Amplification, Cloning And Characterization Of Rna ... · PCR Optimization and reaction conditions Polymerase chain reaction (PCR) was originally intended to amplify the genomic sequences

Article ID: WMC001094 2046-1690

Amplification, Cloning And Characterization Of RnaHelicase Gene From Aedes AegyptiCorresponding Author:Mr. Ahmad Ibrahim,Malaysia, School of Pharmaceutical Sciences, University Sains Malaysia, 11800, Minden, Penang, Malaysia,118000 - Malaysia

Submitting Author:Mr. Ahmad H Ibrahim,Malaysia, School of Pharmaceutical Sciences, University Sains Malaysia, 11800, Minden, Penang, Malaysia,118000 - Malaysia

Article ID: WMC001094

Article Type: Research articles

Submitted on:30-Oct-2010, 10:11:51 AM GMT Published on: 30-Oct-2010, 10:27:56 PM GMT

Article URL: http://www.webmedcentral.com/article_view/1094

Subject Categories:MOLECULAR BIOLOGY

Keywords:Aedes aegypti, RNA Helicase, Amplification, Cloning, Characterization

How to cite the article:Ibrahim A , M. F. F. W , S. Abdul Hameed S , Shah Abdul Majid A . Amplification,Cloning And Characterization Of Rna Helicase Gene From Aedes Aegypti . WebmedCentral MOLECULARBIOLOGY 2010;1(10):WMC001094

Source(s) of Funding:

This research project was fully sponsored by University Sains Malaysia, under grant no S5010800

Competing Interests:

Nil

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Amplification, Cloning And Characterization Of RnaHelicase Gene From Aedes AegyptiAuthor(s): Ibrahim A , M. F. F. W , S. Abdul Hameed S , Shah Abdul Majid A

Abstract

The genomic study of hazardous human parasites forinstant, Aedes aegypti can improve our understandingof the disease vectors and help us to control the lethaldiseases such as dengue fever and other transmitteddiseases. The present study reported the amplification,cloning and determination of nucleotide sequences ofthe gene that encodes for DEAD box ATP-dependentRNA helicase, from Ae. aegypti. Totally, 1415 bplength of DNA segment from Ae. aegypti was amplifiedand cloned, which was found complementary tosequences at the 5´ end of the putative RNA helicasemRNA. Upstream sequences of the PCR primer at the3´ end of our amplified DNA fragment matches exactlywith the first 346 nt of the 5´ end of putative mRNA.The matched sequences consist of 297 bp exon and49 bp untranslated region. Upstream of the matchingsequences were comprised of another untranslatedregion (1048 bp), which may presumably be thepromoter of the gene having nt sequences at -419position, complementary to the TATA box.

Introduction

The mosquito Aedes aegypti is an important vector ofarbovirus pathogens, such as dengue fever and otherdiseases. According to the World Health Organization(WHO, 2006), and reports from recent studies, about2.5 billion people live in regions where transmission ofdengue virus occurs (1). This makes dengue anincreasingly important public health concern for whichno effective therapy currently exists (2).Dengue fever is caused by four closely related virusserotypes designated; DEN-1, DEN-2, DEN-3 andDEN-4 of the genus Flavivirus and family Flaviviridae.Dengue fever (DF) is characterized by fever andbleeding disorders, all of which could progress to highfever, shock and death in extreme cases. DF is a fastgrowing public health problem in tropical andsubtropical countries where the greater part of theworld’s population reside (3). The advent of genomicscan has opened new ways of helping us understandliving organisms better including understanding the

