RESUMO
Micronucleoli are among the structures composing the peculiar scenario of the nucleolus in salivary gland nuclei of dipterans representative of Sciaridae. Micronucleolar bodies contain ribosomal DNA and RNA, are transcriptionally active and may appear free in the nucleoplasm or associated with specific chromosome regions in salivary gland nuclei. This report deals with an extreme case of nucleolar fragmentation/dispersion detected in the salivary gland of Schwenkfeldina sp. Such a phenomenon in this species was found to be restricted to cell types undergoing polyteny and seems to be differentially controlled according to the cell type. Furthermore, transcriptional activity was detected in virtually all the micronucleolar bodies generated in the salivary gland. The relative proportion of the rDNA in polytene and diploid tissues showed that rDNA under-replication did not occur in polytene nuclei suggesting that the nucleolar and concomitant rDNA dispersion in Schwenkfeldina sp. may reflect a previously hypothesised process in order to counterbalance the rDNA loss due to the under-replication. The chromosomal distribution of epigenetic markers for the heterochromatin agreed with early cytological observations in this species suggesting that heterochromatin is spread throughout the chromosome length of Schwenkfeldina sp. A comparison made with results from another sciarid species argues for a role played by the heterochromatin in the establishment of the rDNA topology in polytene nuclei of Sciaridae.
Assuntos
Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Dípteros/genética , Glândulas Salivares/citologia , Animais , Fragmentação do DNA , Replicação do DNA , DNA Ribossômico/metabolismo , Dípteros/metabolismo , Heterocromatina/metabolismo , Cromossomos Politênicos/metabolismo , RNA Ribossômico/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Dipterans exhibit a remarkable diversity of chromosome end structures in contrast to the conserved system defined by telomerase and short repeats. Within dipteran families, structure of chromosome termini is usually conserved within genera. With the aim to assess whether or not the evolutionary distance between genera implies chromosome end diversification, this report exploits two representatives of Sciaridae, Rhynchosciara americana, and Trichomegalosphyspubescens. METHODS: Probes and plasmid microlibraries obtained by chromosome end microdissection, in situ hybridization, cloning, and sequencing are among the methodological approaches employed in this work. RESULTS: The data argue for the existence of either specific terminal DNA sequences for each chromosome tip in T. pubescens, or sequences common to all chromosome ends but their extension does not allow detection by in situ hybridization. Both sciarid species share terminal sequences that are significantly underrepresented in chromosome ends of T. pubescens. CONCLUSIONS: The data suggest an unusual terminal structure in T. pubescens chromosomes compared to other dipterans investigated. A putative, evolutionary process of repetitive DNA expansion that acted differentially to shape chromosome ends of the two flies is also discussed.
Assuntos
Cromossomos de Insetos/genética , Dípteros/genética , Animais , Sequência de Bases , DNA/biossíntese , Biblioteca Gênica , Microdissecção , Plasmídeos/genética , Cromossomos Politênicos/genéticaRESUMO
Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.
RESUMO
The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.
Assuntos
Benzotiazóis/análise , Cromossomos de Insetos/química , DNA/análise , Dípteros/química , Corantes Fluorescentes/análise , Heterocromatina/química , Quinolinas/análise , Animais , Anticorpos/análise , Cromossomos de Insetos/ultraestrutura , Dípteros/ultraestrutura , Drosophila melanogaster/química , Drosophila melanogaster/ultraestrutura , Heterocromatina/ultraestrutura , Imunoquímica/métodos , Microscopia de Fluorescência/métodos , Cromossomos Politênicos/química , Cromossomos Politênicos/ultraestrutura , Coloração e Rotulagem/métodosRESUMO
Nucleoli, nuclear organelles in which ribosomal RNA is synthesized and processed, emerge from nucleolar organizers (NORs) located in distinct chromosomal regions. In polytene nuclei of dipterans, nucleoli of some species can be observed under light microscopy exhibiting distinctive morphology: Drosophila and chironomid species display well-formed nucleoli in contrast to the fragmented and dispersed nucleoli seen in sciarid flies. The available data show no apparent relationship between nucleolar morphology and location of NORs in Diptera. The regulation of rRNA transcription involves controlling both the transcription rate per gene as well as the proportion of rRNA genes adopting a proper chromatin structure for transcription, since active and inactive rRNA gene copies coexist in NORs. Transcription units organized in nucleosomes and those lacking canonical nucleosomes can be analyzed by the method termed psoralen gel retarding assay (PGRA), allowing inferences on the ratio of active to inactive rRNA gene copies. In this work, possible connections between chromosomal location of NORs and proportion of active rRNA genes were studied in Drosophila melanogaster, and in chironomid and sciarid species. The data suggested a link between location of NORs and proportion of active rRNA genes since the copy number showing nucleosomal organization predominates when NORs are located in the pericentric heterochromatin. The results presented in this work are in agreement with previous data on the chromatin structure of rRNA genes from distantly related eukaryotes, as assessed by the PGRA.
Assuntos
Cromatina/genética , Dípteros/genética , Região Organizadora do Nucléolo/genética , RNA Ribossômico/genética , Animais , Chironomidae/genética , DNA Ribossômico/genética , Drosophila melanogaster/genética , Ensaio de Desvio de Mobilidade Eletroforética , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Metilação , Sequências Repetitivas de Ácido NucleicoRESUMO
Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome micro-dissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini.
