Pusar Khidm - Universiti Malaysia Sarawak and... · Pusar Khidm.u MakJumnr k:ld,,·rr,;I . UNIVE SIll MALAYSIA SARA\\Al' Characterization and Expression Study of Pre12a and Pre12b

CHARACTERIZATION AND EXPRESSION STUDY OF PREL2A AND PREL2B ON EARLY VERTEBRATE DEVELOPMENT

Jonathan Chan Chia Jui

(36395)

QL Bachelor of Science with Honours (Resource Biotechnology)

Al 2015 C454 2015

605

Pusar Khidmu MakJumnr k ldmiddotrrI UNIVE SIll MALAYSIA SARAAlshy

Characterization and Expression Study of Pre12a and Pre12b on Early Vertebrate

Development

JONATHAN CHAN CHIA JUI (36395)

A thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of

Science with Honours (Resource Biotechnology)

Faculty of Resource Science and Technology

UNlVERSITI MALAYSIA SARA W AK

2015

I

if if

Acknowledgement

This project was conducted in partial fulfillment for the Degree of Bachelor of Science

with Honors First I would like to express my indebtness appreciation and veneration to

my supervisor Dr Lee Kui Soon for giving me the opportunity to work on project related

with molecular biology under his guidance full support and cherished advices I dedicate

my gratitude and special thanks to Pang Shek Li (Msc) candidate for her persistent advices

experiences well-regarded comments and indelible inspiration patiently guiding me and

teaching me various lab techniques throughout the project I would also like to thank AP

Dr Mohd Hasnain Md Hussain AP Dr Awang Ahmad Sallehin AP Dr Edmund Sim Ui

Hang AP Dr Hairul Roslan and Ng Kher Lee (PhD) for providing necessary equipments

and reagents for my experiments Furthermore I take this opportunity to thank the rest

postgraduates of Animal Biotech Lab and lab assistants including Mr Iskandar for their

assistances and advices on technical troubles as well as supportive atmosphere for learning

Finally I thank to my friends for their encouragement and to my parents for their absolute

moral supports with a wish for their good health

II

UNIVERSITI MALAYSIA SARAWAK

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IV

PusltKhidmru aklumH A middotudcr middot I J r 1 l ~ I Y L- S L WAt

Title and Front Cover

Acknowledgement

Declaration of original work

Table of Contents

List of Abbreviations

List of Tables

List of Figures

Abstract

10 Introduction 2-3

20 Literature Review 4-9

Table of Contents

I

n

III-IV

V-VI

VII

VIII

IX

21 Proline-rich EVH 1 ligand (PREL) family genes 4-5

22 Proline-rich EVH1 ligand 2 (PREL2) or Lamellipodin 5-6

23 EnaV ASP protein family 6-7

24 Zebrafish (Dania rerio) 7-8

25 Gene Duplication 8

26 Duplication -Degeneration- Complementary (DDC) Model 9

30 Methodology 10-15

31 Primer Design and Sequence 10

32 Recovery ofE coli Recombinant Culture from Glycerol Stock 10

33 Colony PCR of Plasmid DNA 10-11

34 Agarose-gel Electrophoresis (AGE) 11

35 Plasmid DNA Extraction using PureYieldtrade Plasmid Miniprep 11-12

36 Plasmid Purification using Wizardreg SV Gel and PCR Clean-Up System 12-13

37 Quantification ofDNA 13

38 Minipreparations of Plasmid DNA using Alkaline Lysis 13-14

39 DNA Sequencing 14

310 Preparation of Digoxigenin-Iabelled RNA in situ Probes 14-15

311 Zebrafish Embryo Fixation and Dehydration 15

312 Embryo Mounting and Microscope Techniques 15

40 Results 16-23

41 Recovery of E coli Recombinant Culture from Glycerol Stock 16

42 Colony PCR 16

43 Plasmid Minipreparations 17

44 Gel Purification of Plasmid DNA 17

45 Plasmid DNA quantification 18

46 Direct DNA Sequencing 18-21

47 Multiple sequence alignment between zebrafish Prel2 peptides from sequenced transcripts with other vertebrates Prel2 peptides 22-25

