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1.
Gene ; 246(1-2): 49-57, 2000 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-10767526

RESUMO

Insects express a battery of potent antimicrobial proteins in response to injury and infection. Recent work from several laboratories has demonstrated that this response is neither stereotypic nor completely nonspecific, and that different pathways are responsible for inducing the expression of antifungal and antibacterial peptides. Here we report the cloning of two closely linked attacin genes from Drosophila melanogaster. We compare their protein coding sequences and find the amino acid sequences to be more highly conserved than the nucleotide sequences, suggesting that both genes are expressed. Like other antimicrobial peptides, attacin expression is strongly induced in infected and injured flies. Unlike others, attacin transcription is uniquely sensitive to mutations in the 18-Wheeler receptor protein, and thus may be regulated by a distinct signaling pathway. The number and organization of binding sites for kappaB and other transcription factors in the promoter regions of both attacin genes are consistent with strong and rapid immune induction. We demonstrate that these promoter regions are sufficient to direct beta-galactosidase expression in transformed Drosophila third-instar larval fat body in a bacterially inducible manner. We present a comparison of the promoter regions of the two attacin genes to those cloned from other antimicrobial peptide genes to assist a better understanding of how antimicrobial genes are differentially regulated.


Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de Insetos/genética , Sequência de Aminoácidos , Animais , Anti-Infecciosos , Sequência de Bases , DNA/química , DNA/genética , Expressão Gênica , Regulação da Expressão Gênica , Genes de Insetos/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
3.
EMBO J ; 16(20): 6120-30, 1997 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-9321392

RESUMO

Mammals and insects employ similar Rel/NF-kappaB signaling cascades in their humoral immune responses. The mammalian interleukin-1 type I receptor (IL-1R) is one way of activating this cascade. The Drosophila Toll protein, whose cytoplasmic domain shows striking similarity to that of the IL-1R, acts in the humoral antimicrobial response. Here we demonstrate that a second IL-1R-related Drosophila protein, 18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12%. Finally, 18-wheeler (18w) expression is induced in response to infection and, in addition to the receptor form, four immune-specific transcripts and proteins are produced.


Assuntos
Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos , Moléculas de Adesão Celular/genética , Proteínas de Drosophila , Drosophila/imunologia , Regulação da Expressão Gênica , Proteínas de Insetos/genética , Proteínas de Membrana/genética , Receptores Imunológicos/genética , Fatores de Transcrição , Animais , Proteínas de Ligação a DNA/metabolismo , Corpo Adiposo/metabolismo , Proteínas de Insetos/biossíntese , Larva , Modelos Genéticos , Mutação , Proteínas Nucleares , Peptídeos/metabolismo , Fosfoproteínas , Proteínas Proto-Oncogênicas , RNA Mensageiro/biossíntese , Receptores de Interleucina-1 , Fator de Transcrição RelB
4.
Development ; 114(1): 99-112, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1576969

RESUMO

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.


Assuntos
Proteínas de Ligação a DNA/genética , Drosophila/genética , Regulação da Expressão Gênica/genética , Expressão Gênica/genética , Genes Reguladores/genética , Genes/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Zíper de Leucina/genética , Dados de Sequência Molecular , Morfogênese/genética , Mutação/genética , Fosforilação
5.
Development ; 111(2): 367-78, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1716553

RESUMO

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.


Assuntos
Drosophila/genética , Expressão Gênica/fisiologia , Genes/fisiologia , Animais , Embrião não Mamífero/ultraestrutura , Microscopia de Interferência , Mutação/genética , RNA/análise
6.
Genes Dev ; 4(1): 111-22, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-2407617

RESUMO

Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted Mr of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr approximately 350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an Mr approximately 23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.


Assuntos
Cílios/análise , Complexo de Golgi/análise , Proteínas/análise , Ouriços-do-Mar/análise , Sequência de Aminoácidos , Animais , Imunofluorescência , Complexo de Golgi/ultraestrutura , Soros Imunes , Immunoblotting , Imuno-Histoquímica , Proteínas de Membrana , Microscopia Eletrônica , Dados de Sequência Molecular , Proteínas/genética , Ouriços-do-Mar/embriologia
7.
EMBO J ; 8(5): 1539-48, 1989 May.
Artigo em Inglês | MEDLINE | ID: mdl-2504582

RESUMO

The segmentation gene, giant, is located in 3A1 within a cloned chromosome region surrounding the zeste locus. Rearrangement breakpoints associated with giant mutations were localized on the genomic clone map, and nearby transcription units were identified. One transcription unit is active during early embryogenesis and its transcripts are spatially localized from blastoderm into extended germband stages, consistent with expected expression patterns predicted by the 'gap' phenotype of giant mutants. Germ line transformation experiments using a 10-kb DNA fragment containing this transcription unit gave complete rescue of the abdominal giant defect but only partial correction of the head defect. The effect of mutations in three other gap loci, Kr, kni and hb, were also analyzed.