Assuntos
Cromossomos de Insetos , Dípteros/genética , Repetições de Microssatélites , Telômero , Animais , Composição de Bases , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Dados de Sequência MolecularRESUMO
The characterisation of sequences at chromosome ends of Rhynchosciara americana was continued with the screening of a genomic library using as a probe a short repeat identified in a previous report (M-22, 22 bp) which was found to be specific for noncentromeric termini of this species. Simple repeats, complex tandem and apparently dispersed repeats were present in the genomic clones analysed. Repetitive sequences do not define individual chromosome tips as they were found in all noncentromeric ends. A novel and unusually short tandem repeat type for dipteran chromosome ends (named M-16) composed of 16 nucleotides and frequently associated with M-22 arrays was characterised in this work. Islands of M-16 and M-22 tandem repeats were found in all the genomic clones analysed. Individual probes representative of each repetitive element hybridised not only to all noncentromeric ends of R. americana chromosomes but also to inter-telomeric bridges. This contrasted with the other repeat types which displayed sub-telomeric localisation as seen by double detection of hybridised probe and telomeric reverse transcriptase. Some stretches composed of M-16 and M-22 tandem repeats localised in different regions of the analysed genomic clones were either identical or showed sequence similarity that was unexpectedly higher than the mean sequence similarity observed among repeats within each of their tandem arrays. The occurrence of segmental duplications, as deduced by sequence analyses involving the two repeats that appeared to reach chromosome ends, might indicate the involvement of this type of duplication process in the chromosome end maintenance in this species.
Assuntos
Cromossomos de Insetos/genética , Dípteros/genética , Repetições de Microssatélites , Animais , Mapeamento Cromossômico , Dados de Sequência Molecular , Duplicações Segmentares GenômicasRESUMO
In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed "RaTART" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.
Assuntos
Cromossomos/química , Dípteros/genética , Retroelementos , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Cromossomos/ultraestrutura , DNA/química , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , Glândulas Salivares/química , Telômero/ultraestruturaRESUMO
Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.
Assuntos
DNA/química , Dípteros/genética , Drosophila melanogaster/genética , RNA/química , Animais , Anticorpos Monoclonais , Cromossomos de Mamíferos/genética , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Hibridização In Situ , Conformação de Ácido Nucleico , Poli A/química , Poli A/genética , Poli A/imunologia , Poli U/química , Poli U/genética , Poli U/imunologiaRESUMO
DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.
Assuntos
Dípteros/genética , Expressão Gênica/genética , Proteínas de Insetos/genética , Proteínas e Peptídeos Salivares/genética , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Sequência de Bases , Western Blotting , Cromossomos/metabolismo , Cromossomos/ultraestrutura , Replicação do DNA/genética , DNA Complementar/química , Dípteros/metabolismo , Feminino , Biblioteca Gênica , Proteínas de Insetos/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Timidina/metabolismo , Fatores de Tempo , Trítio/análiseRESUMO
The characterisation of chromosome end (terminal and sub-terminal) sequences of Rhynchosciara americana chromosomes was continued with the screening of a plasmid library made of amplified DNA fragments from a microdissected chromosome tip. An insert chosen for analysis hybridised to two chromosome ends and contains two microsatellite arrays in close vicinity to a sequence (named M-47), part of which is significantly similar to minisatellites of Salmonidae that are frequently present in the vicinity of microsatellite arrays. PCR results using a single primer representative of M-47 elements suggest that they are also repetitive in Rhynchosciara genomes. In addition, total single primer PCR products hybridised to the non-telocentric end subset of R. americana chromosomes. Another plasmid microlibrary made of chromosome tips amplified by a single M-47 primer was screened for repeats of Rhynchosciara chromosomes. Selected inserts that hybridised strongly to non-telocentric ends of R. americana and R. hollaenderi have a tandem array of 22 bp repeats (M-22). There is sequence divergence among M-22 repeats but their mean similarity is significantly high in relation to the M-22 consensus sequence derived from the cloned tandem array. M-22 elements lie distal to the 414 bp sub-telomeric satellite array characterised previously as suggested by double labelling for M-22 hybridisation and reverse transcriptase. M-47 elements, formerly identified in Salmonidae, thus contribute to specify unusually short repeats composing the sub-telomeric structure of two Rhynchosciara species.
Assuntos
Cromossomos , Dípteros/genética , Repetições de Microssatélites , Animais , Sequência de Bases , DNA Satélite/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNARESUMO
The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.
Assuntos
DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Dípteros/genética , Dípteros/metabolismo , Animais , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromossomos/genética , Cromossomos/metabolismo , Feminino , Hibridização in Situ Fluorescente , Masculino , Sondas RNARESUMO
The chromosomal response to temperature shock in Rhynchosciara americana is similar to that observed in other Diptera. After a 33 degrees C/90 min or a 36 degrees C/30 min shock the reaction for RNA polymerase II (RpII) is enhanced at five loci. The most prominent of these was identified by in situ hybridization as the site of the hsp70 gene. At 33 degrees C, an accumulation of heat shock factor (HSF) and an increase in the level of RpII was observed at some heat shock loci after 5 min and reached a maximum after 15 min at most loci. The pattern of accumulation of HSF and RpII at individual heat shock loci was similar and their increases were generally coordinated among the loci. RpII gradually decreased at sites active prior to shock, the rate of decrease varying with the site. The B2 DNA puff retained RpII for a significant length of time while the histone locus still contained RpII after a shock of 90 min. With a 36 degrees C/30 min shock, the size of the heat shock puffs and the intensities of HSF and RpII peaked at 1-4 h post stress. The level of HSF declined rapidly after 1 h while the level of RpII remained high for an additional 4 h. The reaction of the DNA puffs to heat shock varied. Usually they did not regress completely and retained traces of RpII. BrdU incorporation continued at both amplifying and non-amplifying bands after shock but on average it appeared depressed for about 24 h post stress.