48 Preparation of Digoxigenin-Iabelled RNA in situ Probes 25-26

50 Discussion 27-35

51 Colony PCR and Subculture of Recombinant E coli 27

52 Plasmid Extraction and Purification 27-29

53 Plasmid DNA Quantification 29-30

54 Direct DNA Sequencing 31-32

55 Preparation of Digoxigenin-Iabelled RNA in situ Probes 32-34

56 Zebrafish Embryo Fixation and Dehydration 35

60 Conclusion 36

70 References 37-41

80 Appendices 42-45

VI

I

I

List of Abbreviations

AGE

CME

DDC

EVH

Hoxb

LAIX

LB

Lpd

MRL

PFA

PH

PBST

Prel2

PTU

RA

RE

RIAM

RT

RT-PCR

SDS

SH3

TE buffer

VASP

Agarose gel electrophoresis

Clathrin-mediated endocytosis

Duplication-Degeneration-Complementary

EnaIV ASP homology

Homeobox

LBIAmpicillinlIPTGX-Gal

Luria-BertaniLuria Broth

Lamellipodin

MiglORIAMlLpd

Parafonnaldehyde

Pleckstrin homology

Plrosphate Butfered Saline with Tween-20

Proline-rich EVHl ligand 2

I-Phenyl-2-thiourea

Ras association

Restriction enzyme

Rap I-GTP-interacting adapter molecule

Room temperature

Reverse Transcription-Polymerase Chain Reaction

Sodium dodecyl sulfate

SRC Homology 3

Tris-EDTA buffer

Vasodilator-stimulated phosphoprotein

V

List of Tables

Figure Description Page

Table 331 Setup conditions for colony peR and master mix 11

Table 451 Spectrophotometric analysis of plasmid samples 17

BLASTn significant alignments (~99) for construct Table 461 17-18 Prel2a (1381 letters)

Table 462 BLASTn significant alignments (~99) for construct 19

Prel2b (1171 letters)

I

I

1shy

VIllI

List of Figures

Figure Description Page

Figure 221 Topological features of (A) PH domain (B) RA domain 4

Figure 261 The three possible outcomes of duplicate gene pairs that

contains regulatory regions with distinct functions 8

represented by small boxes

Figure 411 Agar plates of recombinant culture Prel2a (1 amp 2) and 15 Prel2b (3 amp 4) after overnight incubation

Figure 421 Gel result for colony PCR for Prel2a 15

Figure 422 Gel result for colony PCR for Prel2b 15

Figure 431 Plasmid mini prep for Prel2a 16

Figure 432 Plasmid mini prep for Prel2b 16

Figure 441 Purified plasmid for Prel2a 16

Figure 442 Purified plasmid for Prel2b 16

Figure 461 Pairwise alignment bet~een extracted plasmid Prel2a

18and PREDICTED Dania reria sidkeyl h243

(sidkeylh243) transcript variant X6 mRNA

Figure 462 Pairwise alignment between extracted plasmid Prel2b

20and PREDICTED Dania reria Ras association

(RaIGDSAF-6) and pleckstrin homology domains 1

(raphl) transcript variant X6 mRNA

Figure 471 Multiple sequence alignment Prei2a and Pre12b peptide

sequences translated from the construct with Prel2 24

peptides of other vertebrates

Figure 481 Gel result of linearized pGEMT -Easy-PreI2a 25

Figure 482 Gel result oflinearized pGEMT-Easy-PreI2b 25

Figure 483 Gel result of purified linearized pGEMT-Easy-PreI2a 25

Figure 484 Prel2a RNA probes 25

I

Characterization and Expression Study of Prel2a and Prel2b on Early Vertebrate Development

Jonathan Chan Chia Jui

Resource Bioteclmology Faculty of Resource Science and Technology

University Malaysia Sarawak

Abstract

Proline-rich EVHlligand 1 (Prell) and Proline-rich EVHI ligand 2 (PreI2) belong to Prel gene family Both genes are homologous and participate in cellular integrin activation Pre12 interacts with GTPase called Ras in GTP-bound state and is essential in regulating cell adhesion and migration It has been identified to involve in mechanism assisting endocytosis of epidermal growth factor receptor (EGFR) that participates in development of arious diseases such as cancer However the comprehensive and mechanistic model of protein signaling involving cell migration is still unavailable Hence Prel2 is selected as candidate gene with zebrafish (Danio rerio) in this study to examine its function This tudy was done partly to verify the occurrence ofPrel2 gene duplication as well as to fulfill

our understanding on the importance of Prel2 during vertebrate development The methodology involves colony peR plasmid extraction DNA sequencing zebrafish cultivation RNA probe preparation in situ hybridization embryo mounting and microscopy Through these experiments Prel2 genes were characterized and plasmid with the gene construct was isolated purified and ready for RNA probe synthesis for in situ hybridization of zebrafish

