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The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

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Expression analysis of the A. gambiae vasa orthologue AGAP008578. Tissue specific expression analysis using RT-PCR. We used cDNA from dissected adult male and female tissues including the head and thorax (HT), abdomen (AB) ovaries (OV) and testis (TE) to amplify mRNAs of the vasa gene and as control the nanos, beta2-tubulin genes and the S7 genes.
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Figure 1: Expression analysis of the A. gambiae vasa orthologue AGAP008578. Tissue specific expression analysis using RT-PCR. We used cDNA from dissected adult male and female tissues including the head and thorax (HT), abdomen (AB) ovaries (OV) and testis (TE) to amplify mRNAs of the vasa gene and as control the nanos, beta2-tubulin genes and the S7 genes.

Mentions: Using the D. melanogaster Vasa as a template, AGAP008578 was identified as the likely A. gambiae orthologue with an overall amino acid sequence identity of 49%. A likely orthology relationship with vasa was confirmed by a reverse blast of AGAP008578 against the D. melanogaster genome. High level sequence conservation was observed in putative RNA and ATP interacting residues. Regions spanning canonical DEAD-box RNA helicase family domains, including all motifs within the two tandemly repeated RecA-like domains, were the most conserved (see Additional File 1). N-terminal sequences, which are unique to vasa and Dep1p orthologues showed lower levels of conservation. The RNA-interacting residues arginine 403 and glutamine 497 that distinguish Vasa from other Drosophila DEAD box helicases [32] were also present in AGAP008578 within their conserved motifs. To establish the expression profile of AGAP008578 we performed reverse transcriptase-PCR (RTPCR) on total mRNA extracted from dissected adult tissues of wild type A. gambiae (G3 strain). The analysis demonstrated that AGAP008578 transcripts could only be found in testes and ovaries. All somatic tissues tested, including the head, thorax and abdomen from male and female mosquitoes, did not show detectable levels of transcript (Figure 1). We therefore concluded that AGAP008578 is the A. gambiae vasa-like gene; for simplicity this gene will be referred to as vasa for the remainder of this report. We established the organisation of the Anopheles vasa locus by combining available A. gambiae EST clusters (AnoEST), rapid amplification of cDNA ends (RACE) experiments (J. Meredith personal communication) and in-silico exon prediction. Together these data indicated that two alternative vasa transcripts are generated by the alternative utilization of either one of the first two exons both mapping in the 5' untranslated region (Figure 2A). Both types of transcript were found in all tissues of both sexes that show vasa expression (data not shown) and the significance of these alternative transcripts is unknown.


The vasa regulatory region mediates germline expression and maternal transmission of proteins in the malaria mosquito Anopheles gambiae: a versatile tool for genetic control strategies.

Papathanos PA, Windbichler N, Menichelli M, Burt A, Crisanti A - BMC Mol. Biol. (2009)

Expression analysis of the A. gambiae vasa orthologue AGAP008578. Tissue specific expression analysis using RT-PCR. We used cDNA from dissected adult male and female tissues including the head and thorax (HT), abdomen (AB) ovaries (OV) and testis (TE) to amplify mRNAs of the vasa gene and as control the nanos, beta2-tubulin genes and the S7 genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713240&req=5

Figure 1: Expression analysis of the A. gambiae vasa orthologue AGAP008578. Tissue specific expression analysis using RT-PCR. We used cDNA from dissected adult male and female tissues including the head and thorax (HT), abdomen (AB) ovaries (OV) and testis (TE) to amplify mRNAs of the vasa gene and as control the nanos, beta2-tubulin genes and the S7 genes.
Mentions: Using the D. melanogaster Vasa as a template, AGAP008578 was identified as the likely A. gambiae orthologue with an overall amino acid sequence identity of 49%. A likely orthology relationship with vasa was confirmed by a reverse blast of AGAP008578 against the D. melanogaster genome. High level sequence conservation was observed in putative RNA and ATP interacting residues. Regions spanning canonical DEAD-box RNA helicase family domains, including all motifs within the two tandemly repeated RecA-like domains, were the most conserved (see Additional File 1). N-terminal sequences, which are unique to vasa and Dep1p orthologues showed lower levels of conservation. The RNA-interacting residues arginine 403 and glutamine 497 that distinguish Vasa from other Drosophila DEAD box helicases [32] were also present in AGAP008578 within their conserved motifs. To establish the expression profile of AGAP008578 we performed reverse transcriptase-PCR (RTPCR) on total mRNA extracted from dissected adult tissues of wild type A. gambiae (G3 strain). The analysis demonstrated that AGAP008578 transcripts could only be found in testes and ovaries. All somatic tissues tested, including the head, thorax and abdomen from male and female mosquitoes, did not show detectable levels of transcript (Figure 1). We therefore concluded that AGAP008578 is the A. gambiae vasa-like gene; for simplicity this gene will be referred to as vasa for the remainder of this report. We established the organisation of the Anopheles vasa locus by combining available A. gambiae EST clusters (AnoEST), rapid amplification of cDNA ends (RACE) experiments (J. Meredith personal communication) and in-silico exon prediction. Together these data indicated that two alternative vasa transcripts are generated by the alternative utilization of either one of the first two exons both mapping in the 5' untranslated region (Figure 2A). Both types of transcript were found in all tissues of both sexes that show vasa expression (data not shown) and the significance of these alternative transcripts is unknown.

Bottom Line: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes.We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules.

View Article: PubMed Central - HTML - PubMed

Affiliation: Imperial College London, Division of Cell and Molecular Biology, Imperial College Road, London, UK. p.papathanos@imperial.ac.uk

ABSTRACT

Background: Germline specific promoters are an essential component of potential vector control strategies which function by genetic drive, however suitable promoters are not currently available for the main human malaria vector Anopheles gambiae.

Results: We have identified the Anopheles gambiae vasa-like gene and found its expression to be specifically localized to both the male and female gonads in adult mosquitoes. We have functionally characterised using transgenic reporter lines the regulatory regions required for driving transgene expression in a pattern mirroring that of the endogenous vasa locus. Two reporter constructs indicate the existence of distinct vasa regulatory elements within the 5' untranslated regions responsible not only for the spatial and temporal but also for the sex specific germline expression. vasa driven eGFP expression in the ovary of heterozygous mosquitoes resulted in the progressive accumulation of maternal protein and transcript in developing oocytes that were then detectable in all embryos and neonatal larvae.

Conclusion: We have characterized the vasa regulatory regions that are not only suited to drive transgenes in the early germline of both sexes but could also be utilized to manipulate the zygotic genome of developing embryos via maternal deposition of active molecules. We have used computational models to show that a homing endonuclease-based gene drive system can function in the presence of maternal deposition and describe a novel non-invasive control strategy based on early vasa driven homing endonuclease expression.

Show MeSH
Related in: MedlinePlus