Chromatin Structure of Ribosomal RNA Genes in Dipterans and Its Relationship to the Location of Nucleolar Organizers

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.Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paul

The role of repetitive DNA in the chromosome termini of Rhynchosciara americana (Diptera: Sciaridae)

A localização cromossômica do DNA ribossômico (rDNA) foi estudada em cromossomos politênicos e em tecidos diplóides de quatro espécies de sciarídeos: Trichosia pubescens; Rhynchosciara americana; R. milleri e Schwenkfeldina sp.. Resultados de hibridação em cromossomos mitóticos mostraram a existência de um único locus de rDNA; entretanto, sondas ribossomais hibridaram em mais de uma região dos cromossomos politênicos em todas as espécies analisadas devido à adesão de micronucléolos em regiões específicas dos cromossomos. Os micronucléolos são estruturas arredondadas que contêm, provavelmente, DNA extracromossômico transcricionalmente ativo. Em T. pubescens, o rDNA está predominantemente localizado nas secções cromossômicas X-10 e X-8. Em R. americana o rDNA está freqüentemente associado à heterocromatina centromérica dos cromossomos X, C, B e A, e também às secções X-1 e B-13. Sondas ribossômicas em R. milleri hibridaram, em alta freqüência, em regiões teloméricas e pericêntricas de cromossomos politênicos. Schwenkfeldina sp. apresenta uma distribuição incomum do rDNA em núcleos politênicos, caracterizada pela adesão de micronucléolos em muitas regiões cromossômicas. Os resultados mostraram que os micronucléolos estão preferencialmente associados à heterocromatina intercalar ou terminal de todos os sciarídeos analisados e, dependendo da espécie, estão aderidos a um número pequeno (Trichosia), moderado (Rhynchosciara) e grande (Schwenkfeldina sp.) de sítios em cromossomos politênicos. Este trabalho também descreve a caracterização de seqüências presentes nas extremidades cromossômicas de R. americana, que se iniciou através da triagem de uma microbiblioteca plasmidial, feita a partir de uma extremidade microdissecada B-1. Uma repetição do tipo satélite foi identificada e sua composição de bases, estrutura genômica e localização cromossômica são semelhantes às repetições teloméricas complexas de Nematocera que já foram descritas. Contudo, dados obtidos em outras espécies de Rhynchosciara, assim como a localização desse satélite e da transcriptase reversa, sugerem que o elemento repetitivo caracterizado neste trabalho não atinge as extremidades dos cromossomos. A caracterização de seqüências terminais e subterminais presentes nos cromossomos de R. americana foi continuada através da triagem de uma biblioteca de DNA desse díptero clonada em fagos Dash. Escolhemos como sonda para a triagem o clone pRaM47.33, representativo do elemento repetitivo M22, caracterizado em R. americana. Foram analisados cerca de 12kb de um único inserto de fago, que continha, alem das repetições M22, uma nova repetição de 16pb, organizada em tandem e que denominamos de M16. Resultados de hibridações in situ revelaram a presença da repetição M16 nas 5 extremidades cromossômicas não-telocêntricas de R. americana. Essa repetição também foi utilizada como sonda em uma outra triagem da mesma biblioteca genômica, o que permitiu a seleção e análise de aproximadamente 50kb de DNA cromossômico terminal de R. americana. Encontramos também, ao longo dessas 50kb de DNA analisado, repetições de 414pb anteriormente caracterizadas em R. americana; parte de seqüências do transposon Ramar1 e do retrotransposon RaTART . Além disso, foram observadas também seqüências que não apresentam semelhança significativa com seqüências depositadas no banco de dados GenBank, e que tampouco apresentam motivos repetitivos. Os resultados obtidos apontam para a possibilidade de que a região telomérica de R. americana seja composta por mais de um tipo de elemento repetitivo.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. This work also describes the characterization of chromosome end sequences of Rhynchosciara americana, initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome end. We report the identification and sequencing of an R. americana satellite displaying base composition, genomic structure and chromosomal localization similar to the complex telomeric repeats of Nematocera that have previously been characterized. However, data obtained in other Rhynchosciara species, as well as distinct chromosomal localization of satellite and reverse transcriptase loci in R. americana, suggest that the repetitive element characterized does not reach the very end of the chromosome. The characterization of chromosome end sequences of Rhynchosciara americana continued with the screening of a phage library made with its genomic DNA. We choose pRaM47.33, a clone whose insert is a repetitive microsatellite characterized in the subtelomeric region of R. americana chromosomes, as a probe for the screening. We analyzed 12kb of a single phage insert, composed of M22 tandem arrays and a new microsatellite which was 16pb long, arranged in tandem (named M16). In situ hybridization showed the presence of M16 repeats in the five telomeric termini of R. americana chromosomes. The M16 repeat was used as a probe in another screen of the same phage library, which allowed us to analyze approximately 50kb of terminal DNA. We find that repetitive sequences, such as the 414pb repeat previously characterized in R. americana and stretches of Ramar1 and RaTART mobile elements, also characterized in R. americana, compose the subtelomeric region of R. americana chromosomes. Additionally, we find sequences that do not match sequences in the GenBank database and do not present repetitive motifs. Our results suggest that the telomeric regions of R. americana chromosomes are composed of more than one type of repetitive sequence

Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

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.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Unusually short tandem repeats appear to reach chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Ribosomal RNA gene insertions in the R2 site of Rhynchosciara (Diptera: Sciaridae)

Ribosomal RNA genes of most insects are interrupted by R1/R2 retrotransposons. The occurrence of R2 retrotransposons in sciarid genomes was studied by PCR and Southern blot hybridization in three Rhynchosciara species and in Trichosia pubescens. Amplification products with the expected size for non-truncated R2 elements were only obtained in Rhynchosciara americana. The rDNA in this species is located in the proximal end of the X mitotic chromosome but in the salivary gland is associated with all four polytene chromosomes. Approximately 50% of the salivary gland rDNA of most R. americana larval groups analysed had an insertion in the R2 site, while no evidence for the presence of R1 elements was found. In-situ hybridization results showed that rDNA repeat units containing R2 take part in the structure of the extrachromosomal rDNA. Also, rDNA resistance to Bal 31 digestion could be interpreted as evidence for nonlinear rDNA as part of the rDNA in the salivary gland. Insertions in the rDNA of three other sciarid species were not detected by Southern blot and in-situ hybridization, suggesting that rDNA retrotransposons are significantly under-represented in their genomes in comparison with R. americana. R2 elements apparently restricted to R. americana correlate with an increased amount of repetitive DNA in its genome in contrast to other Rhynchosciara species. The results obtained in this work together with previous results suggest that evolutionary changes in the genus Rhynchosciara occurred by differential genomic occupation not only of satellite DNA but possibly also of rDNA retrotransposons.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Funda ao de Amparo a Pesquisa do Estado de Sao Paulo

Chromatin structure of rRNA genes in the salivary gland of <i>D. melanogaster</i>.

<p>(A) Southern-blot hybridization analysis of rDNA fragments obtained by <i>Hae</i>III digestions of genomic DNA from salivary gland chromatin photo-reacted in the presence of psoralen (ps) and from control, untreated DNA (c). The whole insert of the p<i>Dm</i>-238 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044006#pone.0044006-Roiha1" target="_blank">[18]</a> was used as a probe. (B) Each lane from the same image shown on the left was processed for brightness, contrast, and signal intensity in order to improve visualization of radioactive signals. The asterisks indicate the two bands detected when Southern-blot hybridization using the same membrane was performed with the <i>Chironomus</i> 26S rDNA probe <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044006#pone.0044006-Gorab1" target="_blank">[47]</a>.</p

Chromatin structure of rRNA genes in the salivary gland, ovary and testis of <i>Rhynchosciara americana</i>.

<p>Southern-blot hybridization analysis of rDNA fragments obtained by digesting with <i>Eco</i>RI/<i>BglI</i> genomic DNA from salivary gland (sg), ovary (ov) and testis chromatin (te) photo-reacted in the presence of psoralen and also from control, untreated DNA (c). Hybridization bands specific for canonical rDNA units (R2−) and those interrupted by R2 insertions (R2+) are indicated.</p

Chromosomal distribution of trimethyl H3K9 in <i>R. americana</i>.

<p>(A) Indirect immunofluorescence; the insert shows labeling details of chromosome sections A10/12 comprising pericentric heterochromatin of chromosome A. (B) The corresponding phase contrast image. Polytene chromosome sections were identified according to a previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044006#pone.0044006-Madalena1" target="_blank">[8]</a>.</p