The salivary glands of blood-sucking arthropods play an essential role in the adaptation to the haematophagous style of life in virtue of the salivary antihaemosthatic factors which are injected into the host skin during blood feeding. Haematophagy has arisen independently several time in different insects and one of the results of this evolutionary convergence is that a large variety of molecules have evolved to accomplish the same or similar functions. Completely diverse substances act as vasodilators and anti-clotting factors in different blood-feeding arthropods whereas antiplatelet activity is usually provided by the apyrase, an enzyme that hydrolyzing the ADP released by injured cells removes a critical factor promoting aggregation and recruitment of platelets. The only mosquito apyrase characterized so far is the Aedes aegypti gene which is a member of the 5’-nucleotidase family (Champagne DE et al, 1995, Proc Natl Acad Sci USA, 92: 694-698). We have previously identified two short Anopheles gambiae cDNA fragments showing similarity to the Ae. aegypti apyrase and to several members of the 5’-nucleotidase family. Based on their expression profile we proposed that the first one, whose expression was restricted to adult female glands, was the An. gambiae apyrase while the second, that was expressed also in other tissues and in males, represented a 5’-nucleotidase (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). To investigate the evolutionary relationship among these two members of the same protein family we isolated the full-length cDNAs and named the corresponding genes AgApy (An gambiae apyrase) and AgApyL1 (An gambiae apyrase-like 1). The putative AgApy and AgApyL1 proteins are respectively 557 and 570 aminoacids in length and show high degree of conservation to the Ae. aegypti apyrase and to several other members of the 5’-nucleotidase family. The presence of an aminoterminal signal peptide and the absence of transmembrane domains or GPI-anchoring signals suggests that they are both secreted proteins. This prediction is confirmed by the observation that myc-tagged recombinant versions of the proteins are secreted in the cell culture medium after transient transfection of COS7 cells. The developmental expression profile of the AgApy and AgApyL1 genes was analyzed by reverse-transcription PCR. The AgApy mRNA was found only in adult females and, moreover, the transcript abundance increased shortly after blood-feeding, a pattern of expression perfectly consistent with the role of the apyrase in blood feeding. The expression pattern of AgApyL1, instead, seems compatible with a possible role of 5’-nucleotidase. Indeed, the AgApyL1 transcript was found not only in females but also in different larval stages and, at a lower level, in males and in early pupae. Because of our interest in potential applications of salivary gland promoters we have isolated the genomic region encoding the An. gambiae apyrase gene. We placed an 800 bp fragment located immediately upstream of the AgApy gene in front of the Escherichia coli LacZ reporter gene and we inserted it in the Drosophila melanogaster genome by P element-mediated germline transformation. A weak but specific beta-galactosidase activity was detected in the D. melanogaster glands showing that transcriptional regulatory elements responsible for the stage- and tissue-specific expression are conserved between the mosquito An. gambiae and the fruitfly.

The Anopheles gambiae salivary apyrase: promoter sequences of the mosquito gene confer salivary gland expression in Drosophila melanogaster / Lombardo, Fabrizio; M., DI CRISTINA; L., Spanos; C., Louis; COLUZZI BARTOCCIONI, Caio Mario; Arca', Bruno. - In: PARASSITOLOGIA. - ISSN 0048-2951. - STAMPA. - 42 (Suppl. 1):(2000), pp. 136-136. ((Intervento presentato al convegno XXI Congresso SOCIETA' ITALIANA DI PARASSITOLOGIA tenutosi a Padova - Legnaro (Italy) nel 20-24 Giugno 2000.

The Anopheles gambiae salivary apyrase: promoter sequences of the mosquito gene confer salivary gland expression in Drosophila melanogaster.

LOMBARDO, Fabrizio;COLUZZI BARTOCCIONI, Caio Mario;ARCA', Bruno
2000

Abstract

The salivary glands of blood-sucking arthropods play an essential role in the adaptation to the haematophagous style of life in virtue of the salivary antihaemosthatic factors which are injected into the host skin during blood feeding. Haematophagy has arisen independently several time in different insects and one of the results of this evolutionary convergence is that a large variety of molecules have evolved to accomplish the same or similar functions. Completely diverse substances act as vasodilators and anti-clotting factors in different blood-feeding arthropods whereas antiplatelet activity is usually provided by the apyrase, an enzyme that hydrolyzing the ADP released by injured cells removes a critical factor promoting aggregation and recruitment of platelets. The only mosquito apyrase characterized so far is the Aedes aegypti gene which is a member of the 5’-nucleotidase family (Champagne DE et al, 1995, Proc Natl Acad Sci USA, 92: 694-698). We have previously identified two short Anopheles gambiae cDNA fragments showing similarity to the Ae. aegypti apyrase and to several members of the 5’-nucleotidase family. Based on their expression profile we proposed that the first one, whose expression was restricted to adult female glands, was the An. gambiae apyrase while the second, that was expressed also in other tissues and in males, represented a 5’-nucleotidase (Arcà B et al., 1999, Proc Natl Acad Sci USA, 96: 1516-1521). To investigate the evolutionary relationship among these two members of the same protein family we isolated the full-length cDNAs and named the corresponding genes AgApy (An gambiae apyrase) and AgApyL1 (An gambiae apyrase-like 1). The putative AgApy and AgApyL1 proteins are respectively 557 and 570 aminoacids in length and show high degree of conservation to the Ae. aegypti apyrase and to several other members of the 5’-nucleotidase family. The presence of an aminoterminal signal peptide and the absence of transmembrane domains or GPI-anchoring signals suggests that they are both secreted proteins. This prediction is confirmed by the observation that myc-tagged recombinant versions of the proteins are secreted in the cell culture medium after transient transfection of COS7 cells. The developmental expression profile of the AgApy and AgApyL1 genes was analyzed by reverse-transcription PCR. The AgApy mRNA was found only in adult females and, moreover, the transcript abundance increased shortly after blood-feeding, a pattern of expression perfectly consistent with the role of the apyrase in blood feeding. The expression pattern of AgApyL1, instead, seems compatible with a possible role of 5’-nucleotidase. Indeed, the AgApyL1 transcript was found not only in females but also in different larval stages and, at a lower level, in males and in early pupae. Because of our interest in potential applications of salivary gland promoters we have isolated the genomic region encoding the An. gambiae apyrase gene. We placed an 800 bp fragment located immediately upstream of the AgApy gene in front of the Escherichia coli LacZ reporter gene and we inserted it in the Drosophila melanogaster genome by P element-mediated germline transformation. A weak but specific beta-galactosidase activity was detected in the D. melanogaster glands showing that transcriptional regulatory elements responsible for the stage- and tissue-specific expression are conserved between the mosquito An. gambiae and the fruitfly.
2000
XXI Congresso SOCIETA' ITALIANA DI PARASSITOLOGIA
04 Pubblicazione in atti di convegno::04c Atto di convegno in rivista
The Anopheles gambiae salivary apyrase: promoter sequences of the mosquito gene confer salivary gland expression in Drosophila melanogaster / Lombardo, Fabrizio; M., DI CRISTINA; L., Spanos; C., Louis; COLUZZI BARTOCCIONI, Caio Mario; Arca', Bruno. - In: PARASSITOLOGIA. - ISSN 0048-2951. - STAMPA. - 42 (Suppl. 1):(2000), pp. 136-136. ((Intervento presentato al convegno XXI Congresso SOCIETA' ITALIANA DI PARASSITOLOGIA tenutosi a Padova - Legnaro (Italy) nel 20-24 Giugno 2000.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/472822
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