RESUMO
The cell surface receptor Notch is required during development of Drosophila melanogaster for differentiation of numerous tissues. Notch is often required for specification of precursor cells by lateral inhibition and subsequently for differentiation of tissues from these precursor cells. We report here that certain embryonic cells and tissues that develop after lateral inhibition, like the connectives and commissures of the central nervous system, are enriched for a form of Notch not recognized by antibodies made against the intracellular region carboxy-terminal of the CDC10/Ankyrin repeats. Western blotting and immunoprecipitation analyses show that Notch molecules lacking this region are produced during embryogenesis and form protein complexes with the ligand Delta. Experiments with cultured cells indicate that Delta promotes accumulation of a Notch intracellular fragment lacking the carboxyl terminus. Furthermore, Notch lacking the carboxyl terminus functions as a receptor for Delta. These results suggest that Notch activities during development include generation and activity of a truncated receptor we designate NDeltaCterm.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/embriologia , Proteínas de Membrana/metabolismo , Animais , Repetição de Anquirina , Anticorpos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Células Cultivadas , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Imuno-Histoquímica , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Testes de Precipitina , RNA Mensageiro/metabolismo , Receptores Notch , Fatores de Transcrição/genéticaRESUMO
Notch, a cell surface receptor, is required for producing different types of cells during development of Drosophila melanogaster. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to ligand Wingless. The means by which Notch initiates these different intracellular activities was examined in this study. Cultured cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting. Treatment of cells with Delta resulted in accumulation of approximately 120-kDa Notch intracellular domain molecules in the cytoplasmic fraction. This form of Notch did not accumulate in cells treated with Wingless, but the approximately 350-kDa full-length Notch molecules accumulated. These results indicate that N responds differently to binding by Delta and Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Drosophila melanogaster/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Receptores Notch , Transdução de Sinais , Proteína Wnt1RESUMO
The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Dfrizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression; and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during development of Drosophila is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Quinase 3 da Glicogênio Sintase , Proteínas de Insetos/biossíntese , Proteínas de Membrana/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/ultraestrutura , Células Epidérmicas , Epiderme/embriologia , Receptores Frizzled , Proteínas de Insetos/genética , Substâncias Macromoleculares , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Morfogênese/genética , Morfogênese/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/biossíntese , Receptores de Neurotransmissores/genética , Receptores Notch , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteína Wnt1RESUMO
The cloning of double-time (dbt) is reported. DOUBLETIME protein (DBT) is most closely related to human casein kinase Iepsilon. dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. dbtP mutants, which eliminate rhythms of per and tim expression and constitutively overproduce hypophosphorylated PER proteins, abolish most dbt expression. dbt mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. DBT is capable of binding to PER in vitro and in Drosophila cells, suggesting that a physical association of PER and DBT regulates PER phosphorylation and accumulation in vivo.
Assuntos
Relógios Biológicos/genética , Caseína Quinase 1 épsilon , Proteínas de Drosophila , Drosophila/genética , Genes de Insetos , Proteínas Quinases/genética , Sequência de Aminoácidos , Animais , Caseína Quinases , Sequência Conservada , Regulação da Expressão Gênica , Genes Letais , Hibridização In Situ , Proteínas de Insetos/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Fosforilação , Ligação Proteica , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
Notch proteins appear to be involved in cell fate commitments with deep evolutionary roots. Homologues have been shown to play key roles in the development of nematodes, insects, amphibia, and mammals. Activity of the Notch receptor has been observed in the patterning of ectoderm, mesoderm, and endoderm, indicating an origin prior to the functional differentiation of these germ layers. To understand how a single receptor can participate so widely in development, we have been examining the role of specific extracellular segments of Notch. Early studies of mutations affecting widely separated EGF-like elements of Notch first raised the possibility for interaction with multiple ligands. Biochemical approaches, and exhaustive structure function studies in transgenic Drosophila are beginning to reveal how this receptor is activated, and point to a range of physical interactions with other proteins.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Insetos/fisiologia , Proteínas de Membrana/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Proteínas de Drosophila , Drosophila melanogaster/química , Receptores NotchAssuntos
DNA Complementar/síntese química , Reação em Cadeia da Polimerase/métodos , Cromatografia em Gel/métodos , DNA Polimerase I , DNA Complementar/isolamento & purificação , Nucleotídeos de Desoxicitosina , Eletroforese em Gel de Ágar/métodos , Hibridização In Situ/métodos , Indicadores e Reagentes , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/isolamento & purificação , Radioisótopos de Fósforo , Técnica de Diluição de Radioisótopos , Moldes GenéticosRESUMO
The Drosophila genes timeless (tim) and period (per) interact, and both are required for production of circadian rhythms. Here the positional cloning and sequencing of tim are reported. The tim gene encodes a previously uncharacterized protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the PER dimerization motif (PAS) indicates that direct interaction between PER and TIM would require a heterotypic protein association.
Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Proteínas/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Drosophila melanogaster/química , Drosophila melanogaster/fisiologia , Dados de Sequência Molecular , Peso Molecular , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Circadianas Period , Polimorfismo de Fragmento de Restrição , Proteínas/química , Análise de Sequência , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Chromosomal rearrangements constitute a significant feature of genome evolution, and inversion polymorphisms in Drosophila have been studied intensely for decades. Population geneticists have long recognized that the sequence features associated with inversion breakpoints would reveal much about the mutational origin, uniqueness, and genealogical history of individual inversion polymorphisms, but the cloning of breakpoint sequences is not trivial. With the aid of a method for rapid recovery of DNA clones spanning rearrangement breakpoints, we recover and examine the DNA sequences spanning the breakpoints of the cosmopolitan inversion In(3L)Payne in Drosophila melanogaster. By examining the sequence diversity associated with six standard and seven inverted chromosomes from natural populations, we find that the inversion is monophyletic in origin, the sequences are genetically isolated from recombination at the breakpoints, and there is no association with features such as transposable elements. The inverted sequences show 17-fold less nucleotide polymorphism, but there are eight fixed differences in the region spanning both breakpoints. This suggests that this inversion is not recently derived. Finally, Northern analysis and transcript mapping find that the distal breakpoint has disrupted three transcripts that are normally expressed in the standard arrangement. Incidentally, the method introduced here can be used to isolate breakpoint sequences of arrangements associated with many human diseases.
Assuntos
Drosophila melanogaster/genética , Animais , Sequência de Bases , Inversão Cromossômica , Mapeamento Cromossômico , Dados de Sequência Molecular , Polimorfismo Genético , Mapeamento por RestriçãoRESUMO
Notch locus EGF-like element mutations spl, altering eye development, and AxE2, affecting wing and sensilla development, are modified by mutations at Delta. It is shown that two allele-specific suppressors of spl involve single amino acid substitutions in the 4th (Dlsup5) and 9th (Dlsup4) EGF-like elements of the Delta protein. Cultured cells producing spl or AxE2 aggregate with cells producing wild-type Delta or Dlsup5 protein, and Dlsup5-producing cells adhere to cells producing wild-type Notch protein. However, spl,AxE2, and Dlsup5 are each defective in promoting these cell affinities, as none of the mutant proteins can compete with the corresponding wild-type proteins for formation of cell aggregates. Thus, widely separated EGF-like elements of Notch and Delta appear to participate in functional molecular interactions between the proteins. Dlsup5 does not improve adhesiveness of spl in vitro, so suppression in vivo may involve altered developmental signaling by spl-Dlsup5 complexes, rather than modified cell adhesion.
Assuntos
Drosophila melanogaster/genética , Fator de Crescimento Epidérmico/química , Hormônios de Inseto/química , Proteínas de Membrana/química , Mutagênese , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Adesão Celular , Agregação Celular , Linhagem Celular Transformada , Proteínas de Drosophila , Drosophila melanogaster/crescimento & desenvolvimento , Fator de Crescimento Epidérmico/genética , Hormônios de Inseto/genética , Hormônios de Inseto/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Receptores Notch , TransfecçãoRESUMO
A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standard library with the probe to identify and isolate clones carrying sequences homologous to the dissected region. This procedure has the potential to replace the difficult procedure of microcloning, as well as facilitate chromosome walking.