RESUMO
The Drosophila locus embryonic lethal abnormal visual system (elav) encodes a nuclear RNA-binding protein essential for normal neuronal differentiation and maintenance of neurons. ELAV is thought to play its role by binding to RNAs produced by other genes necessary for neuronal differentiation and consequently to affect their metabolism by an as yet unknown mechanism. ELAV structural homologues have been identified in a wide range of organisms, including humans, indicating an important conserved role for the protein. Analysis of elav germline transformants presented here shows that one copy of elav minigenes lacking a complete 3' untranslated region (3' UTR) rescues null mutations at elav, but that two copies are lethal. Additional in vivo experiments demonstrate that elav expression is regulated through the 3' UTR of the gene and indicate that this level of regulation is dependent upon ELAV itself. Because ELAV is an RNA-binding protein, the simplest model to account for these findings is that ELAV binds to the 3' UTR of its own RNA to autoregulate its expression. I discuss the implications of these results for normal elav function.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Drosophila/genética , Regulação da Expressão Gênica/genética , Homeostase/genética , Ribonucleoproteínas/genética , Animais , Animais Geneticamente Modificados , Proteínas ELAV , Feminino , Dosagem de Genes , Genes Letais , Infertilidade/genética , Masculino , Mutação , Mapeamento por Restrição , Ribonucleoproteínas/análise , Deleção de Sequência , Fatores Sexuais , TransgenesRESUMO
Knowledge of multigenic family organization should provide insight into their mode of evolution. Accordingly, we characterized the 5S ribosomal gene family in the Drosophila melanogaster strain ry506. The 5S genes in this strain display a striking HindIII restriction difference compared to the "standard" D. melanogaster 5S genes. The sequence of three ry506 5S genes was determined. We show that the HindIII restriction site heterogeneity within the ry506 5S family most probably results from the same point mutation, suggesting that a single 5S variant was propagated into the 5S cluster of this strain. Furthermore, we demonstrate that the structural organization of the 5S genes in ry506 is a bipartite structure, i.e., that about 40% of the 5S genes constitute a HindIII+/HindIII- mixed cluster, while those remaining constitute an homogeneous HindIII- cluster. The events which might lead to such an heterogeneous pattern are discussed from an evolutionary point of view.
Assuntos
Drosophila melanogaster/genética , Família Multigênica , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Animais , Sequência de Bases , Evolução Biológica , Enzimas de Restrição do DNA , Desoxirribonuclease HindIII , Feminino , Variação Genética , Masculino , Dados de Sequência MolecularRESUMO
The Drosophila gene elav encodes a 483-amino-acid-long nuclear RNA binding protein required for normal neuronal differentiation and maintenance. We molecularly analyzed the three known viable alleles of the gene, namely elavts1, elavFliJ1, and elavFliJ2, which manifest temperature-sensitive phenotypes. The modification of the elavFliJ1 allele corresponds to the change of glycine426 (GGA) into a glutamic acid (GAA). Surprisingly, elavts1 and elavFliJ2 were both found to have tryptophan419 (TGG) changed into two different stop codons, TAG and TGA, respectively. Unexpectedly, protein analysis from elavts1 and elavFliJ2 reveals not only the predicted 45-kD truncated ELAV protein due to translational truncation, but also a predominant full-size 50-kD ELAV protein, both at permissive and nonpermissive temperatures. The full-length protein present in elavts1 and elavFliJ2 can a priori be explained by one of several mechanisms leading to functional suppression of the nonsense mutation or by detection of a previously unrecognized ELAV isoform of similar size resulting from alternative splicing and unaffected by the stop codon. Experiments described in this article support the functional suppression of the nonsense mutation as the mechanism responsible for the full-length protein.
Assuntos
Alelos , Códon/genética , Drosophila melanogaster/genética , Genes de Insetos , Mutação Puntual , Ribonucleoproteínas/genética , Supressão Genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas ELAV , Feminino , Masculino , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribonucleoproteínas/biossíntese , Temperatura , Regiões Terminadoras Genéticas/genética , TriptofanoRESUMO
The advent of high-throughput technologies to analyze RNA expression levels and transcript structure has brought renewed attention to the age-old question of what differentiates males from females. In Drosophila, the characterized somatic sex determination cascade includes proteins implicated in the regulation of pre-mRNA splicing as well as at least 2 transcription factors at its base. Both DNA microarrays and RNA-Seq have been applied in a number of studies to determine the identities, expression levels and structure of transcripts expressed differentially in the 2 sexes, with remarkably divergent results in the number, structure and identity of affected transcripts. We briefly summarize these reports and discuss the reasons for the apparent discrepancies based upon the different conditions used for sample preparation and data analysis.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Especificidade de Órgãos/genética , Diferenciação Sexual/genética , Processamento Alternativo/genética , Animais , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
The fne (found-in-neurons) locus encodes one of the three paralogs of the ELAV gene family of Drosophila melanogaster. Members of this family are found throughout metazoans and encode RNA-binding proteins with primarily neuronal localization, but with remarkably diverse functions given their high level of amino acid sequence conservation. The first identified member of the family, elav of Drosophila is a vital gene. Mutations in the second Drosophila elav paralog, rbp9, are viable but female sterile. No alleles of fne were previously available. FNE protein is normally present in the cytoplasm of all neurons throughout development. Here we describe the generation and characterization of fne(null) mutations by homologous recombination. In contrast to elav and similar to rbp9, fne(null) mutants are viable, but exhibit a specific and fully penetrant fusion of the ß-lobes in their mushroom bodies (MB), a paired neuropil of the central brain involved in a variety of complex behaviors. Mutant males have reduced courtship indices, but normal short- and long-term courtship memory. Our data show that fne has specific functions which are non-overlapping with the other two family members, namely in courtship behavior and in the development of the adult MB. The data further show that courtship memory does not require intact ß-lobes in the MB.