Keywords Prel gene family EVH Danio rerio endocytosis gene duplication

Abstrak

Proline-rich EVHl ligand 1 (Prell) dan Proline-rich EVHl ligand 2 (PreI2) merupakan gen-gen di bawah kategori kumpulan Prel gen Kedua-dua gen tersebut adalah homolog dan menyertai aktivasi integrin sel Prel2 berinteraksi dengan satu GTPase dinamakan Ras yang berikat kepada GTP dan diperlukan dalam regulasi pelekatan dan migrasi sel Gen fer ebut juga dikaitkan menyertai mekanisme endocytosis epidermal growth factor receptor (EGFR) yang penting dalam perkembangan pelbagai penyakit termasuk kanser Walau bagaimanapun model yang komprehensiJ dan mekanistik tentang protein signaling melibati migrasi sel masih tiada Dengan ini Prel2 dipilih sebagai gen calon dan zebrajish (Danio rerio) sebagai model binatang dalpm kajian ini untuk menguji jimgsi gen tersebul Kajian ini dilaksana sebahagiannya menentukan kejadian duplikasi gen Prel2 dan juga menunaikan pemahaman Prel2 gen dalam perkembangan vertebrate Metodologi yang digunakan termasuk colony peR plasmid extraction DNA sequencing kultivasi zebraji h persediaan RNA probe in situ hybridization embryo mounting dan microscopy Sepanjangan eksperimen ini gen-gen Prel2 telah dicirikan dan plasmid yang membawa konstruk gen telah diasingkan ditapiskan dan bersedia untuk sintesis RNA probe untuk kegunaan in situ hybridization zebrajish

Kata kunci Gen Prel EVH Danio rerio endocytosis duplikasi gen

1

10 Introduction

Cell behavior is an exciting field of research in the past two decades They have made

significant progresses in understanding how cells migrate by examining the underlying

molecular mechanisms Dynamic reorganization of actin cytoskeleton which contributes to

celI motility cell shape via structural modifications and cell-cell adhesion via anchoring

ha been intensively studied Cell migration has also been implicated with an a~

heterodimer called integrin in multiple cellular processes such as migration of leukocytes

during immune surveillance tissue in guided regeneration and repair epithelial stem cells

to target regions as wen as migration of cellular sheets during embryonic morphogenesis

including gastrulation (Friedl amp Wolf 20 10 Krause et al 2003 Ridley et at 2003)

The MiglORIAMLpd (MRL) protein family is a group of adaptor molecules that

have known to interact with EnaVASP proteins and conbbute to diverse cellular and

phy iological functions For instance Proline-rich EVHl ligand 2 (PreI2) or known as

Lamellipodin (Lpd) has crucial roles in cell adhesion and cell migration including

lamellipodial protrusion via actin polymerization and Ras GTPase signaling Lofthouse

(2013) mentioned that decreased expression of MRL proteins will cause slowed cell

division increased monomeric (G) filamentous (F) actin ratio and impairs cell migration

Moreover EnaIV ASP protein family is critical for regulating actin assembly and

cell motility in diverse cell types of different orgaisms For instance Drosophila Enabled

(Ena) protein was found in a screen for dominant suppressors whereas vasodilator-

stimulated phosphoprotein (VASP) was detected in platelets in phosphorylated form due to

increased levels of secondary messengers (Krause et aI 2003) Despite huge efforts have

been done on characterizing these molecules there is yet no conc1usi ve evidence for their

effects on actin polymerization and filament elongation linked to profilin

2

Until now many aspects of cell motility such as invasive properties signaling

activities and regulation are very complex and still poorly understood The amount of data

on studying Prel2 genes in zebrafish and their co-relationship with Prel2 genes from other

vertebrates are still limited Hence this study will provide an insight regarding Prel2 as one

f the crucial regulators of cell motility It is also crucial to determine whether Prel2 is an

evolutionarily conserved gene duplicate of the Prel2 gene family

The aim of this project is to characterize and establish the expression profiles of

