Research articles

By Mr. Ahmad Ibrahim , Dr. Wajidi M. F. F. , Mrs. Sawsan S. Abdul Hameed , Dr. Amin Malik Shah Abdul Majid
Corresponding Author Mr. Ahmad Ibrahim
School of Pharmaceutical Sciences, University Sains Malaysia, 11800, Minden, Penang, Malaysia - Malaysia 118000
Submitting Author Mr. Ahmad H Ibrahim
Other Authors Dr. Wajidi M. F. F.
Department of Molecular Biology, - Malaysia

Mrs. Sawsan S. Abdul Hameed
School of Industrial Technology, - Malaysia

Dr. Amin Malik Shah Abdul Majid
Department Of Pharmacology, - Malaysia


Aedes aegypti, RNA Helicase, Amplification, Cloning, Characterization

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 MOLECULAR BIOLOGY 2010;1(10):WMC001094
doi: 10.9754/journal.wmc.2010.001094
Submitted on: 30 Oct 2010 10:11:51 AM GMT
Published on: 30 Oct 2010 10:27:56 PM GMT


The genomic study of hazardous human parasites for instant, Aedes aegypti can improve our understanding of the disease vectors and help us to control the lethal diseases such as dengue fever and other transmitted diseases. The present study reported the amplification, cloning and determination of nucleotide sequences of the gene that encodes for DEAD box ATP-dependent RNA helicase, from Ae. aegypti. Totally, 1415 bp length of DNA segment from Ae. aegypti was amplified and cloned, which was found complementary to sequences at the 5´ end of the putative RNA helicase mRNA. Upstream sequences of the PCR primer at the 3´ end of our amplified DNA fragment matches exactly with the first 346 nt of the 5´ end of putative mRNA. The matched sequences consist of 297 bp exon and 49 bp untranslated region. Upstream of the matching sequences were comprised of another untranslated region (1048 bp), which may presumably be the promoter of the gene having nt sequences at -419 position, complementary to the TATA box.



The mosquito Aedes aegypti is an important vector of arbovirus pathogens, such as dengue fever and other diseases. According to the World Health Organization (WHO, 2006), and reports from recent studies, about 2.5 billion people live in regions where transmission of dengue virus occurs (1). This makes dengue an increasingly important public health concern for which no effective therapy currently exists (2).
Dengue fever is caused by four closely related virus serotypes designated; DEN-1, DEN-2, DEN-3 and DEN-4 of the genus Flavivirus and family Flaviviridae. Dengue fever (DF) is characterized by fever and bleeding disorders, all of which could progress to high fever, shock and death in extreme cases. DF is a fast growing public health problem in tropical and subtropical countries where the greater part of the world’s population reside (3). The advent of genomics can has opened new ways of helping us understand living organisms better including understanding the biology Aedes aegypti. For example, elucidating some of the important features of vector capability can improve our understanding of the mosquito and its association with etiological agents. Characterization of genes in mosquitoes could also help in unravelling the mechanisms involved in resistance that could lead to the development of novel control strategies of the disease vector (4). This makes a vital demand to develop and control these diseases and their vectors.
The access to molecules of potential therapeutic interest has long been a matter of great concern. Up to the present moment, no anti-DENV drug has been reported (5 & 6). The alternative strategy for combating dengue fever is to identify low molecular weight molecules that could selectively block the function of the proteins encoded by these viruses. These molecules control the intracellular traffic of DENV proteins in the infected cell. Currently these strategies are under investigations (7 & 8). Given the difficulties of finding a vaccine for dengue fever, the major method of controlling vector-borne diseases is still by elimination of their vectors. Because of this, epidemiological and entomological studies are needed order to develop and deliver solutions, which can respond to the main risk concerns of dengue.
Extensive studies have identified enzymes that are able to unwind complementary strands of a duplex nucleic acid and it’s known as helicases. RNA helicase proteins are involved in several aspects of RNA metabolism, including transcription, pre-mRNA splicing, ribosome biogenesis and cytoplasm transport (9, 10 and 11). Helicases are a diverse class of enzymes that have the ability to unwind nucleic acid duplexes with a separate directional polarity. The nucleic acids in the cells often have to be unwound so that cellular process may proceed using the information found in these modules (12 & 13). Since the discovery of the earliest DNA helicase in Escherichia coli in 1976 and the earliest detection of eukaryotic ones in Liliaceae (Lily) in 1978, multiple DNA helicases have been isolated from different organisms. A large number of these enzymes have been isolated from both prokaryotic and eukaryotic cells and the number is still increasing (14). The efforts in sequencing of Ae. aegypti DNA were planned to provide new opportunities for research into insecticides and possible genetic modifications to prevent the spread of arboviruses. For Ae. aegypti a lot of research needs to be done to understand the mosquito biology, physiology, biochemistry and behaviour. There is a need to identify those targets, which can be used to modify the insects’ ability to survive; the RNA helicase gene is recommended as one of the targets for genetic manipulation of Ae. aegypti mosquito. Therefore, the aim of this study was to amplify and clone the genomic sequences coding for RNA helicase gene from Ae. aegypti, and to determine the nucleotide sequences RNA helicase from Ae. aegypti.