biology Aedes aegypti. For example, elucidating someof the important features of vector capability canimprove our understanding of the mosquito and itsassociation with etiological agents. Characterization ofgenes in mosquitoes could also help in unravelling themechanisms involved in resistance that could lead tothe development of novel control strategies of thedisease vector (4). This makes a vital demand todevelop and control these diseases and their vectors.The access to molecules of potential therapeuticinterest has long been a matter of great concern. Up tothe present moment, no anti-DENV drug has beenreported (5 & 6). The alternative strategy forcombating dengue fever is to identify low molecularweight molecules that could selectively block thefunction of the proteins encoded by these viruses.These molecules control the intracellular traffic ofDENV proteins in the infected cell. Currently thesestrategies are under investigations (7 & 8). Given thedifficulties of finding a vaccine for dengue fever, themajor method of controlling vector-borne diseases isstill by elimination of their vectors. Because of this,epidemiological and entomological studies are neededorder to develop and deliver solutions, which canrespond to the main risk concerns of dengue.Extensive studies have identified enzymes that areable to unwind complementary strands of a duplexnucleic acid and it’s known as helicases. RNA helicaseproteins are involved in several aspects of RNAmetabolism, including transcription, pre-mRNA splicing,ribosome biogenesis and cytoplasm transport (9, 10and 11). Helicases are a diverse class of enzymes thathave the ability to unwind nucleic acid duplexes with aseparate directional polarity. The nucleic acids in thecells often have to be unwound so that cellularprocess may proceed using the information found inthese modules (12 & 13). Since the discovery of theearliest DNA helicase in Escherichia coli in 1976 andthe earliest detection of eukaryotic ones in Liliaceae(Lily) in 1978, multiple DNA helicases have beenisolated from different organisms. A large number ofthese enzymes have been isolated from bothprokaryotic and eukaryotic cells and the number is stillincreasing (14). The efforts in sequencing of Ae.aegypti DNA were planned to provide newopportunities for research into insecticides andpossible genetic modifications to prevent the spread ofarboviruses. For Ae. aegypti a lot of research needs to



be done to understand the mosquito biology,physiology, biochemistry and behaviour. There is aneed to identify those targets, which can be used tomodify the insects’ ability to survive; the RNA helicasegene is recommended as one of the targets for geneticmanipulation of Ae. aegypti mosquito. Therefore, theaim of this study was to amplify and clone the genomicsequences coding for RNA helicase gene from Ae.aegypti, and to determine the nucleotide sequencesRNA helicase from Ae. aegypti.

Methods

Stocks of Aedes aegypti mosquito and bacteriaAedes aegypti mosquito larvae were kindly providedby Vector Control and Research Unit, Universiti SainsMalaysia. They were kept at 25°C and fed onpowdered bovine liver. The E. coli strain JM109 usedas the host cells for plasmid transformation was grownin Luria-Bertani agar (LA) or LB broth at 37°C. Thisresearch project was conducted from January 2007 toJune 2010.Ae. aegypti DNA extractionDNA extraction was performed using 4th instar larvaeof Ae. aegypt i by a mod i f i ca t ion o f thephenol/chloroform purification method of Jowett (15).Typically, 0.5 g of larvae were treated with 7.5 ml coldcell lysis buffer pH 8.0 ( 0.1M Tris-HCl, 50 mM EDTA,50 mM NaCl, 1% SDS, 0.15 mM Spermine, 0.5 MmSpermidine). The samples were treated initially usingproteinase K to final concentration of 100µg/ml todigest all protein matter followed by furtherhomogenization. RNA was removed by the addition ofRNase to a final concentration 500 µg/ml. DNA wasvisualized by staining with ethidium bromide solution inaccordance with Sambrook et al. (16) standardprocedure.PCR Optimization and reaction conditionsPolymerase chain reaction (PCR) was originallyintended to amplify the genomic sequences thatencode for the Aedes aegypti ´GST 2´ protein. Primersused were designed based on the nucleotidesequence of the GST 2 cDNA deposited at (NCBIGene Bank accession number: AF 384858). Thecomposition of the reaction mixture contained genomicDNA of Aedes aegypti, PCR Buffer, MgCl2, dNTP mix,DNA specific primers and Taq DNA polymerase in avolume 25 µl (Table 1.). Optimizations of PCR wereperformed using 50, 100, 150 and 200 ng DNArespectively. Typically PCR cycles were: First,denaturation at 95°C for 2 minutes to achievecomplete separation of DNA strands, annealingtemperature (between 40 – 5 50°C) for 30 seconds