Pre12a and Prel2b of Prel gene family in selected embryonic stages of Danio rerio The

pres nce of two copies of Prel2 genes has helped to prove the occurrence of gene

duplication in zebrafish genome This duplicative event is a key mechanism of evolving

novel gene function during evolution Once characterized the data was helpful to

structurally and functionally distinguish Prel2a from other gene families The expression

patterns of Prel2a homologs in other mammals will also be compared to reveal functional

and expression relationships between them This will provide a deeper insight into how

Prel2a expression and the recruitment of EnaiV ASP proteins to cell leading edge are being

regulated

3

20 Literature Review

21 Proline-rich EVHl ligand (Prel) family genes

Members of Prel gene family include Prell and Prel2 Both molecules belong to the MRL

(Mig- 10RIAMILamellipodin) protein family Prell is also known as Rap 1-GTPshy

interacting adaptor molecule (RIAM) as it interacts with Ras-associated protein 1 (Rap 1)

and was identified from a yeast-two hybrid system while hunting for Rap I-interacting

molecules in Jurkat T-cell line (Lafuente 2004) It has a molecular weight of 74 kDa and

carries 665 amino acids It possesses binding motifs for Profilin EVH 1 SH3 and WW

domain-containing molecules (Holt amp Koffer 2001 Lafuente 2004) On the other hand

Prel2 or called Lamellipodin (Lpd) directly interacts with a small GTPase called Ras in

GTP-bound state Both RIAM and Lpd share about 36 of RA domain homology with

Grb proteins and a homology section of 300 amino acids called Grb7-MIG-IO homology

region is detected in both MRL and Grb proteins (Jenzora et at 2005 Manser

Roonprapunt amp Margolis 1997) However SH2 and BPS domains are absent in MRL

while baving several proline-rich motifs at C-terminus as well as differences in Ras

association (RA) and pleckstrin homology (PH) compared to Grb proteins (Lafuente 2004)

Prell and Prel2 are both involved in cellular integrin activation (Lee et at 2008)

The activation relies on the formation of scaffolds by the MRL proteins which link the Ras

GTPase membrane-targeting sequences to talin and recruit it to membrane integrins The

change in affinity of cellular integrins is crucial to physiological processes which include

association of extracellular matrix cell migration immune functions and homeostatic

regulation (Hynes 2002) RIAM acts as one of the Rap 1 effector molecules that possesses

two domains RA and PH (Hynes 2002) The PH domain allows RIAM to recruit proteins

onto membrane directing them to cellular compartments and participate in signal

transduction pathway (Hirata Kanematsu Takeuchi amp Yagisawa 1998) RIAM IS

actively expressed in hematopoietic cells whereas LpdPrel2 is particularly active as

4

Pus Khidm 1 a umat d UNI VERSITf MALAYSIA SARAVAj~

paralogue in fibroblasts and somatic cells (Krause et al 2004) Together with EnaIV ASP

proteins Prell directly localizes at lamellipodial and fillopodial tips as well as involving in

the direct binding to activated Ras via phosphoinositide pathway (Jenzora et al 2005)

Targeting ofPrell to focal adhesions and lamellipodia was proven through immunolabelling

experiment with green fluorescent protein (Jenzora et al 2005)

22 Proline-rich EVHl ligand 2 (Prell) or Lamellipodin (Lpd)

Prel2 or Lpd constitutes of 1250 amino acids much shorter than RIAM and contains eight

SH3 binding motifs 6 EVHI binding motifs and three Profilin binding motifs Lpd is a key

EnaIVASP binding protein in regulating cell migration It shares significant homology with

RIAM through the conservation of RA and PH domains and possess binding sites for

Profilin EVHI and SH3 domains Lpd and RIAM differ in amino acid identity at both C

and N termini with RIAM contains more EVHl binding regions than Lpd The N terminus

of Lpd is constituted of approximately 50 amino acids and is favorably polar followed by

RA and PH domains whereas the C terminJls is proline-rich The Prell PH domain binds at

great affinity to phosphatidylinositol monophosphates PI(3)P and PI(5)P

~ Figure 221 Topological features of (A) PH domain (B) RA domain (Images adapted from (A) httpllwwwcellsignalcoml Ferguson et al 1995 (B) httpwwwebiacukpdbe)

