RESUMO
Recent results suggest that the Drosophila transcriptional activator known as GAGA factor functions by influencing chromatin structure.
Assuntos
Cromatina/fisiologia , Cromatina/ultraestrutura , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Drosophila melanogaster/metabolismo , Dados de Sequência MolecularAssuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Heterocromatina/fisiologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Genes Supressores , Dados de Sequência Molecular , Complexo Repressor Polycomb 1 , Proteínas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismoRESUMO
Cyclodiene resistance represents 60% of the reported cases of insecticide resistance and is also present in vertebrates. Resistance is due to insensitivity of the cyclodiene/picrotoxinin binding site on the gamma-aminobutyric acid subtype A (GABAA) receptor-chloride ionophore complex. Following isolation of cyclodiene-resistant Drosophila mutants, we report the cloning of the locus conferring resistance via a "chromosomal walk" and rescue of the susceptible phenotype by P-element-mediated germ-line transformation. Amino acid sequence analysis of a cDNA from the locus reveals homology with vertebrate GABAA subunits. To our knowledge, this represents the first cloning of an invertebrate GABA receptor and also allows us to manipulate the resistance status of an insect via germ-line transformation. This gene may be useful as a selectable marker in other insect systems.
Assuntos
Drosophila/genética , Resistência a Medicamentos/genética , Rearranjo Gênico , Inseticidas/farmacologia , Receptores de GABA-A/genética , Transformação Genética , Sequência de Aminoácidos , Animais , Bovinos , Passeio de Cromossomo , Clonagem Molecular/métodos , Cosmídeos , Cruzamentos Genéticos , DNA/genética , DNA/isolamento & purificação , Elementos de DNA Transponíveis , Dieldrin/farmacologia , Drosophila/fisiologia , Feminino , Substâncias Macromoleculares , Masculino , Dados de Sequência Molecular , Fenótipo , Conformação Proteica , Ratos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Over 120 kb of contiguous genomic DNA sequence derived from the 99C-99D region of the Drosophila melanogaster third chromosome were isolated by molecular cloning. Sequences within this region required for the expression of the lysosomal gene-enzyme system acid phosphatase-1 (Acph-1) were identified by both P element-mediated germline transformation and transient expression and lie within a single 5 kb fragment. Acph-1 is encoded by a 2.1 kb poly(A)+ RNA transcript, which is expressed throughout development. Enzyme activity peaks also correlate with increases in RNA abundance. The ca-74 deletion, which exhibits position effect variegation at the Acph-1 gene (Frisardi and MacIntyre 1984), was also partially characterized. The variegating ca-74 breakpoint is located approximately 20 kb proximal to the Acph-1 gene. Results suggest that the heterochromatin at this breakpoint comprises highly repetitive or satellite DNA.
Assuntos
Fosfatase Ácida/genética , Cromossomos/metabolismo , Drosophila melanogaster/genética , Expressão Gênica , Fosfatase Ácida/metabolismo , Animais , Northern Blotting , Southern Blotting , Clonagem Molecular , DNA/isolamento & purificação , Elementos de DNA Transponíveis , Drosophila melanogaster/enzimologia , Genes , Lisossomos/enzimologia , Hibridização de Ácido Nucleico , Mapeamento por Restrição , Transcrição Gênica , Transformação GenéticaRESUMO
The claret (ca) locus in Drosophila encodes products that are needed both for wild-type eyecolor and for correct meiotic chromosome segregation. Mutants described previously provide evidence that two mutationally independent coding regions are present at ca. We have recovered six new P element-induced and one spontaneous ca mutant. Four of these new mutants affect both eyecolor and chromosome segregation. The high frequency of co-mutation of these two functions suggests that the corresponding genes are closely adjacent to one another. We recovered genomic DNA sequences corresponding to the ca locus by chromosome walking, and showed using revertant analysis that the cloned region encodes ca+. Transformation experiments demonstrate that the mutant effect resulting in meiotic chromosome non-disjunction (nd) and loss is fully rescued by DNA from the cloned region. Two RNAs of 7.4 and 2.2 kb have been identified by Northern blot analysis as the putative eyecolor and segregational products. Expression of the RNAs with respect to males and females, and their presence or absence in ca and nd mutants indicate that the 7.4 kb RNA corresponds to the product needed for wild-type eyecolor and the 2.2 kb RNA is the product required for normal chromosome segregation. These RNAs are transcribed in opposite directions to one another. Alleles that affect both eyecolor and chromosome segregation are deletion mutants that affect both transcripts. Thus, the putative eyecolor and segregational products are encoded by separate genes. Mutants that affect both eyecolor and chromosome segregation apparently do so because they delete essential regions of both genes.