and elongation at 72 °C for 1 minute. The PCR wasperformed for 35 cycles. Concentration of primers wasoptimized between 20 pmol to 40 pmol to obtain bestresult. MgCl2 concentration was varied between 0.5mM to 5 mM to optimize the PCR condition.Cloning of PCR-amplified DNA fragment The PCR product DNA was ligated to pGEM-T Easyvector (Promega) using the molar ratio 3:1 (insert:vector). The ligation mixture consisted of 2X rapidligation buffer, 3 Weiss units T4 ligase, 50 ng pGEM-TEasy vector DNA, 25 ng insert DNA and deionizedwater to make the volume 10 µl according to thestandard procedure. The transformation mixture wasplated to screen for colonies on LB agar, 0.5 mM IPTG,80 µg/ml X-Gal and 100µg/ml ampicillin. Singlecandidate colony was isolated and the plasmid fromcandidate colonies verified. The purified plasmidswere sent for sequencing to 1st Base laboratory.Digestion of plasmid DNA using restrictionendonucleases Restriction endonucleases were purchased fromPromega. The final volume for the digestion used was20 µl. One tenth volume of 10X reaction buffer, onetenth volume of BSA (1 mg/ml) and 0.5 µg of the DNAto be digested together with 19.5 µl of water wasadded to make the final volume of 20 µl were placedinto a 1.5 ml microcentrifuge tube. Finally 0.5 µl or 1 µlof enzyme was added to the reaction mixture.Components were mixed and the mixture was thenincubated at an optimal temperature of 37°C for 4.0hour according to the supplied instruction.The sequencing analysis of DNA fragmentsBasic alignment search tool Blast was used tocompare query sequences with those contained inNational Center for Biotechnology informationdatabases (NCBI), using BLASTN. In the subsequentstep a comparative analysis of the amino acidsequence of the desired fragments was done using aBLASTX comparison of the sequences in the NCBIdatabase. Because full-length sequences are notavailable, thus amino acid provided by NCBIdatabases was used as predicted template foralignment with homology proteins. The sequencetranslated amino acid sequence was obtained usingNCBI Blast Fasta. The helicases gene was searchedwith ( Inparanoid-gene search) al ignmenthttp://inparanoid51.sbc.su.se/cgi-bin/blast_search.cgiin 5/29/2009.