Conversely the PH domain of Lpd is phosphatidylinositol (34) bisphosphate

(PI(34)P2) -specific (Krause et aI 2004) In addition the interaction between Lpd and

EnaIV ASP molecule was determined by immunoprecipitation of mouse cortical neurons

5

with high expressivity of mena (Krause et a 2004) In vivo study of Lpd also proves the

need of ScarlW A VE complex for regulating cell migration (Law et al 2013)

In human the corresponding gene is situated on a region of chromosome 10 which

is linked to amyotrophic lateral sclerosis (ALS) or called Lou Gehrigs disease (Hadano et

at 2001) ALS is implicated as a common destructive progressive motor neuron disorder

Krause and his team have hypothesized that combined role of EVH1 and EVH2 are

essential for leading edge localization of EnaV ASP molecules on lamellipodia and

filopodia However EVH2-containing proteins were first discovered before EVH1shy

containing Lpd was eventually screened from databases using FPPPP sequence In genetic

knockdown of Lpd lamellipodial formation and velocity were reduced in Lpd knock-down

cell lines (Krause et al 2004) On the other hand along with the dorsal ruffling of

lamellipodia in Lpd-overexpressed c-Abl-coexpressed fibroblasts the results suggested

Lpd regulatory role in lamellipodial dynamics (Krause et at 2004 Michael et a 2010)

Study of Lpd knock-down human podocytes (LPDkd Hupo) revealed Lpd importance

in lamellipodia generation and focal adhesion which is mediated by the Nephrins ability to

recruit Lpd (Venkatareddy et a 2011) The role of Lpd is also associated in neuronal

development by fostering axonal morphogenesis via EnaIV ASP-Lpd interaction (Michael

et at 2010) In addition Lpd is vital for endophilin recruitment at clathrin-coated pits and

mediates endophihn downstream to regulate F-actio polymerization necessary for clathrinshy

mediated endocytosis (CME) (Vehlow et a 2013) In neuronal cells endophilin will

gather synaptojanin to assist in membrane fission during vesicle invagination and In

uncoating ofclathrin-bound vesicles (Schuske et a 2003 Verstreken et at 2003) I ~

23 EnaNASP protein family

EnaNASP homology (EVH) protein family comprises of Enabled proteins of Drosophila

(Ena) in Caenorrhabditis elegans (Unc-34) Dictyostelium (DdV ASP) and three proteins

6

(VASP Mena and EVL) in vertebrates (Krause et aI 2003) The domains shared among

these proteins include EVHl domain at N-terminus the proline-rich domain and EVH2

domain at C-terminus EVH 1 domain is required for successful focal adhesions by

interacting with FPPPP sequence motifs in Lpd vinculin and zyxin (Boeda et al 2007)

Based on Lafuente et al (2004) and Krause et al (2003) studies RIAM

independence over EnaVASP proteins suggested a positive regulatory effect as opposed to

the negative regulation of cell adhesion by Lpd When EnaV ASP proteins are reduced

lamellipodiaI protrusion is retarded tollowed by improved cell translocation (Bear 2002)

24 Zebrafish (Danio rerio)

Zebrafish (Danio rerio) is a member of Super-Order Teleost family native to the hurtling

streams of southeastern Himalayan region (Mayden et al 2007) It becomes an excellent

animal model for studying gene function in the embryogenic and physiological processes

of vertebrates and for temporal comparison of gene expression between different

vertebrates to determine evolutionary rates (Weinberg 1992) As a cold-water fish

zebrafish lives at growth conditions of 18degC to 24 degC and pH 65 to pH 70 The oviparous

fish can lay around 300 translucent eggs that hatch into larvae around 2 days after

fertilization and capable of swimming and feeding independently after Day 5 At third

month the feeds and growing temperature are critical in determining the fish maturity

It is an ideal model system in scientificresearch for its optical transparency of

embryos a relatively rapid growth and short generation time to facilitate genetic

experiments less difficulty encountered in breeding fish and producing gametes and the

effectiveness of inducing mutations (Kimmel 1989 Weinberg 1992) The transparent

embryos are oviparous to allow easy access and monitoring development and cell growth