Stocks of Aedes aegypti mosquito and bacteria
Aedes aegypti mosquito larvae were kindly provided by Vector Control and Research Unit, Universiti Sains Malaysia. They were kept at 25°C and fed on powdered bovine liver. The E. coli strain JM109 used as the host cells for plasmid transformation was grown in Luria-Bertani agar (LA) or LB broth at 37°C. This research project was conducted from January 2007 to June 2010.
Ae. aegypti DNA extraction
DNA extraction was performed using 4th instar larvae of Ae. aegypti by a modification of the phenol/chloroform purification method of Jowett (15). Typically, 0.5 g of larvae were treated with 7.5 ml cold cell lysis buffer pH 8.0 ( 0.1M Tris-HCl, 50 mM EDTA, 50 mM NaCl, 1% SDS, 0.15 mM Spermine, 0.5 Mm Spermidine). The samples were treated initially using proteinase K to final concentration of 100μg/ml to digest all protein matter followed by further homogenization. RNA was removed by the addition of RNase to a final concentration 500 μg/ml. DNA was visualized by staining with ethidium bromide solution in accordance with Sambrook et al. (16) standard procedure.
PCR Optimization and reaction conditions
Polymerase chain reaction (PCR) was originally intended to amplify the genomic sequences that encode for the Aedes aegypti ´GST 2´ protein. Primers used were designed based on the nucleotide sequence of the GST 2 cDNA deposited at (NCBI Gene Bank accession number: AF 384858). The composition of the reaction mixture contained genomic DNA of Aedes aegypti, PCR Buffer, MgCl2, dNTP mix, DNA specific primers and Taq DNA polymerase in a volume 25 μl (Table 1.). Optimizations of PCR were performed using 50, 100, 150 and 200 ng DNA respectively. Typically PCR cycles were: First, denaturation at 95°C for 2 minutes to achieve complete separation of DNA strands, annealing temperature (between 40 – 5 50°C) for 30 seconds and elongation at 72 °C for 1 minute. The PCR was performed for 35 cycles. Concentration of primers was optimized between 20 pmol to 40 pmol to obtain best result. MgCl2 concentration was varied between 0.5 mM to 5 mM to optimize the PCR condition.
Cloning of PCR-amplified DNA fragment
The PCR product DNA was ligated to pGEM-T Easy vector (Promega) using the molar ratio 3:1 (insert: vector). The ligation mixture consisted of 2X rapid ligation buffer, 3 Weiss units T4 ligase, 50 ng pGEM-T Easy vector DNA, 25 ng insert DNA and deionized water to make the volume 10 μl according to the standard procedure. The transformation mixture was plated to screen for colonies on LB agar, 0.5 mM IPTG, 80 μg/ml X-Gal and 100μg/ml ampicillin. Single candidate colony was isolated and the plasmid from candidate colonies verified. The purified plasmids were sent for sequencing to 1st Base laboratory.
Digestion of plasmid DNA using restriction endonucleases
Restriction endonucleases were purchased from Promega. The final volume for the digestion used was 20 μl. One tenth volume of 10X reaction buffer, one tenth volume of BSA (1 mg/ml) and 0.5 μg of the DNA to be digested together with 19.5 μl of water was added to make the final volume of 20 μl were placed into a 1.5 ml microcentrifuge tube. Finally 0.5 μl or 1 μl of enzyme was added to the reaction mixture. Components were mixed and the mixture was then incubated at an optimal temperature of 37°C for 4.0 hour according to the supplied instruction.
The sequencing analysis of DNA fragments
Basic alignment search tool Blast was used to compare query sequences with those contained in National Center for Biotechnology information databases (NCBI), using BLASTN. In the subsequent step a comparative analysis of the amino acid sequence of the desired fragments was done using a BLASTX comparison of the sequences in the NCBI database. Because full-length sequences are not available, thus amino acid provided by NCBI databases was used as predicted template for alignment with homology proteins. The sequence translated amino acid sequence was obtained using NCBI Blast Fasta. The helicases gene was searched with (Inparanoid-gene search) alignment in 5/29/2009.


