RESUMO
The ability to react to unfavorable environmental changes is crucial for survival and reproduction, and several adaptive responses to stress have been conserved during evolution [1-3]. Specific immune and heat shock responses mediate the elimination of invading pathogens and of damaged proteins or cells [4-6]. Furthermore, MAP kinases and other signaling factors mediate cellular responses to a very broad range of environmental insults [7-9]. Here we describe a novel systemic response to stress in Drosophila. The Turandot A (TotA) gene encodes a humoral factor, which is secreted from the fat body and accumulates in the body fluids. TotA is strongly induced upon bacterial challenge, as well as by other types of stress such as high temperature, mechanical pressure, dehydration, UV irradiation, and oxidative agents. It is also upregulated during metamorphosis and at high age. Strikingly, flies that overexpress TotA show prolonged survival and retain normal activity at otherwise lethal temperatures. Although TotA is only induced by severe stress, it responds to a much wider range of stimuli than heat shock genes such as hsp70 or immune genes such as Cecropin A1.
Assuntos
Drosophila/fisiologia , Regulação da Expressão Gênica , Transtornos de Estresse por Calor/fisiopatologia , Animais , Fenômenos Fisiológicos Bacterianos , Drosophila/genética , Transtornos de Estresse por Calor/genéticaRESUMO
The genomic region containing the genes encoding the first leukotriene B(4) receptor, BLTR, as well as the recently cloned second leukotriene B(4)-activated receptor, BLTR2, was mapped by (a) sequence analysis of a human bacterial artificial chromosome (BAC) library containing a 15-kb segment corresponding to chromosome 14q11. 2-12 where the BLTR/BLTR2 genes were previously shown to be located, together with (b) sequence analysis of 83 expressed sequence tags (ESTs) from this region. The BLTR gene includes four different 5' untranslated regions (UTRs) and a mutual acceptor site for the exon containing the intronless open reading frame. The BLTR2 gene is intronless and overlapped by a 5' UTR splice version of BLTR and, on the reverse strand, of the apoptosis-related CIDE-B gene. This indicates a complex posttranscriptional gene regulation. Further adding to the complexity of the region is evidence of a fourth putative and novel gene, most homologous to the rat adenylyl cyclase IV gene.
Assuntos
Cromossomos Humanos Par 14/genética , Mapeamento Físico do Cromossomo , Receptores do Leucotrieno B4/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Processamento Alternativo/genética , Proteínas Reguladoras de Apoptose , Cromossomos Bacterianos/genética , DNA Complementar/análise , DNA Complementar/genética , Etiquetas de Sequências Expressas , Biblioteca Genômica , Humanos , Proteínas/genética , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
By homology screening using BLAST searches of expressed sequence tags (ESTs), we have found a previously unidentified cDNA encoding a putative seven-transmembrane receptor with highest similarity to the leukotriene B(4) receptor, BLTR. Analysis of calcium flow in transfected cells, along with sequence analysis, revealed that the EST encoded a functionally inactive protein, lacking the segment corresponding to the C-terminal part of the putative receptor protein. The missing segment was obtained by PCR amplification of a human leukocyte cDNA library and ligated to the truncated EST cDNA. The novel cDNA encodes a full-length receptor with 39% identity to the previously cloned BLTR. Studies of intracellular calcium flow of transfected HeLa cells exposed to various leukotrienes showed that also the novel BLTR-like receptor can be activated by leukotriene B(4), and it is therefore tentatively named BLTR2.
Assuntos
DNA Complementar/genética , Receptores do Leucotrieno B4/genética , Receptores de Leucotrienos/genética , Equorina/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Cálcio/metabolismo , Cromossomos Humanos Par 14/genética , Clonagem Molecular , Etiquetas de Sequências Expressas , Genes Reporter , Células HeLa , Humanos , Leucotrieno B4/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
We have isolated cDNA clones encoding the full-length Drosophila melanogaster cysteine proteinase 1 (CP1). The clones were isolated from the Drosophila melanogaster haemocytic mbn-2 cell line, where the gene is relatively strongly expressed, giving a transcript of 1.6 kb in size. We present the sequence encoding the full-length protein, and deduced the genomic organization of the gene by comparison to previously published genomic partial sequence data. Immunofluorescence shows that CP1 is localized in small granules, probably lysosomes, in mbn-2 cells. The data presented suggest a role for cysteine proteinase in immune functions in insects. It is likely to participate in the degradation of internalized material in phagocytic cells.
Assuntos
Catepsinas/genética , Cisteína Endopeptidases/genética , Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Endopeptidases , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L , Linhagem Celular , Clonagem Molecular , Cisteína Endopeptidases/classificação , Cisteína Endopeptidases/metabolismo , DNA Complementar , Drosophila melanogaster/genética , Expressão Gênica , Genes de Insetos , Hemócitos/citologia , Hemócitos/enzimologia , Proteínas de Insetos/classificação , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.
Assuntos
Calpaína/biossíntese , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/metabolismo , Expressão Gênica , Genes de Insetos , Sequência de Aminoácidos , Animais , Southern Blotting , Calmodulina/genética , Calpaína/genética , Clonagem Molecular , Sequência Conservada , Sistema Digestório/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Éxons , Immunoblotting , Hibridização In Situ , Dados de Sequência Molecular , Sistema Nervoso/metabolismo , Especificidade de Órgãos , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Transcrição Gênica , VertebradosRESUMO
Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.