Additionally the pattern of cell cleavage and their migration can be clearly observed and

characterized

7

As vertebrate zebrafish shares certain major biological characters with human

Also the developmental behaviors particularly neural crests are analogous to vertebrates

which can be clearly labelled and altered by ablation or heterotopic transplantation (Eisen amp

Weston 1993) Genetic manipulations including morpholino (modified anti-sense

oligonucleotides) knock-down have been extensively used to study individual gene

functions in vivo (Amores et at 1998 Corey amp Abrams 2001) Besides that one study on

the teleost genetic complexity implied that the fish genome has been duplicated over 100

million years ago giving rise to morphological diversity within such group of vertebrates

25 Gene Duplication

Gene duplication is crucial for the emergence of new gene functions that are essential to

allow organisms to adapt to their living environment (Hughes 1994 Lynch amp Conery

2000) It i well documented that genes in large portion of every species genome are

functionally constrained as nucleotide substitutions are deleterious to the organisms

fitness (Hurles 2004) In these protein coding genes a base change would most likely alter

the corresponding amino acid and subsequently changes the phenotype However gene

duplication provides a mean of making another copy of particular gene such that both

evolve separately without functionally interfering one another

Duplicated genes are believed to be naturally selected if they acquire early and late

functions or specificities or otherwise would be eliminated during evolution Several

mechanisms ofhow genes duplicate have been described (Hurles 2004) In retrotransposition

mRNA transcripts of individual genes transcribe reversely and the products integrate into

the genome randomly to become intron-free poly-A tail-containing retrogenes which are

functional in most genomes (Yu et al 2004) Moreover segmental duplication either due

to homologous or non-homologous recombination generate tandem fragments of genome

(Samonte amp Eichler 2002) In rare instances whole genome may also be duplicated via

polyploidization as seen in many plants to aid in diversification (Soltis et at 2008)

g

26 DupUcation-Degeneration-Complementary (nDC) Model

The functional divergence outcomes of duplicate gene pairs theorized under this model

include nonfunctionalization where one copy is silenced through degenerative mutations

neofunctionalization where one copy obtained a new beneficial function and is naturally

selected and subfunctionalization where functions of both copies are partially compromised

by neutral mutation producing paralogs with subfunctions different from the ancestral

function (Semon amp Wolfe 2008) In other words the ancestral genes of such model have

neutrally mutable regions or alleles that tend to loss portion of its functions For instance

the expres ion patterns of two copies of hoxb5 hoxb5a and hoxb5b indicate

subfunctionalization of ancestral hoxb5 gene (Jarinova et at 2008) Besides a conjecture

was given by Lee (2008) that Prel2 genes might have evolved due to subfunctionalization

producing Pre12a and Prel2b with early and late functions

Furthermore degenerative loss (nonfunctionalization) of gene sub functions from

duplicated genes due to mutatjons at gene regulatory elements can increase the chance of

preserving the duplicates (Force et at 1999) As a result of degenerative loss of duplicated

copies this has allowed the late vertebrates or mammals to evolve complex gene functions

such that each might be partitioned complementarily by two zebra fish homologues

- PuJl fwIcu ~ Dude n IW fllllilLQIl

---shy +COOCH

--shymiddotmiddotmiddotmiddotmiddotmiddotmiddot middotmiddot middotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddottmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddotmiddot__middotmiddotmiddot -- ---~ + -

JIlIl i n

-shy -shyshyt Imiddotj middot t

c _ o

Figure 261 The three possible outcomes of duplicate gene pairs that contains regulatory regions

with distinct functions represented by small boxes

9

3 Methodology

31 Primer Design and Sequence

Prel 2a and Prel 2b primer pairs previously designed usmg Primer3

(httpbiot 1 umassmededuibioappsprimer3_ wwwcgi) were used for PCR amplification

Forward 5-TGG TGG ATG AAA GAC AGA CG-3 Prel2b

Reverse 5-GCT TCC CAT ACT TGG CAA TG-3

32 Recovery of E coli Recombinant Culture from Glycerol Stock

E coli strain JM109 cells containing the recombinant DNA for Prel2a and Prel2b were

recovered from -80degC storage An inoculum of each recombinant culture in the Eppendorf

tube was streaked on LB agar plate containing ampicillin (100 Ilglml) Next the plates were

sealed with parafllm tapes followed by overnight incubation at 37degC

33 Colony PCR of Plasmid DNA

Individual white colonies from each plate were gently swabbed using sterile 10 III pipette

tip and inoculated into the bottom of sterile 15 ml microcentrifuge tubes Next 25 III PCR

reaction was prepared from 100 IlM forward primer 100 IlM reverse primer 10 mM dNTP

mix 5 x PCR buffer 25 mM MgCz 05 U Taq DNA Polymerase (Invitrogen USA) and

ddH20 Mastercycler Gradient Thermal Cycler was set for PCR Then 10 agarose gel

electrophoresis was performed to check for positive clones which were then recultured

