A cytoskeletal actin gene in the mosquito Anopheles gambiae

Five actin genes have been identified in the mosquito Anopheles gambiae , and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster , and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.     

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.     

DNA organization and length polymorphism at the 2L telomeric region of Anopheles gambiae

The molecular structure of the telomeric region at the left arm of the second chromosome of the mosquito Anopheles gambiae has been determined in the transformed strain G418 that contains a pUChsneo transgene attached at the 2L chromosome end, and in the Pink eye laboratory strain (PE). Both strains contain the same complex satellite positioned distal to a unique region. FIGE mapping of the telomeric region of the PE strain revealed distinct DNA fragment lengths that segregated with individual chromosomes. Genomic DNA fragments were cloned from the 2L telomeric region, which accounted for about half of 2L chromosomes in the PE population. In all three cases studied, long fragments of different middle repetitive sequences were found attached to the distal ends of the 2L satellite. We propose that random fragments of DNA may be occasionally added during recombination between complex satellite repeats at the chromosome ends.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

Optimization of the Gal4-UAS system in an Anopheles gambiae cell line

The development of the bipartite Gal4-UAS system in Anopheles gambiae would improve the functional characterization of genes in this important malaria vector. Towards this aim, we used Gal4 driver plasmids to successfully activate expression of the reporter gene, luciferase, from UAS responder plasmids when cotransfected into an An. gambiae cell line. To optimize Gal4-regulated gene expression in mosquitoes, we compared the efficiency of a series of alternative Gal4 transactivators to drive reporter gene expression from responder plasmids incorporating different numbers of tandemly arrayed Gal4 binding sites or upstream activation sequences (UAS). The results indicated that the native Gal4 is only weakly active in these cells. Modified forms of Gal4, including those carrying minimal VP16 activation domains, as well as a deleted form of Gal4, give up to 20-fold greater activity than the native protein, when used in conjunction with a responder plasmid having 14 UAS repeats. The identification of Gal4-UAS vectors that are efficiently expressed in a mosquito cell line should facilitate the transfer of this versatile expression system to An. gambiae, and potentially to other insects of medical importance.     

Molecular characterization of the Anopheles gambiae 2L telomeric region via an integrated transgene

A Drosophila P-element derivative (pUChsneo) integrated into the telomeric region of the left arm of the second chromosome of Anopheles gambiae was used to clone the proximally flanking An. gambiae sequences. Molecular analyses revealed that the pUChsneo construct was partially duplicated and had integrated into a subterminal minisatellite. This satellite has a repeat unit of 820 bp and is located exclusively at the tip of 2L. No sequence similarity to subterminal minisatellites from other dipterans was detected, but some structural features such as tandem subrepeats are shared. The end of the chromosome was mapped with respect to restriction sites in pUChsneo at approximately generation 100 after the integration event. Considering inevitable terminal nucleotide loss due to incomplete DNA replication, we conclude that the chromosome end must have undergone a dramatic elongation process since it was mapped in generation 23.     

The core polypeptide of cystic fibrosis tracheal mucin contains a tandem repeat structure. Evidence for a common mucin in airway and gastrointestinal tissue.

A cystic fibrosis trachea cDNA library was constructed and probed with a synthetic oligonucleotide containing a consensus sequence recently identified in human intestinal mucin. One of the isolated clones, AMN-22, has been characterized extensively. The cDNA sequence of this 884-bp fragment was determined, and revealed a tandem repeat structure rich in threonine and proline residues. The repeating sequence of AMN-22 was similar but not identical to that determined for gut mucin. When examined by Northern analysis, the mRNA hybridizing to AMN-22 is extremely polydisperse in cystic fibrosis (CF) trachea, with apparent message length varying from approximately 2 kb to greater than 10 kb. A similar pattern was observed, with less abundant message, in CF bronchiectatic lung parenchyma. The lung cDNA hybridized to a similarly polydisperse message in ulcerative colitis colon RNA, but did not hybridize to control RNA from U937 lymphoma cells or stomach RNA. Pedigree analysis of restriction digests of genomic DNA revealed a pattern indicating a single polymorphic locus for the mucin gene expressed in the lung and the intestine. Southern analyses of human:mouse somatic cell hybrid cell lines allow a chromosomal localization for the mucin gene to human chromosome II, within the region 11p13-11pTer. Taken together, these data demonstrate that a polymorphic gene encodes a mucin core polypeptide expressed in both lung and intestine.     