Results

Optimization of PCR conditions



Temperature optimization Several PCR reactions were performed to ascertainthe optimum PCR conditions to obtain amplification oftarget DNA. The annealing temperature was optimizedin the range between 40-50°C. Fig. 1A and B showedPCR cared out at 41°C and 42°C and showed severalbands and appearance of smears. PCR performed atother annealing temperatures did not improve theappearance of amplification products. However,observation of smears on the agarose gelelectrophoresis showed that other parameter like theMgCl2 concentrations had to be optimized. Theamplification product of 1400 bp was observed moreclearly at 41°C and 42°C temperature, indicating that itwas the optimum temperature for obtaining amplifiedproduct.Determination of suitable primers for DNAamplificationSeveral PCR reactions were performed using differentcombinations of primers. Eventually the best resultwas obtained using Aegt 083 and Aegt 916r primers ata concentration of 30 pmol and 10 pmol respectively(Figure 2.A). Reaction using Aegt 083 and Aegt 916rproduced two main fragments estimated at 1.4 kb and0.6 kb. The amount of DNA used was also varied tooptimize PCR conditions. It was found that using 150ng DNA amount produced the best results (Figure2.B). Other combination of primers either producednon-specific bands or smearing (Figure 3.).Optimization of MgCl2 concentration in PCRPrevious optimization of PCR conditions showed thatthe optimum annealing temperature was 40°C and theopt imum amount of DNA was 150ng. Theconcentration of MgCl2 was also optimized by using0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM,3.5 mM, 4.0 mM, 4.5 mM and 5.0 mM as shown inFigure 4. Two fragments 1.4 and 0.6 kb were clearlyvisible in the PCR product (lane 9) with 2.5 mM ofMgCl2. Only a faint band of the fragment 1.4 kb wasseen in lane 13 using 4.5 mM of MgCl2. Lanes 6, 7, 12and 14 with 1.5, 2.0, 4.0 and 5.0 mM of MgCl2respectively did not produce any DNA fragments.Lanes 4, 5, 10 and 11 resulted in smearing with 0.5,1.0, 3.0 and 3.5 mM of MgCl2 respectively as shownFigure 4.Sequence analysis of the 1400 bp DNA fragmentThe sequences containing the 5´ terminus, 3´ terminusof the full sequence of the 1.4 kb amplified fragment(Figure 5.). The actual size of the DNA fragment is1415nt. Analysis using BLASTN and BLASTX toolsrevealed that the alignments of the sequence showedthat it exactly matches 102 amino acids with the cDNAencoding Aedes aegypti RNA helicase (EMBL:AAEL004456-RA). The 3´ end sequence of the

amplified fragment exactly matches the first 346 nt ofthe 5´ end of the Aedes aegypti gene encoding theDEAD box ATP-dependent RNA helicase (Figure 6.).The matched sequences consist of 279 exonsequences and 49 5’ untranslated sequences. Inaddition upstream of the matching sequence of RNAhelicase gene, there are 1048 untranslated sequencesthat presumably include the promoter of the gene. Thepromoter does not appear to contain a TATA box atposition -50 that is observed for many genescontaining TATA box. However, at position -419 thereis a sequence that matches a TATA box. The twosymmetric elements motif sequences (CAAC andCTTC) appeared at six positions in the promoter viz.-432, -344, -331, -276, -183 and -98 (Figure 6.). Thesymmetric elements are probably binding siteselements that help promoter activities. The GC- box(CCGGCC) showed at position -232 and +38respectively, is most probably the Sp1 binding site. Inaddition another GC- box (CCGCCCG) appeared atthe position -74. Aedes aegypti RNA helicase geneputative GC-boxes motifs could help promotermachinery in transcription.

Discussion

Cloning the Aedes aegypti RNA helicase geneThe original aim of this study was to clone andcharacterize the Aedes aegypti GST-2 gene. Primerswere designed from the sequence of the GST-2 cDNA(NCBI Gene Bank accession number: AF 384858).Two DNA fragments of approximate size 1.4 kb and0.6 kb were amplified. The reaction product wassuccessfully cloned into the TA cloning vector pGEM-Teasy vector and sequenced. However, BLASTanalysis of the nucleotide sequence of the 1.4 kb DNAfragment showed that a 300 bp of the cloned DNAmatches exactly the 5´ end of a cDNA suggested asRNA helicase gene of Aedes aegypti. This suggestedthat a partial sequence of the Aedes aegypti RNAhelicase gene was serendipitously discovered. To datethere is no record of any publication on the Aedesaegypti RNA helicase gene. However, the primer Aegt0 8 3(5’-CTCGCAGCTCTACCTGATCC-3’)-(5’-GGATCAGGTAGAGCTGCGAG-3’) were found in both direction.To have explanation of the process above; one of theimportant factors of a PCR system are the primers,which designed to bind to desired segment of the DNAand amplify the target region. During the PCRannealing cycle, PCR primers anneal to thecomplementary region of the DNA. The polymerase