10

Table 331 Setup conditions for colony peR and master mix

Composition Prel2a Piel2b

1x (f1I) 3x (f1I) 1x (f1I) 3x (f1I)

5x PCR buffer-Mg 50 150 50 150

10 mM dNTP mix 05 15 05 15

25mMMgCh 15 45 15 45

100 jlM Forward primer of Pre12a 10 30 - -

100 jlM Rever primer of Pre12a I

10 30 - -100 J1M Forward primer of Pre12b - - 10 30

100 J1M Rever primer of Pre12b - - I 10 30

Template DNA 10 30 I I 10 30

(colony) (colonies) (colony) (colonies)

Go-Taq DNA polymerase 02 06 02 06

Double-distilled water (ddH20) 148 444 148 444

Final volume 250 750 250 750 I

34 Agarose-gel Electrophoresis (AGE)

1 agarose gel was prepared from 05 g of agarose powder added into 50 ml of 1 x T AE

buffer (0040 M Tris-acetate and 0001 M EDTA) in a 200 m1 bottle (Schott Duran) and

followed by heating in Sharp R-315JS Carousel 12 CF Microwave Oven for 40 seconds

with rotating ring activated and left cooled to about 55degC 1 jll of ethidium bromide (EtBr)

was added by using 05 to 100 jll BIOHIT pipettor The solution was poured into 50 ml

gel tray cooled and then loading dye (6x) was mixed into each sample on a clean parafilm

before loading to wells The electrophoresis tank was connected to Enduro Power Supply

(max at 300 V) and electrophoresed at 90 V for 45 minutes

35 Plasmid DNA Extraction using PureYieldtrade Plasmid Miniprep

Plasmid minipreparation was performed at 20 reactions for each gene 15 ml of bacterial

culture grown in LB medium was transferred to a 15 ml microcentrifuge tube The tube

as centrifuged at RT for 2 minutes followed by discarding the broth Another 15ml of

culture was added and centrifugation was repeated After pouring off supernatant the

11

pellet was resuspended in 600 )ll ultrapure water Then 100 )ll of Cell Lysis Buffer was

added and the content was mixed by inverting the tube 6 to 10 times until the solution

turned into clear blue entirely After that 350 )ll of chilled (4degC) Neutralization Solution

was added and mixed thoroughly by inverting the tube 10 to 12 times Once the content

turned yellow and precipitate formed the tube was centrifuged at 13000 rpm for 3 minutes

Approximately 900 )ll of the supernatant was transferred to a PureYieldtrade

minicolumn as embled on a collection tube and centrifuged for 30 seconds The

flowthrough wa discarded and the minicolumn was placed back into the collection tube

Next 200 )ll of Endotoxin Removal Wash was added to the mini column followed by

centrifugation at 13000 rpm for 30 seconds After that 400 )ll of Column Wash Solution

(diluted with EtOH) was added to the mini column and spun for another 1 min The

minicolumn was transferred to a clean 15 ml microcentrifuge tube followed by adding 30

Jl1 of Elution Buffer directly to the minicolumn matrix The assembly was incubated for 2

minutes at RT before spinning at max speed for 30 seconds followed by keeping at -20degC

36 Plasmid Purification using Wizardreg SV Gel and PCR Clean-Up System

Prior to purification 07 agarose gel with two wells taped was set up 100 to 200 Jl1 of

plasmid was then loaded into the well and electrophoresed After electrophoresis DNA

band from the gel was excised and the gel slice was placed in a pre-weighed 15 ml microcentrifuge tube After weighing on a balance the weight of gel slice is determined

