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A retrotransposon named Lian-Aa1 was discovered in an intron of an AaHR3-1 gene of the yellow fever mosquito, Aedes aegypti. This retrotransposon contained a long open reading frame with 1,219 amino acids that included endonuclease, reverse transcriptase, and RNase H domains. It was shown that in the Rock strain of Ae. aegypti, there were up to 1,380 copies of Lian elements, equivalent to 0.8% of the entire genome. Five additional copies of Lian elements were isolated, mapped by restriction digestion, and partially sequenced. The 5' and 3' ends of the Lian family were determined by comparing the terminal sequences of the six copies and were subsequently confirmed by the identification of putative target duplications flanking Lian-Aa1 and Lian-Aa2. The Lian family is likely a novel family of non-long-terminal-repeat (non-LTR) retrotransposons that terminate in a repeat of (CTGA-TAC)2. On average, the six copies of Lian elements showed only 0.6% sequence divergence at the nucleotide level in both a 735-bp region at the 5' end and a 1,124-bp coding region. Genomic Southern blots also revealed a very high degree of similarity among hundreds of Lian elements, suggesting very recent activity of Lian. Furthermore, all six analyzed Lian elements were closely associated with one or more different families of repetitive elements. It is possible that these associations could reflect the complex relationship between Lian elements and the rest of the Ae. aegypti genome. Phylogenetic analyses based on the reverse transcriptase, domains of 36 non-LTR retrotransposons including Lian-Aa1 identified five major subgroups that were supported by bootstrap replications. In contrast to the majority of non-LTR retrotransposons, Lian-Aa1 has an RNase H domain that is similar to a few other non-LTR retrotransposons and some retroviruses, which is consistent with the previously proposed independent assortment of different domains during the evolution of retroelements.
Mol Biol Evol 1998 Jul
PMID:Structural, genomic, and phylogenetic analysis of Lian, a novel family of non-LTR retrotransposons in the yellow fever mosquito, Aedes aegypti. 965 85

The biosynthesis of lipophorin of the yellow fever mosquito, Aedes aegypti, was investigated. Fat bodies were incubated in vitro with radiolabeled methionine and cysteine, and radiolabeled proteins secreted into the medium were analyzed by density gradient ultracentrifugation, SDS-PAGE and fluorography. Lipophorin was synthesized in the fat body and secreted into the medium. Its density was 1.114 g/ml, similar to that of lipophorin circulating in hemolymph. Three peptides of a tryptic digest of apolipophorin II were sequenced and degenerate oligonucleotide primers were designed based on the amino acid sequences. With these primers, a cDNA product of 1.2 kb was amplified by RT-PCR using as template RNA extracted from adult female mosquitoes 24 h after ingestion of a blood meal. This cDNA was cloned, sequenced and used as a probe for Northern blot analysis, which revealed that the apoproteins of lipophorin were coded for by a single mRNA of approximately 10 kb. The expression of the apolipophorins was induced by blood feeding. From the data presented we concluded that Aedes aegypti lipophorin is synthesized in the fat body and that the expression of its apolipophorins is induced by blood feeding.
Insect Biochem Mol Biol 1998 Oct
PMID:Biosynthesis of Aedes aegypti lipophorin and gene expression of its apolipoproteins. 980 20

Early trypsin is a female-specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first 4-6 h after ingestion of a blood meal. Transcription of the early trypsin gene occurs after adult emergence under control of juvenile hormone, but the transcript remains untranslated before feeding. Early trypsin was in vitro translated using mRNA extracted from midguts of unfed and fed females, indicating that there are not structural features in the early trypsin mRNA that impede translation in vitro. Eight single protein meals exhibiting different molecular weights and amino acid composition, as well as ingestion of several amino acid mixtures of different complexity, had the ability to prompt early trypsin translation. In contrast, ingestion of saline, latex or midgut-filling sugars were unable to induce early trypsin mRNA translation. In addition intra-thoracic injection of an amino acid solution induced early trypsin translation, while injection of saline or albumin failed. In summary an increase in the size of the midgut amino acid pool by feeding or injection of an amino acid solution was sufficient to induce translation of early trypsin mRNA; 35S-labeled amino acids, fed with a protein meal, were incorporated into newly synthesized early trypsin; the first phase of trypsin synthesis is likely induced by an initial rapid increase in the concentration of amino acids in the midgut cells after ingestion of a blood meal.
Insect Biochem Mol Biol 1999 Mar
PMID:Increase in the size of the amino acid pool is sufficient to activate translation of early trypsin mRNA in Aedes aegypti midgut. 1031 37

