RESUMO
In anautogenous mosquitoes, a blood meal is required for activation of genes encoding yolk protein precursors (YPP). Vitellogenin (Vg), the major YPP gene, is transcribed at a very high level following blood meal activation. It is expressed exclusively in the female fat body, the tissue producing most of mosquito hemolymph and immune proteins. In this paper, we analyzed the upstream region of the Aedes aegypti Vg gene in order to identify regulatory elements responsible for its unique expression pattern. To achieve this goal, we analyzed the gene using transgenic Drosophila and Aedes as well as DNA-binding assays. These analyses revealed three regulatory regions in the 2.1 kb upstream portion of the Vg gene. The proximal region containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh is required for the correct tissue- and stage-specific expression at a low level. The median region carrying sites for early ecdysone response factors E74 and E75 is responsible for hormonal enhancement of Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic of the Vg gene. The present work elucidates the molecular basis of blood meal-dependent expression of this mosquito gene, laying the foundation for mosquito-specific expression cassettes with predictable stage and tissue specificity.
Assuntos
Aedes/genética , Vitelogeninas/genética , Região 5'-Flanqueadora/genética , Aedes/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Sangue , Clonagem Molecular , Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Ensaio de Desvio de Mobilidade Eletroforética , Corpo Adiposo/crescimento & desenvolvimento , Corpo Adiposo/metabolismo , Comportamento Alimentar , Feminino , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transformação Genética , Vitelogeninas/metabolismoRESUMO
Screening of Drosophila melanogaster genomic library was carried out using mouse brain polysomal poly(A)+RNA. As a result, 100 clones were selected, among which 14 clones were picked up after hybridization with fly head poly(A)+RNA. It follows therefore, that these clones contain evolutionary conserved sequences which are expressed in Drosophila fly heads. Analysis of these 14 clones revealed RNA-coding fragments. Comparison of their expression in heads and bodies of Drosophila was carried out. Using in situ hybridization we determined the localization of selected 14 sequences on polytene chromosomes. The possibility of further analysis of some clones to study developmental and functional processes in neural system of Drosophila is discussed.
Assuntos
Drosophila melanogaster/genética , Animais , Autorradiografia , Evolução Biológica , Southern Blotting , Clonagem Molecular , DNA/análise , DNA/genética , Regulação da Expressão Gênica , Cariotipagem , Hibridização de Ácido NucleicoRESUMO
A cDNA copy of the Nc70F gene which is specifically expressed in Drosophila neural tissue was cloned and characterized. The gene has an open reading frame for the protein of 384 amino acids. The protein contains dimerization, DNA binding, activation and repression domains which are common for the eucaryotic transcription factors. However, the domain organization of the Nc70F protein has some peculiarities. The primary structure of the Nc70F product and other transcription factors were compared. High level of homology of Nc70F protein with the mouse delta transcription factor was found. The in situ hybridization on tissue section showed that the Nc70F gene expression is restricted to the central nerve system at all stages of Drosophila ontogenesis. By using Drosophila genomic and cDNA clones of Nc70F genes as probes, homologous transcripts were identified in the human poly(A) +RNA. The evolutionary conservative portion of this gene was localized in the 5-exons.
Assuntos
Evolução Biológica , Sistema Nervoso Central/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Código Genético , Fatores de Transcrição/genética , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Fatores de Ligação de DNA Eritroide Específicos , Expressão Gênica , Hibridização In Situ , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator de Transcrição YY1RESUMO
Molecular-genetic analysis of the Nc (neural conserved) genome sequence of Drosophila melanogaster located at the position 73EF of Drosophila melanogaster was performed. The Nc73EF sequence was shown to be expressed in the nervous system of Drosophila. We constructed the restriction map of this sequence and revealed the main RNA-coding fragment in the 5'-3' orientation. The RNA-dot analysis data demonstrated that expression of the Nc73EF transcripts took place mainly in the nervous system. Hybridization with the human brain poly(A)+RNA confirmed the basic RNA-coding fragment to be evolutionary conservative. Southern blot analysis showed this fragment to be unique in the Drosophila genome. Northern blots detected three transcripts of this DNA fragment.
Assuntos
Evolução Biológica , Drosophila melanogaster/genética , Expressão Gênica , Sistema Nervoso/metabolismo , Animais , Sequência de Bases , Sequência Conservada , DNA/genética , Humanos , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
The primary structure of the cDNA copy of the evolutionary conserved gene Nc73EF from the region 73EF of Drosophila melanogaster was determined. Gene Nc73EF was shown to encode a protein highly homologous to the E1 subunit of human 2-oxoglutarate dehydrogenase, which catalyzes one of the key reactions of the Krebs cycle.
