RESUMO
In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.
Assuntos
Drosophila melanogaster/genética , Genes Homeobox , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Proto-Oncogene Mas , Homologia de Sequência de AminoácidosRESUMO
Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately 5-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut1 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5' end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect.
Assuntos
Adenilil Ciclases/genética , Drosophila melanogaster/genética , Genes , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Calmodulina/farmacologia , Mapeamento Cromossômico , Aprendizagem , Memória , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de SequênciaRESUMO
Biochemical, immunological, and molecular cloning studies have suggested the existence of multiple forms of adenylyl cyclase (EC 4.6.1.1). An adenylyl cyclase cDNA clone (type II) was isolated from a rat brain library and found to encode a protein of 1090 amino acids that was homologous to but distinct from the previously described Ca2+/calmodulin-stimulated adenylyl cyclase from bovine brain. Expression of the type II cDNA in an insect cell line resulted in an increased level of adenylyl cyclase activity that was insensitive to Ca2+/calmodulin. Addition of activated Gs alpha protein to type II-containing membranes increased enzyme activity. The mRNA encoding the type II protein was expressed at high levels in brain tissue and at low levels in olfactory epithelium and lung. The existence of multiple adenylyl cyclase enzymes may provide for complex and distinct modes of biochemical regulation of cAMP levels in the brain.
Assuntos
Adenilil Ciclases/genética , Encéfalo/enzimologia , Isoenzimas/genética , Adenilil Ciclases/isolamento & purificação , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cálcio/farmacologia , Calmodulina/farmacologia , Linhagem Celular , Expressão Gênica , Biblioteca Gênica , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Especificidade de Órgãos , Conformação Proteica , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Complementary DNA's that encode an adenylyl cyclase were isolated from a bovine brain library. Most of the deduced amino acid sequence of 1134 residues is divisible into two alternating sets of hydrophobic and hydrophilic domains. Each of the two large hydrophobic domains appears to contain six transmembrane spans. Each of the two large hydrophilic domains contains a sequence that is homologous to a single cytoplasmic domain of several guanylyl cyclases; these sequences may represent nucleotide binding sites. An unexpected topographical resemblance between adenylyl cyclase and various plasma membrane channels and transporters was observed. This structural complexity suggests possible, unappreciated functions for this important enzyme.
Assuntos
Adenilil Ciclases , Proteínas de Transporte , Canais Iônicos , Adenilil Ciclases/genética , Adenilil Ciclases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/enzimologia , Bovinos , Linhagem Celular , Clonagem Molecular , DNA/genética , Eletroforese em Gel de Poliacrilamida , Proteínas de Membrana , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Conformação Proteica , TransfecçãoRESUMO
Odorant-binding protein (OBP) is found in nasal epithelium, and it selectively binds odorants. Three complementary DNAs encoding rat odorant-binding protein have now been cloned and sequenced. One clone contains an open reading frame predicted to encode an 18,091-dalton protein. RNA blot analysis confirms the localization of OBP messenger RNA in the nasal epithelium. This OBP has 33 percent amino acid identity to alpha 2-microglobulin, a secreted plasma protein. Other members of an alpha 2-microglobulin superfamily bind and transport hydrophobic ligands. Thus, OBP probably binds and carries odorants within the nasal epithelium to putative olfactory receptors.