Five short interspersed repetitive elements (SINEs) were found fortuitously in the introns of a steroid hormone receptor AaHR3-2 gene of the yellow fever mosquito, Aedes aegypti, constituting a novel family of tRNA-related SINEs named Feilai. In addition, nine other Feilai elements were found in currently available sequences in Ae. aegypti, six of which were also near genes. Approximately 5.9 x 10(4) copies of Feilai were present in Ae. aegypti, equivalent to 2% of the entire genome. An additional 35 Feilai elements were isolated from a genomic library. Of the total 49 Feilai elements, 20 were full-length. Sequence comparisons and phylogenetic analyses of the full-length elements strongly suggest that there are at least two subfamilies within the Feilai family. There is a high degree of conservation within the two subfamilies. However, sequence divergence between the subfamilies, along with the presence of highly degenerate Feilai elements, suggests that Feilai is likely a diverse family of SINEs that has existed in Ae. aegypti for a long time. Many Feilai elements were closely associated with other transposons, especially with fragments of non-LTR retrotransposons and miniature inverted-repeat transposable elements. The 500-bp sequences immediately flanking a Feilai element were highly A + T-rich, which is consistent with the fact that no Feilai has been found in the coding regions of genes. It is likely that the highly reiterated and interspersed Feilai elements are partially responsible for the pattern of short-period interspersion of the Ae. aegypti genome. The evolutionary relationship between Feilai and the Ae. aegypti genome is likely complex.
Mol Biol Evol 1999 Jun
PMID:Genomic and evolutionary analysis of Feilai, a diverse family of highly reiterated SINEs in the yellow fever mosquito, Aedes aegypti. 1036 54

The Lepidopteran transposable element piggyBac is being recognized as a useful vector for genetic engineering in a variety of insect species. This transposon can mediate transformation in the Dipteran species Ceratitis capitata, and can potentially serve as a versatile vector for transformation of a wide variety of insect species. Using a plasmid-based interplasmid transposition assay, we have demonstrated that this transposon, of the short inverted terminal repeat type, is capable of transposition in embryos of three different insect species, Drosophila melanogaster, the yellow fever mosquito Aedes aegypti, and its host of origin, Trichoplusia ni. This assay can confirm the potential utility of piggyBac as a gene transfer tool in a given insect species, and provides an experimental model for assessing molecular mechanisms of transposon movement.
Mol Gen Genet 1999 Jun
PMID:Transposition of the piggyBac element in embryos of Drosophila melanogaster, Aedes aegypti and Trichoplusia ni. 1039 18

The steroid hormone ecdysone controls genetic regulatory hierarchies underlying insect molting, metamorphosis and, in some insects, reproduction. Cytogenetic and molecular analysis of ecdysone response in Drosophila larval salivary glands has revealed regulatory hierarchies including early genes which encode transcription factors controlling late ecdysone response. In order to determine whether similar hierarchies control reproductive ecdysone response, we have investigated ecdysone-regulated gene expression in vitellogenic mosquito ovaries and fat bodies. Here, we identify the homologue of the Drosophila E75 early ecdysone inducible gene in the yellow fever mosquito Aedes aegypti, and show that, as in Drosophila, the mosquito homologue, AaE75, consists of three overlapping transcription units with three mRNA isoforms, AaE75A, AaE75B, and AaE75C, originating as a result of alternative splicing. All three AaE75 isoforms are induced at the onset of vitellogenesis by a blood meal-activated hormonal cascade, and highly expressed in the mosquito ovary and fat body, suggesting their involvement in the regulation of oogenesis and vitellogenesis, respectively. Furthermore, in vitro fat body culture experiments demonstrate that AaE75 isoforms are induced by 20-hydroxyecdysone, an active ecdysteroid in the mosquito. These findings suggest that related ecdysone-triggered regulatory hierarchies may be used reiteratively during developmental and reproductive ecdysone responses.
Mol Cell Endocrinol 1999 Apr 25
PMID:E75 expression in mosquito ovary and fat body suggests reiterative use of ecdysone-regulated hierarchies in development and reproduction. 1041 2

