RESUMO
The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Ecdisona/metabolismo , Genes de Insetos , Hormônios de Inseto/genética , Regiões Promotoras Genéticas , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/genética , DNA/metabolismo , Hormônios de Inseto/metabolismo , Dados de Sequência MolecularRESUMO
Previous work has demonstrated expression of the larval serum protein-2 gene, Lsp-2, uniquely during the late larval and pupal stages of development in Drosophila melanogaster. Here we report that the LSP-2 polypeptide accumulates in the hemolymph throughout adult life as well. Western blot analysis using an LSP-2 antiserum reveals notable differences in the molecular weight of the larval and adult polypeptides. Lsp-2 transcription results in a unique mRNA of 2.3 kb, exhibiting the same 5' end in both larvae and adults. However, adult Lsp-2 mRNa is only expressed at 1% of the high level detectable in late third-instar larvae. Whereas Lsp-2 mRNA accumulates uniformly in all fat body cells of third-instar larvae, over 80% of the adult Lsp-2 transcript is expressed in the adipose tissue of the head. These results suggest a differential regulation for expression of the Drosophila Lsp-2 gene in adults and larvae.
Assuntos
Proteínas de Drosophila , Drosophila/genética , Hormônios de Inseto/genética , RNA Mensageiro/metabolismo , Animais , Corpo Adiposo/metabolismo , Expressão Gênica , Hemolinfa/metabolismo , Hormônios de Inseto/biossíntese , Larva/metabolismoRESUMO
The larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is expressed at a very high level in the fat body of third-instar larvae. Here we report that Lsp-2 transcription in adult flies produces a unique mRNA localized in the adult adipose tissue of the head in both sexes. To identify regulatory regions of this Drosophila gene, Lsp-2 5'-flanking DNA sequences were fused to the E. coli beta-galactosidase gene (lacZ). Transient expression of the hybrid gene in third-instar larvae indicates that 230 bp just upstream from the 'TATA box' of the Lsp-2 gene are sufficient for larval fat body-specific expression.