binding enable the synthesis of DNA to continue. PCRamplification needs two primers matching to thebeginning and the end of the DNA segment of thetemplate in opposite direction. When genomic DNA isused for PCR amplification, the complementaryprimers bind the template with perfect match. Ifcomplementary template is not present, the primersmay bind the genomic DNA with mismatch, andhomoeologous sequences from different related DNAsegment may be amplified (17).Degree of homology among helicasesBased on the cluster homology of gene conservedregion sequences, analysis of genomic sequence datahas allowed the identification of considerable numbersof open reading frames containing some or all of thecharacteristic helicase motifs and has allowedclassification of the respective gene products into oneof the helicase classes. Until now, the question of thestructure of the RNA helices genes belonging toAedes aegypti, and subgroping of these genesaccording to the distribution of their intron length hasyet to be elucidated.The alignment of all annotated sequences of DEADbox proteins in SwissProt (from all species) hasrevealed the presence of nine conserved sequencemotifs with very little variation. Each of the familiescould have specific variations on the conservedsequence motifs. Subsets of helicase proteins familiesshare short conserved amino acid sequencefingerprints (18). A study of all sequenced genomesincluding those of human, Drosophila, Caenorhabditis,Saccharomyces and Arabidopsis revealed that eachspecies has large number of genes belonging to theDEAD-box superfamily (19).A study of RNA helicase families in three speciesnamely Arabidopsis thaliana, Caenorhabditis elegansand Drosophila melanogaster showed that they have55, 32, and 29 helicase genes respectively (20).These genes were found by analyzing the ESTdatabase of each species. A large majority of RNAhelicase genes in these three species weretranscribed while others may be pseudogenes.Subgroups of homologous genes were clearlyidentified based on their intron patterns. It would beinteresting to see if Aedes aegypti RNA helicase genewould fall into the subgroups like the genes from thethree organisms above.Helicases in Drosophila and Aedes aegyptiThe RNA helicases of Drosophila are obviously a largeand complex family. Campbell et al (21) suggestedthat model organisms, such as Drosophila, and otherorganisms like Aedes aegypti together need to befurther investigated, and to find the relationship in RNAhelicase genes across mosquito species and

Drosophila. For some members of the RNA helicase family,genetic analysis has allowed a function to be assumed.In Drosophila there is a group of dif ferentATP-dependent RNA helicase genes, such as Vasa.This gene is similar to the eukaryotic initiation factor4A (eIF4A) (22 and 23). A separation between ATPhydrolysis and RNA unwinding was observed for theprotein product of D. melanogaster Vasa (24). Othergenes coding for ATP-dependent RNA helicases arealso found in Drosophila, such as genes encoding foran RM62 protein, which is similar to the humannuclear antigen p68 (25). De-Valoir et al. (26) isolateda Drosophila ME31B gene that has an mRNAexpression pattern to some extent similar to that ofVasa and also encodes a DEAD box protein. Thisgene ME31B reflected its maternal (ovarian germ-line)expression.In this study partial Aedes aegypti RNA helicase genesequences were obtained. The gene was identified byBLAST analysis. Sequence alignment acrossmembers of homologous proteins predicted that theRNA helicase gene AAEL004456-PA [Aedes aegypti]is a member of the mitochondrial DEAD BOX 28 family.The family DEAD BOX 28 contains DDX28EC_3.6.1.-mitochondrial DEAD BOX28 [Drosophilamelanogaster] and the DDX28 [Homo sapiens]. Thesetwo proteins are probably ATP dependant RNAhelicase. This DEAD box protein family is named asDDX28 DEAD (Asp-Glu-Ala-Asp) box polypeptide 28(27). The genes in this family are intronless, it encodesan RNA-dependent ATPase. The sequence of theAedes aegypti RNA helicase gene cloned in thecurrent study also does not possess any intron thus far,suggesting that it may also belong to the family DEADBox DDX28 family. The Drosophila and humanproteins have been localized in the mitochondria andthe nucleus, which indicates that the protein istransported between the mitochondria and the nucleus.Based on their distribution patterns, some membersare assumed to be concerned in embryogenesis,spermatogenesis, and cellular growth and division (27)