Assuntos
Corte , Proteínas de Drosophila/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Animais , Encéfalo/anatomia & histologia , Encéfalo/fisiologia , Drosophila melanogaster/genética , Deleção de Genes , Recombinação Homóloga , Masculino , Memória , Corpos Pedunculados/anatomia & histologia , Mutação , Neurópilo/citologia , Comportamento Sexual AnimalRESUMO
A Drosophila gene encoding a 351-amino acid-long predicted arginase (40% identity with vertebrate arginases) is reported. Interestingly, the third intron of the arginase gene includes the elav locus, whose coding sequence is on the complementary DNA strand to that of the arginase. Terrestrial vertebrates produce two arginases from duplicated genes. One form, essentially present in the liver, is a key enzyme of the urea cycle and eliminates excess ammonia through the excretion of urea. The function of the extrahepatic arginase, more ubiquitous, is not well understood. In macrophages, arginase competes with nitric-oxide synthase, which converts arginine into nitric oxide. Most organisms, including insects, produce only one type of arginase, whose function is not centered on ammonia detoxification. A Drosophila cDNA encoding a predicted arginase was isolated. It produces a 1.3-kilobase transcript present with highest levels toward the end of embryogenesis and thereafter. During embryogenesis, the arginase transcripts localize to the fat body. The first mutant allele of the Drosophila arginase gene was identified. It is predicted to produce a 199-amino acid-long C-terminally truncated protein, likely to be inactive. Preliminary characterization of the mutation shows that this recessive allele causes a developmental delay but does not affect viability.
Assuntos
Arginase/biossíntese , Arginase/genética , Drosophila/enzimologia , Drosophila/genética , Íntrons , Alelos , Sequência de Aminoácidos , Animais , Northern Blotting , Aberrações Cromossômicas , Cruzamentos Genéticos , DNA Complementar/metabolismo , Drosophila/embriologia , Corpo Adiposo/metabolismo , Biblioteca Gênica , Hibridização In Situ , Fígado/metabolismo , Macrófagos/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Óxido Nítrico Sintase/metabolismo , Fases de Leitura Aberta , Biossíntese de Proteínas , RNA/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Ureia/metabolismoRESUMO
The P-element-mediated gene transfer system was used to introduce Drosophila teissieri 5S genes into the Drosophila melanogaster genome. Eight transformed D. melanogaster strains that carry D. teissieri 5S mini-clusters consisting of 9-21 adjacent 5S units were characterized. No genetic exchanges between D. melanogaster and D. teissieri 5S clusters were detected over a 2-year survey of the eight strains. The occurrence of small rearrangements within the D. melanogaster 5S cluster was demonstrated in one of the transformed strains.
Assuntos
Drosophila/genética , Família Multigênica , RNA Ribossômico 5S/genética , RNA Ribossômico/genética , Animais , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Rearranjo Gênico , Técnicas Genéticas , Filogenia , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Transformação GenéticaRESUMO
The 5S genes of the eight species of the D. melanogaster subgroup have been mapped. The spacers, in contrast with coding regions, differ markedly between most species. One 5S gene unit has been sequenced for both D. simulans and D. teissieri. The mature 5S RNA region in these two species is identical to the corresponding region of D. melanogaster. Only 5 nucleotide variations occur between the D. melanogaster and D. simulans 5S gene spacers. The spacer in D. teissieri is very different. Only two segments, located one at each side of the coding region, are clearly homologous to corresponding sequences of D. melanogaster and D. simulans.
Assuntos
DNA/genética , Drosophila melanogaster/genética , Drosophila/genética , RNA Ribossômico/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA/isolamento & purificação , Enzimas de Restrição do DNA , DNA Ribossômico , Hibridização de Ácido Nucleico , Plasmídeos , Especificidade da EspécieRESUMO
The Ubx and abd-A gene products are required for proper development of thoracic and abdominal structures in Drosophila. We expressed LexA-Ubx and LexA-abdA fusion proteins in yeast. These proteins activated expression of target genes that carried either upstream LexA operators or upstream Ubx binding sites. Both proteins contain homeodomains. Experiments with mutant fusion proteins show that the homeodomain is not required for the proteins to form dimers or enter the nucleus, and that, when DNA binding is provided by the LexA moiety, the homeodomain is not required for gene activation. Our results suggest that the homeodomain is necessary for these proteins to bind Ubx sites, but that the homeodomain does not contact DNA exactly like bacterial helix-turn-helix proteins. Finally, our data suggest that gene activation by these proteins is a simple consequence of their binding to DNA, while negative gene regulation requires that these proteins act together with other Drosophila gene products.
Assuntos
Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes Homeobox , Serina Endopeptidases , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Clonagem Molecular , Análise Mutacional de DNA , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5'-flanking region (54-55 bp), the 3'-flanking region (21-22 bp), and the internal spacer (149-206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6-17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps.
Assuntos
Drosophila/genética , Evolução Molecular , Genes de Insetos , RNA Ribossômico 5S/genética , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Ribossômico , Dados de Sequência Molecular , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Regulated gene activity is crucial to the formation and function of the nervous system. It is well known that gene regulation can occur at the transcriptional, post-transcriptional, translational, and post-translational levels. In this review our focus has been on the post-transcriptional regulation in neurons and on neural-specific RNA binding proteins that may be involved in post-transcriptional modulation of gene activity. We have taken advantage of this opportunity to review our work on the elav gene of Drosophila melanogaster which encodes a neural-specific RNA binding protein and relate it to other members of this elav-like gene family. We report new data that suggests that elav is post-transcriptionally regulated and we demonstrate that below-threshold levels of ELAV protein severely affects neuronal differentiation.