A novel family of non-long-terminal-repeat (non-LTR) retrotransposons, named MosquI, was discovered in the yellow fever mosquito, Aedes aegypti. There were approximately 14 copies of MosquI in the A. aegypti genome. Four of the five analyzed MosquI elements were truncated at the 5' ends while one of them, MosquI-Aa2, was full-length. All five MosquI elements ended with 4-10 TAA tandem repeats, as the Drosophila I factors do. Interestingly, MosquI elements were often found near genes and other repetitive elements. The 6,623-bp MosquI-Aa2 contained two open reading frames (ORFs) flanked by a 404-bp 5' untranslated region and a 326-bp 3' untranslated region. The two ORFs code for nucleocapsids, endonuclease, reverse transcriptase, and RNase H domains. Although overall structural and sequence comparisons suggest that MosquI is highly similar to the Drosophila I factors, phylogenetic analysis based on the reverse transcriptase domains of 40 non-LTR retrotransposons indicate that MosquI and I factors are likely paralogous elements which may have been separated before the split between the ancestors of mollusca and arthropoda. Pairwise comparisons between the four truncated MosquI elements showed 96.7%-99.5% identity at the nucleotide level, while comparisons between the full-length MosquI-Aa2 and the truncated copies showed only 80.2%-81.8% identity. These comparisons and preliminary phylogenetic analyses suggest that the full-length and truncated MosquI elements may belong to two subfamilies originating from two source genes that diverged a long time ago. In contrast to the defective I factors in Drosophila melanogaster, which are likely very old components of the genome, the truncated MosquI elements seem to have been recently active. Finally, the genomic distribution and evolution of MosquI elements are analyzed in the context of other non-LTR retrotransposons in A. aegypti.
Mol Biol Evol 1999 Dec
PMID:MosquI, a novel family of mosquito retrotransposons distantly related to the Drosophila I factors, may consist of elements of more than one origin. 1060 10

We report here the use of the enhanced green fluorescent protein (EGFP) from the jellyfish, Aequorea victoria, as a genetic marker for the genetic transformation of mosquitoes. The EGFP gene, under the control of the actin5C promoter of Drosophila melanogaster was inserted into the Hermes transposable element. Preblastoderm embryos of a wild-type strain of the yellow fever mosquito, Aedes aegypti, were microinjected with this plasmid, together with a helper plasmid containing the Hermes transposase placed under the control of the D. melanogaster hsp70 promoter. Somatic EGFP expression was observed during early instars in approximately one-half of all G0 individuals. Two G1 individuals arising from a G0 female displayed high levels of EGFP gene expression during all stages of development. EGFP was transmitted in a Mendelian fashion to the G2 and G3 generations and molecular analysis confirmed the presence of the Hermes[actin5C:EGFP] gene in these insects. These results clearly demonstrate that EGFP can be used as an effective genetic marker in wild-type Ae. aegypti and most likely in other mosquito species as well.
Insect Mol Biol 2000 Feb
PMID:Green fluorescent protein as a genetic marker in transgenic Aedes aegypti. 1067 65

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8-bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut-and-paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT-like element in the mosquito genome.
Insect Mol Biol 2000 Feb
PMID:Structure of hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti. 1067 66

The study of the changes in the steady-state levels of the early trypsin (ET) messenger RNA (mRNA) was used as a sensitive assay for measuring the effects of recombinant juvenile hormone esterase (rJHE) on juvenile hormone (JH)-dependent gene expression in mosquitoes. ET is a female-specific protease present in the midgut of the yellow fever mosquito Aedes aegypti during the first few hours after ingestion of a blood meal. Transcription of the early trypsin gene is part of the normal postemergence maturation of the midgut in the adult female, and it is regulated by JH. JHE was cloned from Heliothis virescens and expressed in a baculovirus vector. Injection of rJHE into mosquitoes resulted in an increase of JHE activity in the haemolymph. Injection of rJHE into newly emerged adult females delayed the normal increase in steady-state levels of ET mRNA observed in controls. Topically applied methoprene (a JH analogue) reversed the effect of rJHE. Injection of increasing concentrations of rJHE into 3-day-old unfed females resulted in a dose-dependent decrease in the steady-state levels of ET mRNA after 24 h. The effect of rJHE was transient, once the enzyme was cleared (72 h after injection), the steady-state levels of ET mRNA were restored. The injection of rJHE is an effective tool for modifying JH-dependent expression of the early trypsin gene in mosquitoes.
Insect Mol Biol 2000 Feb
PMID:Recombinant juvenile hormone esterase, an effective tool for modifying juvenile hormone-dependent expression of the early trypsin gene in mosquitoes. 1067 68


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