(100 to 200 mg) 10 ~ll of Membrane Binding Solution pet 10 mg of gel slice was then

added to the tube The content was briefly vortexed and incubated at 55degC until gel slice is

completely dissolved Next the melted gel was transferred to SV Minicolumn assembled

n a collection tube followed by adding 700 Jl1 of Membrane Wash Solution (diluted with

tOR) The assembly was centrifuged at 13000 rpm for 1 minute The flowthrough was

12

discarded and the minicolumn was reinserted into collection tube Washing was repeated

with 500 J11 Membrane Wash Solution and centrifuged at 13000 rpm for 5 minutes In

elution minicolumn was transferred to new microcentrifuge tube and 30 J11 of ultrapure

H20 was added to mini column followed by incubating for 2 to 3 minutes Next the

assembly was spun at 13000 rpm for 1 minute Similar elution step was repeated twice

using 20 J11 ultrapure H20 The purified plasmids were then stored at -20degC

37 Quantification of DNA

Unpurified and purified plasmid DNAs were recovered from -20oe storage Each aliquot (1

J1l of plasmid + 999 J11 of dH20 at dilution 1 1 000) was quantified using UV -VIS-

Spectrophotometer at 260 nm wavelength (Aranda et aI 2009) Purity can be determined

from A260-320A280-320 ratio and concentration was calculated as follow

DNA (J1g1J11) = [A260 x (40 )lg RNAml)(1 A 260 unit) x (dilution factor)]11 000

38 Minipreparations of Plasmid DNA using Alkaline Lysis

The step was performed as described by Sambrook et al (2001) Individual white colonies

were picked up and grown in 15 ml Eppendorf tubes for 6 hours Next 200 )ll of each

culture were inoculated into 50 ml Falcon tubes containing ampicillin and incubated

overnight at 37degC in shaking incubator at 160 rpm In next day 3 ml bacterial cells were

harvested by centrifugation in each 15 ml tube removing liquid traces resuspending pellet

in 100 )ll resuspension buffer Lysis buffer was added and the tubes were gently inverted

until turning into clear viscous liquid before chilled for 5 minutes 150 )ll Neutralisation

solution (3M NaOAc pH 48) was added followed by inverting and chilling the mixture for

3 minutes before centrifugation for 5 minutes Next equal volume of PCI (2524 1) was

added to supernatant followed by spinning for 3 minutes The upper layer was transferred into

new tube followed by adding 2x volumes of 95 ethanol to precipitate plasmid for 3

minutes The content was spun at max speed for 5 minutes and the supernatant was

discarded The pellet was washed with 70 ethanol by spinning for 3 minutes The ethanol

was removed and the pellet was air-dried The plasmid was then redissolved in 30 III

ultrapure H20 before storing at storage at -20dege

39 DNA Sequencing

Samples (zPrel2a amp zPrel2b) were sequenced using single pass DNA sequencing method

at the 1 st BASE DNA Sequencing Facility (Selangor Malaysia) with Applied Biosystem

3700 DNA Analyzer and BigDyereg Terminator v31 cycle sequencing kit M13 primer pairs

(F 20 and R -20) were used in forward and reverse reactions

310 Preparation of Digoxigenin-Iabelled RNA in situ Probes

Once insert orientation was checked 5 Ilg of plasmid DNA incorporated with the gene of

interest was linearized with IOU of appro~riate RE to a total of 50 Ill An aliquot was

loaded on gel to check for complete digestion The remaining sample was purified using

Wizardreg SV Gel and PCR Clean-Up Kit (Promega USA) and concentration was estimated

by comparing with a quantitative ladder Probes were synthesized using Digoxigenin (Dig)

RNA Labelling Kit (Roche GmbH Mannheim Germany)

1 Ilg of linearized plasmid was then combined with 2 III 10 x transcription buffer 2

III 10 x Dig-NTP Ipbelling mixture 2 III RNA polymerase and 1 111 RNase inhibitor

followed by adding dH20 to make up a volume of20 111 The mixture was incubated at 37dege

for 2 hours 2 111 aliquot of content was kept at -200 e and 1 III DNase was added to remaining

reaction During RNA cleaning 5 111 of 4M LiCl and 140 111 of 100 ethanol were added

the sample and incubated overnight The sample was spun at 14000 rpm for 15 minutes

followed by washing with 300 111 of 70 ethanol The sample was pelleted by spinning at

14000 rpm and air-dried for 5 minutes After that 15 111 of ultrapure water was added to

t 14