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On-line version ISSN 1678-8060
Mem. Inst. Oswaldo Cruz vol.100 no.6 Rio de Janeiro Oct. 2005
VECTOR GENETIC STUDIES
C GentileI; JBP LimaII,III; AA PeixotoI,1
IDepartamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brasil
IIDepartamento de Entomologia, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brasil
IIIInstituto de Biologia do Exército, Rio de Janeiro, RJ, Brasil
The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.
Key words: Anopheles aquasalis - Aedes aegypti - Culex quinquefasciatus - Drosophila constitutive gene - rp49 - rpL32 - mosquitoes
The ribosomal protein 49 gene (rp49) of the fruitfly Drosophila (O'Connell & Rosbash 1984), also known as rpL32 (http://flybase.bio.indiana.edu), has been widely used as an endogenous constitutive control in gene expression studies (e.g. Glossop et al. 1999, Goodwin et al. 2000, Kurapati et al. 2000, Stanewsky et al. 2002). The sequence of rp49 is also available for the mosquito Anopheles gambiae (Holt et al. 2002), the most important Afrotropical malaria vector, but until recently this gene had not been sequenced from other species of mosquitoes. As part of our molecular studies of insect vectors of tropical diseases, we attempted to clone a rp49 homologous fragment in An. aquasalis, a widespread Neotropical malaria vector that is associated with coastal habitats (Consoli & Lourenço-de-Oliveira 1994, Fairley et al. 2002, Forattini 2002).
Specimens used in this work were derived from a laboratory colony of An. aquasalis established in 1993 with around 200 females collected in a farm in Paracambi, Rio de Janeiro, Brazil (Carvalho et al. 2002). Using kits supplied by Amersham Biosciences, genomic DNA was isolated by means of the GenomicPrep Cells & Tissue DNA isolation kit; mRNAs with the QuickPrep Micro mRNA purification kit and cDNAs were synthesized using the First-Strand cDNA synthesis kit. PCR was performed in 40 µl using Tth DNA polymerase (Biotools) according to manufacturer's directions using various cycling conditions (see below) and the primers listed in the Table. PCR products were purified using either the Micro Spin S-400 HR Column (Amersham Biosciences) or the Wizard SV Gel and PCR Clean-up System (Promega), and cloned using the pGEM-T Easy Vector Kit (Promega). DNA sequencing was carried out in an ABI377 Sequencer using the Big Dye 3.1 Kit (Applied Biosystems).
The first rp49 fragment from An. aquasalis was obtained using cDNA as template and degenerate primers based on conserved regions identified by comparison between the putative protein sequences of D. melanogaster and An. gambiae. Initially, we conducted PCR using primers 5rp49deg1 and oligo-d(T)20 and the following cycling conditions: 94ºC for 5 min; 15 cycles at 94ºC for 1 min, 55ºC (minus 1ºC each cycle) for 1 min and 72ºC for 2 min; then 20 more cycles at 94ºC for 1 min, 50ºC for 1 min and 72ºC for 2 min. Although no amplification products could be detected by 2% agarose gel electrophoresis, a fragment of ~320 base pairs was observed after reamplification of 1 µl of the first reaction using primers 5rp49deg1 and 3rp49deg3 (Fig. 1, Table), and 35 cycles at 94ºC for 1 min, 50ºC for 1 min, and 72ºC for 2 min. This product was purified, cloned and sequenced as described above. Homology to rp49 was confirmed by comparison to the Drosophila sequence database using BlastX (http://www.ncbi.nlm.nih.gov/).
Based on the An. aquasalis rp49 cDNA sequence obtained, a specific primer (5aquaRP1, Fig. 1, Table) was designed and used with degenerate primer 3rp49deg3 in a new PCR (94ºC for 5 min, followed by 35 cycles of 94ºC for 30 s, 50ºC for 30 s, and 72ºC for 1 min) to amplify an An. aquasalis rp49 400 bp genomic sequence. This fragment was cloned and sequenced. Comparison with the cDNA sequence confirmed the presence of an 88 bp intron, located in the same position as that of D. melanogaster.
Because primer 5aquaRP1 is in a conserved region, we tried to see if it could be used to isolate fragments of rp49 in other mosquito species. Aedes aegypti is a highly competent vector of dengue and urban yellow fever in Brazil and elsewhere (Lourenço-de-Oliveira et al. 2004); this mosquito originated from Africa and is now synanthropic (Alonso et al. 2003, Braga et al. 2004, Cunha et al. 2005) throughout the tropics (within the 20ºC isotherm) between latitudes 45º N and 35º S approximately (Christophers 1960, Forattini 2002). The tropical house mosquito Culex quinquefasciatus is the urban vector of lymphatic filariasis (White & Nathan 2002) and transmits arboviruses such as St. Louis and West Nile (Service 2001). The primer 5aquaRP1 was used, together with oligo-d(T)20, to amplify rp49 cDNA sequences from these two species as described above. As before, products were observed only after a reamplification reaction, using primers 5aquaRP1 and 3rp49deg3. Fragments of ~ 300 bp obtained from Ae. aegypti and Cx. quinquefasciatus were purified, cloned and sequenced. Homology to rp49 was again confirmed by comparison to the Drosophila sequence database.
Fig. 1 gives the alignment of RP49 proteins from D. melanogaster and An. gambiae compared to the deduced amino acid sequences obtained from the fragments we isolated from An. aquasalis, Ae. aegypti and Cx. quinquefasciatus (sequences submitted to the GenBank, accession numbers AY539746 to AY539748). As shown, the amplified region is highly conserved with only a few substitutions observed among the five sequences.
To illustrate the use of rp49 as an endogenous control in mosquitoes we designed a primer (5aquaexpRP) across the intron-exon boundary to specifically amplify cDNA sequences of An. aquasalis (Fig. 1, Table). Fig. 2 shows the results of PCR carried out using this and other primers. Lanes 1 and 2 show the amplification products obtained for An. aquasalis cDNA and genomic DNA, respectively, using the primers 5aquaRP1 and 3aeaquaRP1, flanking the intron that accounts for the size difference between the two fragments. Lane 3 is the negative control for these reactions. Lanes 4 and 5 show the results of PCR using primers 5aquaexpRP and 3aeaquaRP1 with the same templates and lane 6 is their respective negative control. Note that although some primer-dimer formation is observed in all three lanes, a 190 bp fragment corresponding to the expected size is amplified when cDNA, but not genomic DNA, is used as template. Similar results were obtained with Ae. aegypti using primers 5aeexpRP and 3aeaquaRP1b (data not shown).
Since rp49 has been frequently used as an endogenous constitutive control in gene expression studies in Drosophila, its homologues from An. aquasalis, Ae. aegypti, Cx. quinquefasciatus, and other vector species might provide useful tools in molecular studies of these medically important mosquitoes.
To Dr Denise Valle for comments on the manuscript, Paulo Roberto de Amoretty for his technical assistance, and Robson Costa da Silva for his help with the DNA sequencing.
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Received 14 July 2005
Accepted 24 August 2005
Financial support: Howard Hughes Medical Institute, Guggenheim Foundation, CNPq, Faperj, Fiocruz