Assuntos
Sequência Conservada , Drosophila melanogaster/genética , Genes de Insetos , Complexo Cetoglutarato Desidrogenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Catálise , Ciclo do Ácido Cítrico , Código Genético , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Insect oocytes are extraordinarily specialized for receptor-mediated endocytosis of yolk protein precursors. The clathrin heavy chain (CHC) is the major structural protein of coated vesicles, the principal organelles of receptor-mediated endocytosis. To understand the role of clathrin in the development of the oocyte's powerful endocytotic machinery we determined the structure of the mosquito chc gene. The gene spans approximately 45 kilobases and its coding region is divided into seven exons, five of which encode the protein. Three distinct mature transcripts of this gene were identified in mosquito tissues. Two of them code isoforms of the CHC polypeptide differing in their NH2-terminal sequences, and are specifically expressed in female germ-line cells. The third transcript has a 3'-untranslated region about 1 kilobase longer than the other variants, and is found only in the somatic cells. Tissue-specific 5'-exon splicing and alternative polyadenylation of the primary transcript combine to give rise to these mRNAs. We identified two alternative promoters, distal and proximal, separated by approximately 10 kilobases involved in tissue-specific regulation of mosquito chc gene expression. Our data provide the first molecular evidence for complex structure and regulation of a chc gene, in this case occurring at both the transcriptional and post-transcriptional levels.
Assuntos
Processamento Alternativo , Clatrina/genética , Éxons , Regulação da Expressão Gênica no Desenvolvimento , RNA Mensageiro/metabolismo , Aedes , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cadeias Pesadas de Clatrina , Feminino , Dados de Sequência Molecular , Ovário/química , Transcrição GênicaRESUMO
We have deduced the amino acid sequences of clathrin heavy chain (CHC) polypeptides based on cDNA and genomic clones from the mosquito, Aedes aegypti. Two isoforms which differ in the very beginning of the N-terminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were identified, characterized and compared to one another as well as to CHC polypeptides from different species. The 1682 amino acid sequence of the AaCHCa isoform predicts a molecular mass (M[r]) of 191,743 daltons and an isoelectric point of 5.80, whereas the 1674 amino acid sequence of the AaCHCb isoform predicts a M(r) of 191,033 daltons and an isoelectric point of 5.71. Both mosquito AaCHC isoforms are highly conserved, with full-sequence identities of 88% to Drosophila melanogaster, 81% to mammal (rat, cow and human), 71% to C. elegans, 58% to Dictyostelium discoideum, and 49% to yeast CHC polypeptides. The highest degree of conservation is in the middle portion of the mosquito CHC molecule which includes the linker region and extended triskelion arm, with decreasing conservation through the N-terminal domain, trimerization domain, and the relatively diverged C-terminal region. The protein domains do not directly correspond to specific exons of the mosquito AaCHC gene, with the exception of exon 6 which encodes the C-terminal domain of the CHC polypeptide. Polyclonal antibodies raised against a bacteria-expressed AaCHC fusion protein recognized one major band of about 180 kDa in vitellogenic ovary whole-lysate. Immunogold labelling of the AaCHC polypeptide localized it to the coat of coated pits and coated vesicles in oocytes from vitellogenic follicles. Northern blot and in situ hybridization analyses suggest that regulation of AaCHC gene expression in the ovary is complex, and it likely involves both developmental and hormonal signals.
Assuntos
Aedes/genética , Clatrina/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Vitelogênese/genética , Aedes/fisiologia , Sequência de Aminoácidos , Animais , Clatrina/análise , Clatrina/química , Cadeias Pesadas de Clatrina , Invaginações Revestidas da Membrana Celular/química , Vesículas Revestidas/química , Sequência Conservada/genética , Feminino , Genes de Insetos/genética , Dados de Sequência Molecular , Oócitos/química , Ovário/química , Estrutura Terciária de Proteína , RNA Mensageiro/análise , Homologia de Sequência de AminoácidosRESUMO
The mosquito (Aedes aegypti) vitellogenin receptor (AaVgR) is a large membrane-bound protein (214 kDa when linearized) that mediates internalization of vitellogenin, the major yolk-protein precursor, by oocytes during egg development. We have cloned and sequenced two cDNA fragments encompassing the entire coding region of AaVgR mRNA, to our knowledge the first insect VgR sequence to be reported. The 7.3-kb AaVgR mRNA is present only in female germ-line cells and is abundant in previtellogenic oocytes, suggesting that the AaVgR gene is expressed early in oocyte differentiation. The deduced amino acid sequence predicts a 202.7-kDa protein before posttranslational processing. The AaVgR is a member of the low density lipoprotein receptor superfamily, sharing significant homology with the chicken (Gallus gallus) VgR and particularly the Drosophila melanogaster yolk protein receptor, in spite of a very different ligand for the latter. Distance-based phylogenetic analyses suggest that the insect VgR/yolk protein receptor lineage and the vertebrate VgR/low density lipoprotein receptor lineage diverged before the bifurcation of nematode and deuterostome lines.