Conclusion(s)

Ae. aegypti has been and will stay as one of the mostintensely studied mosquito species because of itsassociation with human diseases. A sequence of oneof the Aedes aegypti RNA helicase gene has beenobtained. This gene is new and has not been clonedbefore and does not appear in any of the geneticdatabases such as NCBI and EMBL. Although, a 1.4



kb fragment of this gene has been cloned andcharacterized, sequences flanking the 3’ and 5’ endsof this gene need to be studied. The 3’ endssequences will reveal the complete coding sequencesand also the intron-exon structure of this gene.Cloning and characterization of the 5’ ends will allowcharacterization of the gene promoter. Until now, thequestions of the expression pattern and structure ofthe RNA helicase genes belonging to Aedes aegyptiare not completed. Further studies are needed tocharacterize other genes in the RNA helicases familiesand to determine their functions.

Abbreviation(s)

LIST OF ABBERVIATIONS

λ Lambda

acc. no. Accession number

BLAST Basic Local Alignment and Search Tool

ATP Adenosine triphosphate

bp Base pair

dNTP Deoxynucleotide triphosphate

EDTA Ethylendiamineteraacetic

IPTG Isopropyl β-thiogalactopyranoside

kb Kilo base pair

M Molar

ntORF

NucleotideOpen reading frame

PCR Polymerase chain reaction

RNA Ribonucleic acid

Acknowledgement(s)

We would like to thank School of Distance Education,School of Biological Sciences and School ofPharmaceutical Sciences in USM to the greatassistance.

Authors Contribution(s)

1.Mr. Ahmad H. Ibrahim was responsible forconducting the experimental lab work as a part of hisM.Sc. project, and also writing the paper. Ph.D studentat School of Pharmaceutical Sciences Universiti SainsMalaysia 11800, Minden, Penang, Malaysia, Mp:+60174492335, Qualifications: B.Sc. (BaghdadUniversity), M.Sc. (Universiti Sains Malaysia), e-mail:[email protected]. Mustafa F. F. Wajidi was the main supervisor.Department of Molecular Biology, Distance EducationSchool, Universiti Sains Malaysia, Malaysia, 11800Minden, Penang, Malaysia. B.Sc. (Nottingham) Ph.D.(Newcastle upon Tyne), e-mail: [email protected] Tel :+604-653231.3.Dr. Amin M. S. Abdul Majid has contributed in editing

the manuscript and also assisted partially in thefinancial support. School of Pharmaceutical SciencesUniversiti Sains Malaysia 11800, Minden, Penang,Malaysia, Tel: +6046534582, Fax: +6046534582, Mb:+60124230842. Qualifications: B.Sc. (Auckland),M.Sc., Ph.D. (New South Wales), e-mai l :[email protected] 4.Sawsan S. Abdul Hameed, School of IndustrialTechnology, Universiti Sains Malaysia, Malaysia,11800 Minden, Penang. Qualifications: B.Sc.(Baghdad University), (M.Sc.) from Universiti SainsMalays ia , Te l : +6017-449-2933, emai l :[email protected] research project was conducted from January2007 to June 2010

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1

Figures

A)

B)Figure 1. PCR temperature optimization. The PCR products were electrophoresed in

1.0 % agarose gel. The PCR product appears with smearing and manyunspecific products available.

Illustrations

Illustration 1

Figures



2

A) PCR amplification was performed at 42 °C.Lane 1: 1 kb marker (Promega).Lanes 2 and 3: The negative controls without DNA andprimers respectively.Lanes 4, 5 and 6 are PCR with 200 ng, 150 ng and 100 ng

DNA respectively.Lane 7: PCR with 50 ng DNA.B) PCR amplification was performed at 41 °C. Lane 1: marker (Promega).Lanes 2 and 3 are the negative controls without DNA andprimers respectively.Lane 4: PCR with 50 ng DNA.Lanes 5, 6 and 7 are PCR with 100ng , 150ng and 200 ngrespectively.