Optimization of PCR conditions
Temperature optimization
Several PCR reactions were performed to ascertain the optimum PCR conditions to obtain amplification of target DNA. The annealing temperature was optimized in the range between 40-50°C. Fig. 1A and B showed PCR cared out at 41°C and 42°C and showed several bands and appearance of smears. PCR performed at other annealing temperatures did not improve the appearance of amplification products. However, observation of smears on the agarose gel electrophoresis showed that other parameter like the MgCl2 concentrations had to be optimized. The amplification product of 1400 bp was observed more clearly at 41°C and 42°C temperature, indicating that it was the optimum temperature for obtaining amplified product.
Determination of suitable primers for DNA amplification
Several PCR reactions were performed using different combinations of primers. Eventually the best result was obtained using Aegt 083 and Aegt 916r primers at a concentration of 30 pmol and 10 pmol respectively (Figure 2.A). Reaction using Aegt 083 and Aegt 916r produced two main fragments estimated at 1.4 kb and 0.6 kb. The amount of DNA used was also varied to optimize PCR conditions. It was found that using 150 ng DNA amount produced the best results (Figure 2.B). Other combination of primers either produced non-specific bands or smearing (Figure 3.).
Optimization of MgCl2 concentration in PCR
Previous optimization of PCR conditions showed that the optimum annealing temperature was 40°C and the optimum amount of DNA was 150ng. The concentration of MgCl2 was also optimized by using 0.5 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 in Figure 4. Two fragments 1.4 and 0.6 kb were clearly visible in the PCR product (lane 9) with 2.5 mM of MgCl2. Only a faint band of the fragment 1.4 kb was seen in lane 13 using 4.5 mM of MgCl2. Lanes 6, 7, 12 and 14 with 1.5, 2.0, 4.0 and 5.0 mM of MgCl2 respectively 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 shown Figure 4.
Sequence analysis of the 1400 bp DNA fragment
The sequences containing the 5´ terminus, 3´ terminus of the full sequence of the 1.4 kb amplified fragment (Figure 5.). The actual size of the DNA fragment is 1415nt. Analysis using BLASTN and BLASTX tools revealed that the alignments of the sequence showed that it exactly matches 102 amino acids with the cDNA encoding Aedes aegypti RNA helicase (EMBL: AAEL004456-RA). The 3´ end sequence of the amplified fragment exactly matches the first 346 nt of the 5´ end of the Aedes aegypti gene encoding the DEAD box ATP-dependent RNA helicase (Figure 6.).
The matched sequences consist of 279 exon sequences and 49 5’ untranslated sequences. In addition upstream of the matching sequence of RNA helicase gene, there are 1048 untranslated sequences that presumably include the promoter of the gene. The promoter does not appear to contain a TATA box at position -50 that is observed for many genes containing TATA box. However, at position -419 there is a sequence that matches a TATA box. The two symmetric elements motif sequences (CAAC and CTTC) appeared at six positions in the promoter viz. -432, -344, -331, -276, -183 and -98 (Figure 6.). The symmetric elements are probably binding sites elements that help promoter activities. The GC- box (CCGGCC) showed at position -232 and +38 respectively, is most probably the Sp1 binding site. In addition another GC- box (CCGCCCG) appeared at the position -74. Aedes aegypti RNA helicase gene putative GC-boxes motifs could help promoter machinery in transcription.


