incubation time to allow the proper expression of
leucine in yeast cells. The results were successful as shown in panel E of figure 6, where colony
growth occurred and all the colonies were positive for interaction of bait and prey proteins.
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Visualization of DracI transcript in specific tissues or whole organism of Drosophila via
RNA in-situ hybridization can contribute in studying of where the protein is produced and active in
Drosophila. UAS-Gal system such as ptc-GAL4 allows for control of spatio-temporal expression of
DracI gene to study how the protein acts during the developmental stages of Drosophila. To obtain
the riboprobe (antisense of DracI mRNA) for the RNA in-situ hybridization experiment done in the
lab, DracI cDNA cloned in pBluescript plasmid was digested out by BamHI restriction enzyme at
the 5’ end that resulted in 5’ overhang to not allow transcription into the internal sequences of
pBluescript vector. Following the removal of the restriction enzymes via QIAprep column, an invitro transcription reaction was setup to incorporate DIG labels (DIG-UTP) into the resulting
riboprobe. The T7 RNA polymerase was used for antisense transcription from the 3’ end of DracI
cDNA (3’ to 5’). The DIG labelled DracI riboprobe was hybridized to either mRNA of wild-type
DracI expressing embryo or mRNA of ptc-GAL4 DracI expressing embryo. Washes would follow
after the hybridization step to remove any unbound riboprobes. To visualize the expression of DracI
in the two different embryos, anti-DIG-alkaline phosphatase (AK) conjugate was incorporated into a
wash step, which would bind to DIG-labelled targets. The anti-DIG-AK conjugate would produce
bluish brown staining of the embryo upon addition of BCIP/x-phosphate and NBT that create a
colometric reaction through redox reaction.
The results of RNA in-situ hybridization shown in figure 7, display the successful
hybridization of DIG-labelled DracI riboprobe to the DracI mRNA of the two different embryos.
The microscopy image, shown in panel A, displays the staining after 10 minutes of incubation with
the NBT/BCIP chemical compounds during the early developmental stages of the ptc-GAL4 DracI
expressing embryo. Many of the ptc-GAL4 DracI expressing embryos did not have stripes since the
patch gene (ptc) is expressed after the early developmental stages. Although, the ptc-GAL4 DracI
expressing embryo shown in panel A was one of the very few embryos that expressed the ptc gene in
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its development stage. Longer incubation of the embryos in the last AP wash that contains
NBT/BCIP chemical compounds allowed for observation of many ptc-GAL4 DracI expressing
embryo with stripe patterns. Panel B in figure 7 also shows the wild-type DracI expressing embryo
that is in the same developmental stage as the embryo shown in panel A but the staining occurring
on the whole embryo that is more concentrated at the dorsally located amnioserosa (embryonic
tissue) of the embryo. The wild-type DracI expressing embryo (B) shows that the DracI mRNA is
expressed in every most tissues of the embryo since the staining did not produce any unique patterns
similar to the ptc-GAL4 DracI expressing embryo. The observation of DracI expression in the wildtype embryo shows that the DracI gene can be expressed in the early developmental stages of
embryo in some tissues except tissues expressing the patch gene.
Fig 7. Investigation of DracI expression in ptc-GAL4 and wild-type embryo via RNA in-situ hybridization: RNA in-situ
hybridization via DIG-labelling was utilized to determine the spatio-temporal expression pattern of DracI in wildtype
embryos via AS-GAL4 (B) and DracI expressing ptc-GAL4 embryos (A). To visualize the DracI mRNA transcripts, the
staining method was used via colormetric reaction between anti-DIG-AK conjugate and BCIP/x-phosphatase chemical
compound with a redox reaction by NBT. Note: both embryos shown in panel A and B are in the same embryonic
developmental stage.
The vast information gathered in the lab about DracI protein can be used in experiments such
as sequencing to identify the binding proteins found in the yeast two-hybrid interaction or using GFP
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protein fusion experiment to determine the in-vivo activities of DracI protein and other proteins that
are involved with DracI signalling similar to the cadherin-GFP fusion demonstaration done in the
lab. The transgene fusion of the mutated DracI can be used in RNAi experiments to study reduced
in-vivo expression of DracI in Drosophila. Site-specific recombination technique based on Gateway
cloning technology could also be used in identifying how hairpin RNAi of DracI can phenotypically
affect Drosophila. The advantage of using recombination technique based on Gateway cloning
technology is the ease and efficiency of recombining DracI cDNA to many different destination
vectors that have different properties for use in different experiments such as reduced in-vivo
(RNAi), ectopic expression (GFP) and spatio-temporal expression.
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References:
1. Hakeda S. et. al. Rac function and regulation during drosophila development. Nature. 2002.
416: 438-442
2. Andjelkovic, M., et al. Developmental regulation of expression and activity of multiple forms
of the Drosophila RAC protein kinase. J. Biol. Chem. 1995. 270 (8): 4066-4075.
3. Harden N., Kim A., Langmann C., Sinclair D. and Sanny J. MBB432: Advanced Molecular
Biology Technique. Lab Manual. 2009. 3-56
4. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning, A Laboratory Manual,
Second Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
(1989).
5. Hutchinson C., Phillips S., Edgell M. Mutagenesis at a specific position in a DNA sequence.
The journal of biolo. Chem. 1978. 253 (18): 6551-6560
6. Storici F., Resnick MA. The delitto perfetto approach to in vivo site-directed mutagenesis
and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol.
2006. 409: 329-45.
7. Vogelstein, B., and Gillespie, D. Preparative and analytical purification of DNA from
agarose. Proc. Natl. Acad. Sci. 1979. 76, 615–619.
8. Manabu Tsuda, Ki-Hyeon Seong et. al. POSH, a scaffold protein for JNK signaling, binds to
ALG-2 and ALIX in Drosophila. FEBS Letters. 2006 580: 3269-3300
9. Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D "Green fluorescent protein as a marker
for gene expression". Science. 1994. 263 (5148): 802–5
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Appendix I: Vectors
25
26
27
28
Appendix II: BLASTN & BLASTP Data
Sequencing results:
8A2 sample: (no site directed mutagenesis) wild-type (BLASTP data)
8A4 sample: (no site directed mutagenesis) wild-type (BLASTP data)
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8A3 sample: site-directed mutagenesis, Drac1V12, mutant oligos RV1 and RV2 (BLASTP data)
8A3 sample DNA alignment with the wild-type Drosophila melanogaster Rac1 mRNA (BLASTN
data) (GGA  GTA)
Appendix III: Raw sequencing data
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Sample 8A2:
Sample 8A3:
Sample 8A4:
31