3

(A) (B)

Figure 2. Determination of suitable primers for DNA amplification. The annealingtemperature used was 40°C. The PCR products were electrophoresed in1.0 % agarose gel. The combination primers used were Aegt 083 & Aegt916r.



4

A) The combination of primers at concentration 30 pmol Aegt 083& 10pmol Aegt 916r respectively. Lanes 1 and 8 are 1 kb and 100bp markers respectively (Fermentas).Lanes 2 and 3 are the negative controls without DNA and primersrespectively.Lane 4 is PCR with 50 ng DNA.Lanes 5, 6 and 7 are PCR with 100, 150 and 200 ng DNA respectively

B) The combination of primers at concentration 10pmol Aegt 083 & 30pmol Aegt 916r respectively.

Lanes 1 and 2 are 1 kb and 100bp markers respectively (Fermentas).Lanes 3, 4, 5 are PCR products with 50, 100 and 150 ng DNA respectively.Lane 6 is PCR with 200 ng DNA.Lanes 7 and 8 are the negative controls without DNA and primers

respectively.



5

Figure 3. Determination of suitable primers for DNA amplification. The annealingtemperature was 40°C. The PCR products were electrophoresed in 1.0 %agarose gel. The combination of primers was Aegt 083 & Aegt 916r.



6

The combination of primers used at concentration 40 pmol & 20 pmolrespectively. Lanes 1& 8: 1kb the molecular size marker (Fermentas). Lanes 2 and 3: the negative controls without DNA and primersrespectively. Lanes 4, 5, 6 and 7: PCR with 50, 100, 150 and 200 ng DNArespectively.

The combination of primers used at concentrations of 20 pmol & 40pmol respectively.Lanes 9 and 10: the negative controls without DNA and primersrespectivelyLanes 11, 12, 13and 14: PCR with 50, 100, 150 and 200 ng DNArespectively.



7

Figure 4. The effect of MgCl2 concentration on the specificity of PCR.



8

Lanes 1 and 8 is 1kb molecular weight marker (Fermentas).Lanes 2 and 3 are negative controls without DNA and primersrespectively.Lane 9: PCR reaction with 2.5 mM MgCl2. Lane13: PCR with 4.5 mM MgCl2.Lanes 4, 5, 10 and 11 concentration of MgCl2. Lanes 6, 7, 12 and 14 concentration of MgCl2.



9

Sequence of DNA insert with 1415 nucleotides.