Cloning the Aedes aegypti RNA helicase gene
The original aim of this study was to clone and characterize the Aedes aegypti GST-2 gene. Primers were 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 and 0.6 kb were amplified. The reaction product was successfully cloned into the TA cloning vector pGEM-T easy vector and sequenced. However, BLAST analysis of the nucleotide sequence of the 1.4 kb DNA fragment showed that a 300 bp of the cloned DNA matches exactly the 5´ end of a cDNA suggested as RNA helicase gene of Aedes aegypti. This suggested that a partial sequence of the Aedes aegypti RNA helicase gene was serendipitously discovered. To date there is no record of any publication on the Aedes aegypti RNA helicase gene. However, the primer Aegt 083 (5’-CTCGCAGCTCTACCTGATCC-3’)-(5’-GGATCAGGTAGAGCTGCGAG-3’) were found in both direction. To have explanation of the process above; one of the important factors of a PCR system are the primers, which designed to bind to desired segment of the DNA and amplify the target region. During the PCR annealing cycle, PCR primers anneal to the complementary region of the DNA. The polymerase binding enable the synthesis of DNA to continue. PCR amplification needs two primers matching to the beginning and the end of the DNA segment of the template in opposite direction. When genomic DNA is used for PCR amplification, the complementary primers bind the template with perfect match. If complementary template is not present, the primers may bind the genomic DNA with mismatch, and homoeologous sequences from different related DNA segment may be amplified (17).
Degree of homology among helicases
Based on the cluster homology of gene conserved region sequences, analysis of genomic sequence data has allowed the identification of considerable numbers of open reading frames containing some or all of the characteristic helicase motifs and has allowed classification of the respective gene products into one of the helicase classes. Until now, the question of the structure of the RNA helices genes belonging to Aedes aegypti, and subgroping of these genes according to the distribution of their intron length has yet to be elucidated.
The alignment of all annotated sequences of DEAD box proteins in SwissProt (from all species) has revealed the presence of nine conserved sequence motifs with very little variation. Each of the families could have specific variations on the conserved sequence motifs. Subsets of helicase proteins families share short conserved amino acid sequence fingerprints (18). A study of all sequenced genomes including those of human, Drosophila, Caenorhabditis, Saccharomyces and Arabidopsis revealed that each species has large number of genes belonging to the DEAD-box superfamily (19).
A study of RNA helicase families in three species namely Arabidopsis thaliana, Caenorhabditis elegans and Drosophila melanogaster showed that they have 55, 32, and 29 helicase genes respectively (20). These genes were found by analyzing the EST database of each species. A large majority of RNA helicase genes in these three species were transcribed while others may be pseudogenes. Subgroups of homologous genes were clearly identified based on their intron patterns. It would be interesting to see if Aedes aegypti RNA helicase gene would fall into the subgroups like the genes from the three organisms above.
Helicases in Drosophila and Aedes aegypti
The RNA helicases of Drosophila are obviously a large and complex family. Campbell et al (21) suggested that model organisms, such as Drosophila, and other organisms like Aedes aegypti together need to be further investigated, and to find the relationship in RNA helicase 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 different ATP-dependent RNA helicase genes, such as Vasa. This gene is similar to the eukaryotic initiation factor 4A (eIF4A) (22 and 23). A separation between ATP hydrolysis and RNA unwinding was observed for the protein product of D. melanogaster Vasa (24). Other genes coding for ATP-dependent RNA helicases are also found in Drosophila, such as genes encoding for an RM62 protein, which is similar to the human nuclear antigen p68 (25). De-Valoir et al. (26) isolated a Drosophila ME31B gene that has an mRNA expression pattern to some extent similar to that of Vasa and also encodes a DEAD box protein. This gene ME31B reflected its maternal (ovarian germ-line) expression.
In this study partial Aedes aegypti RNA helicase gene sequences were obtained. The gene was identified by BLAST analysis. Sequence alignment across members of homologous proteins predicted that the RNA helicase gene AAEL004456-PA [Aedes aegypti] is a member of the mitochondrial DEAD BOX 28 family. The family DEAD BOX 28 contains DDX28 EC_3.6.1.-mitochondrial DEAD BOX28 [Drosophila melanogaster] and the DDX28 [Homo sapiens]. These two proteins are probably ATP dependant RNA helicase. This DEAD box protein family is named as DDX28 DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 (27). The genes in this family are intronless, it encodes an RNA-dependent ATPase. The sequence of the Aedes aegypti RNA helicase gene cloned in the current study also does not possess any intron thus far, suggesting that it may also belong to the family DEAD Box DDX28 family. The Drosophila and human proteins have been localized in the mitochondria and the nucleus, which indicates that the protein is transported between the mitochondria and the nucleus. Based on their distribution patterns, some members are assumed to be concerned in embryogenesis, spermatogenesis, and cellular growth and division (27)