A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae

Four genes expressed in the Anopheles gambiae adult female salivary glands and similar in sequence to the Aedes aegypti D7 gene were identified. The genes, called D7-related (D7r), are included in a single cluster encompassing approximately six kilobases on chromosome arm 3R. The deduced proteins contain secretory signals and they are probably injected by the mosquito into the host with the saliva during blood feeding. The region of similarity to D7 encompasses the carboxy-terminal part of the Ae. aegypti protein and the different An. gambiae D7r show a degree of similarity to each other, varying from 53% to 73%. The weak but significant similarity to members of a wide family of insect proteins, including odourant- and pheromone-binding proteins, raises the possibility that the D7r-encoded proteins may bind and/or carry small hydrophobic ligands.     

Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element

Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) injected into mosquito embryos. Two G1 generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event in the injected G0 embryos. Genetic cross data suggest that the insertion site of the element either resulted in, or is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios in outcrosses to non-transformed individuals and failure to establish a pure-breeding line. The insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement in the injection technique at the end of the studies resulted in increased G0 hatching, transient expression and EGFP-expression rates among G1 progeny.     

The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti . Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae , but not in the vinegar fly, Drosophila melanogaster . The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.     

Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short-hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.     

The rate of terminal nucleotide loss from a telomere of the mosquito Anopheles gambiae

Using a single copy pUChsneo transgene insertion at the Anopheles gambiae 2L telomere, this chromosome end was monitored by genomic Southern blots for forty-four mosquito generations. During this time, the chromosome end lost terminal nucleotides at an apparently constant rate of 55 bp/generation, which can be accounted for by incomplete DNA replication and does not imply exonuclease activity. No telomere elongation events were detected, suggesting that a previously described gene conversion event at this transgene does not occur very frequently. Moreover, no evidence for elongation by transposable elements was found, as described in Drosophila melanogaster. These results are consistent with the proposal that gene conversion between complex terminal satellite repeats that are present at natural telomeres, represents the major telomere elongation mechanism in A. gambiae. Such recombination events between repetitive sequences would occur more frequently than between the single copy pUChsneo transgene on the 2L homologues.     

Immune activation upregulates lysozyme gene expression in Aedes aegypti mosquito cell culture

After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedes aegypti (Aag-2 cells) secreted an induced protein with a mass of approximately 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anopheles gambiae and Anopheles darlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12-36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells.     

Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle

We have characterized four new families of homologous genes of the mosquito, Anopheles gambiae , all of which include members shown by previous work to be cuticular in nature. The CPLCG, CPLCW, CPLCP, and CPLCA families (where CPLC is 'cuticular protein of low complexity') encode proteins with a high proportion of low-complexity sequence. We have also annotated the An. gambiae Tweedle genes, a family of cuticular protein genes first described in Drosophila , and additional ungrouped An. gambiae cuticular proteins identified by proteomics. Our annotations reveal multiple gene-family expansions that are specific to Diptera or Culicidae. The CPLCG and CPLCW families occur within a large and dynamic tandem array on chromosome 3R that includes sets of concertedly evolving genes. Most gene families exhibit two or more different expression profiles during development.     

The Anopheles gambiae alpha-tubulin-1b promoter directs neuronal, testes and developing imaginal tissue specific expression and is a sensitive enhancer detector

A knowledge gap in mosquito functional genetic analysis is the dearth of characterized regulatory regions that can target tissue specific transgene expression. To broaden the tools available, a promoter region of the Anopheles gambiaeα-tubulin1b gene has been assayed following fusion to the green fluorescent protein (GFP) reporter gene and stable transformation of An. gambiae. In eight transgenic lines, the Angtub α1b regulatory region directed a core profile of tissue specific expression in the head, chordotonal organs, ventral nerve cord and testes. This profile overlaps those seen for α2-tubulin expression in Drosophila melanogaster and Bombyx mori. In addition, widespread position dependant expression was observed in other specific tissues that were unique to each line. For example, in different lines, expression was observed in larval and adult muscles, fatbody, cuticle and midgut secretory cells. The majority of genomic transgene insertions were mapped to within 10 kb of a gene, suggesting that the Angtub α1b basal promoter is particularly sensitive to enhancers and may be suitable to form the basis of a sensitive enhancer trapping construct, in combination with a binary expression system such as Gal4-UAS.