-1047 CTCGCAGCTCTACCTGATCCAAGCTTGGCGTTGATAACCGTTCGGTT

-1000 CAATTGCTCCTCTATTTTCTATCTCCGTATTGAACTGCAGCCGTGAATGA

-951 TTTTCTGAAAACTTTAATGATTAATTCCTTTAAATATAATGGAATTTAAG

-901 AATATTTACCTGAGAACACAGAAGAAAAACGAGAAAAACTAGCCGACGC

-851 AATTAGCAACAAAACTGAAGTTTTGACGTCTTCAATGAATACACCGAACA

-801 AAATTACCTCGCATTAATCGGGCTTAGTTTAGAATACTAATATGAAATTG

-751 TATACTTCGAAAACATGACCAAGTATACCTTAACATAATATCAAAAATGA

-701 GTGAATTTTAAACATTTCACGATTTATATTATTTCTGAGTGCACTATGGC

-651 CAAATGTTTGCACAATTAAGCTAGGTATGCTGTTCCGCTCAAGAAAAAAA

-601 AATCTGCTCAAGAAATGGTCAAGAAATTTCCTACAGTGAGCTCCATTTGA

-551 ATTGACATTTCTTTTCAACTGCGTACTGCGATCAAAGAAAAATGCTTTTC

-501 TTGAGCATTTCCTGAAAGAAATTTCCCACGCATCCGAGATAGGCGATTAC

-451 TATTCTGTTGAAGTGCATACTTCGCTTTATTCTATAAAATCAGTGAAAAT

-401 TTATTCACATTCTATTATTTTCACAATTTTGATGATCATTCTTTCGTAGT

-351 AACTTTAACAACCACAAAGGTCTTCAAAAAAGCTTATACAAAATGTCAAT

-301 AATTTATAGTTCAATAAACTATCCTTCATTGTCTACAAGATGCCTCCAGG

-251 ATAGGCCAAGAAATTTACTTTTAGCTGAGAATCCGGCCTAATATATTCCA

-201 AAAAGTTCACGTAATTCCAACCTGTGGGGTTATGATACACACAGGAACCAWebmedCentral > Research articles Page 16 of 20


10

Figure 5. Total sequences of 1.4 kb DNA fragment insert. The primer Aegt 083(5’-CTCGCAGCTCTACCTGATCC-3’)-(5’-GGATCAGGTAGAGCTGCGAG-3’)served as forward and reverse primers. The highlighted sequences representsequences that match the canonical sequences of a TATA-box. Putative GC-boxesare shown in bold.



11

(A)

E ScoreSequences producing significant alignments: (Bits) Value

gi|108879921|gb|EAT44146.1| DEAD box ATP-dependent RNA helicase 138 8e-31gi|19920478|ref|NP_608544.1| CG3561-PA [Drosophila melanogast... 40.0 0.30gi|24581136|ref|NP_722809.1| CG31684-PA [Drosophila melanogas... 37.7 1.5gi|68128913|emb|CAJ06141.1| hypothetical protein, conserved [Lei 37.0 2.5gi|54645062|gb|EAL33802.1| GA17520-PA [Drosophila pseudoobscura 37.0 2.5gi|68479999|ref|XP_716047.1| hypothetical protein CaO19_2207 ... 35.8 5.7gi|83273476|ref|XP_729415.1| hypothetical protein PY01631 [Pl... 35.0 9.7



12

(B)

Figure 6:A) BlastXresults forthe 1.4 kbamplifiedfragment.

B) Amino acid sequence similarity between the 1.4 kb amplified DNA fragment anda cDNA encodes a RNA helicase in Aedes aegypti.

gi|108879921|gb|EAT44146.1| DEAD box ATP-dependent RNA helicase [Aedes aegypti]Length=508Score = 138 bits (347), Expect = 8e-31 Identities = 83/100 (83%), Positives = 85/100 (85%), Gaps = 1/100 (1%) Frame = +3

Query 1098 MLKTVKVLFGFRTNFSSTAIFTTTTPRKPIISCRRAQHNLFVEHYPLGTEGGGGKKFGDV 1277 MLKTVKVLF FRTNFSSTAIFTTTTPRKPIISCRRAQHNLFVEHYPLGTEGGGGKKFGDVSbjct 1 MLKTVKVLFSFRTNFSSTAIFTTTTPRKPIISCRRAQHNLFVEHYPLGTEGGGGKKFGDV 60

Query 1278 PLASAGWRHRKSVGDYFIIRCSNHTRHIH-LAMFPFKVSG 1394 PLASAGWRHRKSVGDYFII H L+ PF+ GSbjct 61 PLASAGWRHRKSVGDYFIIHPVQQPYETHSLSDVPFQSLG 100

Score = 52.4 bits (124), Expect = 6e-05 Identities = 23/23 (100%), Positives = 23/23 (100%), Gaps = 0/23 (0%) Frame = +2

Query 1331 HPVQQPYETHSLSDVPFQSLGIR 1399 HPVQQPYETHSLSDVPFQSLGIRSbjct 80 HPVQQPYETHSLSDVPFQSLGIR 102



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