Assuntos
Drosophila melanogaster/genética , Transcrição Gênica , Alelos , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica , Rearranjo Gênico , Genes Letais , Fenótipo , Mapeamento por Restrição , Transformação Genética
8.
Genes Dev ; 1(10): 1280-92, 1987 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-2828169

RESUMO

We have characterized the temporal and spatial expression of Spec3 mRNA in embryos of the sea urchin, Strongylocentrotus purpuratus. This mRNA, 2.0 kb in length, is present at low levels in unfertilized eggs but accumulates rapidly during cleavage, increasing 50-fold by hatching blastula stage. Message levels then decline abruptly, remain constant during mesenchyme blastula and gastrula stages, and increase again during prism and pluteus stages. This accumulation pattern is quite similar to that of the ectodermally expressed beta-tubulin mRNAs described recently by Harlow and Nemer (1987a). In situ hybridization shows that although Spec3 message accumulates in all blastomeres at early blastula stages, it later becomes restricted to ectoderm. By late blastula stage, hybridization is strongest in the animal hemisphere. At gastrula, signals are variable over ectoderm, and by pluteus, grains are concentrated in the ciliary band, though present in other ectodermal cells as well. Deciliation and regeneration of cilia in gastrula-stage embryos results in a four- to fivefold increase in Spec3 mRNA levels, implying that the Spec3 gene product is associated with ciliogenesis. Spec3 mRNA is encoded by a single gene in the haploid genome, and characterization of the gene shows that it contains three exons that encode an open reading frame for a hydrophobic protein of 21.6 kD. The reading frame reveals that the carboxy-terminal part of the protein contains two long hydrophobic stretches, 31 and 37 residues long, separated by short hydrophilic regions of six to eight residues. The presence of these two distinct hydrophobic stretches suggests that the Spec3 protein contains two alpha-helical domains that either span the lipid bilayer or are associated with some other hydrophobic environment.


Assuntos
Cílios/fisiologia , RNA Mensageiro/metabolismo , Ouriços-do-Mar/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Blastocisto/fisiologia , Enzimas de Restrição do DNA , Ectoderma/fisiologia , Gástrula/fisiologia , Regulação da Expressão Gênica , Genes , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Ouriços-do-Mar/embriologia
9.
Nucleic Acids Res ; 10(23): 7829-42, 1982 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-6296792

RESUMO

Two closely related cDNA clones, pSpec1 and pSpec2, specifying two developmentally regulated tissue specific mRNAs from sea urchin embryos were used to probe a sea urchin genomic lambda library. Screening 10,000 phage by plaque hybridization yielded several hundred positive signals. With more stringent wash procedures, only two to three phage were positive. Three of these phage, one isolated by stringent wash procedures and two isolated by standard wash procedures were further investigated by restriction analysis, RNA gel blots, and DNA sequencing. The phage isolated by the stringent wash procedure appears to be a gene coding for the Specl mRNA. The other phage contain only partial homology to pSpec1 and pSpec2, 150 to 200 base pairs of the 3' untranslated region of the Spec1 and Spec2 mRNAs. It is concluded that the Spec1 and Spec2 mRNAs contain a highly repetitive element near their 3' end. The element is present at 2000 to 3000 copies per genome and may be transcribed at some sites other than those coding for the Spec1 and Spec2 genes. The possible function and evolutionary origin of the repetitive element is discussed.


Assuntos
Clonagem Molecular , Genes , RNA Mensageiro/genética , Ouriços-do-Mar/genética , Animais , Sequência de Bases , DNA/metabolismo , Enzimas de Restrição do DNA , Embrião não Mamífero/metabolismo , Hibridização de Ácido Nucleico , Biossíntese de Proteínas
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