Ae. aegypti has been and will stay as one of the most intensely studied mosquito species because of its association with human diseases. A sequence of one of the Aedes aegypti RNA helicase gene has been obtained. This gene is new and has not been cloned before and does not appear in any of the genetic databases such as NCBI and EMBL. Although, a 1.4 kb fragment of this gene has been cloned and characterized, sequences flanking the 3’ and 5’ ends of this gene need to be studied. The 3’ ends sequences will reveal the complete coding sequences and also the intron-exon structure of this gene. Cloning and characterization of the 5’ ends will allow characterization of the gene promoter. Until now, the questions of the expression pattern and structure of the RNA helicase genes belonging to Aedes aegypti are not completed. Further studies are needed to characterize other genes in the RNA helicases families and to determine their functions.





acc. no.

Accession number


Basic Local Alignment and Search Tool


Adenosine triphosphate


Base pair


Deoxynucleotide triphosphate




Isopropyl β-thiogalactopyranoside


Kilo base pair






Open reading frame


Polymerase chain reaction


Ribonucleic acid


We would like to thank School of Distance Education, School of Biological Sciences and School of Pharmaceutical Sciences in USM to the great assistance.

Authors Contribution(s)

1.Mr. Ahmad H. Ibrahim was responsible for conducting the experimental lab work as a part of his M.Sc. project, and also writing the paper. Ph.D student at School of Pharmaceutical Sciences Universiti Sains Malaysia 11800, Minden, Penang, Malaysia, Mp: +60174492335, Qualifications: B.Sc. (Baghdad University), M.Sc. (Universiti Sains Malaysia), e-mail:
2.Dr. Mustafa F. F. Wajidi was the main supervisor. Department of Molecular Biology, Distance Education School, Universiti Sains Malaysia, Malaysia, 11800 Minden, Penang, Malaysia. B.Sc. (Nottingham) Ph.D. (Newcastle upon Tyne), e-mail: Tel : +604-653231.
3.Dr. Amin M. S. Abdul Majid has contributed in editing the manuscript and also assisted partially in the financial support. School of Pharmaceutical Sciences Universiti Sains Malaysia 11800, Minden, Penang, Malaysia, Tel: +6046534582, Fax: +6046534582, Mb: +60124230842. Qualifications: B.Sc. (Auckland), M.Sc., Ph.D. (New South Wales), e-mail:
4.Sawsan S. Abdul Hameed, School of Industrial Technology, Universiti Sains Malaysia, Malaysia, 11800 Minden, Penang. Qualifications: B.Sc. (Baghdad University), (M.Sc.) from Universiti Sains Malaysia, Tel : +6017-449-2933, email:
This research project was conducted from January 2007 to June 2010


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Source(s) of Funding

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

Competing Interests



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