RESUMEN
The diptera Rhynchosciara americana (sciaridae) is an important model organism in polyteny and gene amplification research, but up to now a limited amount of data regarding DNA sequences and molecular aspects of this species is available. Considering the importance of going further on the DNA puffs biological meaning, we proposed to generate EST sequences from a DNA library constructed from salivary glands. After their categorization in gene ontology terms, they were used to construct an 'electronic Northern' that represents a general view of the salivary gland metabolic status in an important phase of larval development: the spinning of communal cocoon. In this phase occurs the last polytene DNA replication cycle concomitantly with the specific loci amplification related to protein secretion.
Asunto(s)
Dípteros/genética , Etiquetas de Secuencia Expresada , Secuencia de Aminoácidos , Animales , Codón , ADN Complementario , Dípteros/metabolismo , Regulación del Desarrollo de la Expresión Génica , Insectos/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , ARN Mensajero , Glándulas Salivales/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.
Asunto(s)
Escarabajos/enzimología , Escarabajos/genética , Luciferasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Escherichia coli/genética , Luminiscencia , Datos de Secuencia Molecular , Filogenia , Homología de Secuencia de AminoácidoRESUMEN
We have mapped the only transcription unit known to be present in the C-8 DNA puff of Rhynchosciara americana and describe the isolation and sequence of a cDNA clone, pRa C-8-22, which contains a nearly complete copy of the mRNA transcribed from this DNA puff and part of the sequence of genomic clone BSC8-0.9, which contains the promotor region and the remainder of the transcription unit. The characteristics of the protein predicted from the ORF present in the cDNA indicate that it is unique and secreted.
Asunto(s)
ADN/genética , Dípteros/genética , Proteínas de Insectos , Proteínas y Péptidos Salivales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas y Péptidos Salivales/metabolismo , Transcripción GenéticaRESUMEN
The synchronous development of a sibling group of Rhynchosciara larvae enables us to follow the relationship between the local transcription and extrareplication of C3 and C8 DNA puffs. Although DNA amplification at these two loci takes place during the last cycle of DNA duplication in salivary gland chromosomes, a different timing of puff expression was observed for the two regions analysed. C3 puff transcription is a late event in relation to the C8 counterpart. We present evidence that this might be a consequence of the different firing of DNA amplification in both loci. No signs of DNA rearrangements were detected with probes that extend the previously analysed regions.
Asunto(s)
Replicación del ADN/fisiología , Dípteros/genética , Amplificación de Genes/fisiología , Transcripción Genética/fisiología , Animales , Northern Blotting , Southern Blotting , ADN/fisiología , Sondas de ADN , Drosophila/genética , Biblioteca de Genes , Larva , Glándulas SalivalesRESUMEN
We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.
Asunto(s)
Replicación del ADN , Dípteros/genética , Amplificación de Genes , Animales , Cromosomas/ultraestructura , Clonación Molecular , Dípteros/ultraestructura , Larva , Secuencias Repetitivas de Ácidos Nucleicos , Replicón , Saccharomyces cerevisiae/genética , Glándulas Salivales/ultraestructuraRESUMEN
Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.
Asunto(s)
Cromosomas/ultraestructura , Dípteros/genética , Amplificación de Genes , Glándulas Salivales/ultraestructura , Animales , ADN , Dípteros/crecimiento & desarrollo , Larva , Hibridación de Ácido NucleicoRESUMEN
Electrophoretic analysis of 3H-RNA obtained from the proximal sections of Rhynchosciara salivary glands at two distinct developmental periods, one characterized by the presence and the other by the absence of the giant B-2 DNA puff, revealed that the appearance of a 14S poly(A)+ RNA is correlated with the opening of this puff. That this RNA species is transcribed from this puff is indicated by the fact that it is found in RNA extracted from B-2 puffs obtained by microdissection. This confirmed by the specific hybridization of the 14S poly(A)+ RNA to the B-2 locus. Our data indicate that the polyadenylation process takes place at the chromosome level, and that the nuclear sap is not an important compartment in the transport of polyadenylated RNA from the chromosome to the cytoplasm. The kinetics of migration of the 14S species to the cytoplasm were studied; the data indicate that this process is very rapid and, in addition, that the 14S RNA is unstable.
Asunto(s)
ADN/metabolismo , Dípteros/genética , Marcadores Genéticos , Transcripción Genética , Animales , Citoplasma/metabolismo , Dípteros/metabolismo , Amplificación de Genes , Poli A/metabolismo , ARN/metabolismo , Glándulas Salivales/ultraestructuraRESUMEN
The distribution of fast, intermediate and slow renaturing fractions of Rhynchosciara americana DNA was examined in the polytene salivary gland chromosomes by in situ hybridization. Heterochromatic areas readily hybridized but hybrid formation in the euchromatin depended more on the repetitiveness of the RNA probe.
Asunto(s)
Cromosomas/ultraestructura , ADN/genética , Dípteros/genética , Animales , Secuencia de Bases , Cromatina/ultraestructura , Heterocromatina/ultraestructura , Hibridación de Ácido Nucleico , Glándulas Salivales/ultraestructuraRESUMEN
A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and alpha-amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs. Autoradiographic studies carried out in the presence or absence of the toxin alpha-amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in alpha-amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.