Eduardo Gorab

Reverse transcriptase-related proteins in telomeres and in certain chromosomal loci of Rhynchosciara (Diptera: Sciaridae)

The localization of reverse transcriptase-related proteins in polytene chromosomes of dipterans was investigated using previously characterized antibodies to a recombinant polypeptide containing conserved motifs of insect reverse transcriptases. The immunoreactions were carried out with polytene chromosome squashes of eight sciarids, one chironomid and three Drosophila species. Telomeric staining was regularly observed on chromosomes of the sciarid Rhynchosciara americana under normal growth conditions. Five of eight chromosomal tips were labelled except for the heterochromatic ends that are occasionally found associated forming a chromocentre in the salivary gland. Reverse transcriptase-related proteins were detected at chromosomal tips of young larvae and remained bound to the telomeres throughout larval development. As in salivary gland chromosomes, five non-telocentric ends of the chromosomes from Malpighian tubules of R. americana appeared clearly stained with anti-reverse transcriptase. The occurrence of telomeric reverse transcriptase in R. americana correlates with the presence of RNA in addition to an unusual enrichment with homopolymeric dA/dT DNA associated with the telomeric heterochromatin. The antibodies also reacted with a few interstitial sites in chromosomes of four Rhynchosciara species, one band overlapping the histone gene locus of three species in the americana -like group. The results provide evidence for a reverse transcriptase-related protein as a constitutive component in telomeres of R. americana and also in certain interstitial loci of Rhynchosciara species in which RNA was immunologically detected in the form of RNA:DNA hybrids.

Antibodies against the D-domain of a Chironomus ecdysone receptor protein react with DNA puff sites in Trichosia pubescens

Abstract. An antiserum (called AScE/D) against the semiconserved D-domain of a Chironomus tentans ecdysone receptor protein (cEcR) gave indirect immunofluorescence signals at DNA puff sites in Trichosia pubescens. The signals varied in maximum intensity at different DNA puff sites. Control experiments using the secondary rhodamine-labeled anti-rabbit IgG alone, preimmune serum, affinity purified AScE/D (called pABcE/D) and AScE/D preabsorbed with expressing bacterial extract or highly purified bacterially expressed cEcR indicated that the signals obtained at these chromosomal sites were likely to be due to specific interaction between an endogenous sciarid EcR and antibodies against cEcR. This conclusion was supported by observation of signals at certain Ec-inducible primary RNA puff sites. AScE/D signals began to appear at DNA puff sites during L3, the stage when amplification initiates, but at most sites their mean intensity was low and not statistically significant. Sites with AScE/D signals of significant mean intensity at this stage already showed evidence of transcription. The number and strength of transcription signals increased during L4. Comparison of the developmental course of signals for AScE/D, DNA synthesis, RNA presence/synthesis, and puff size for several DNA puffs during late larval- prepupal development showed a closer relationship of AScE/D signals with the initiation of RNA synthesis than with the initiation of DNA synthesis. Therefore, although we cannot absolutely eliminate a direct involvement of EcR in the amplification process at some sites, this investigation gives stronger support for its direct involvement in transcription. Since AScE/D signals are observed at DNA puff sites from the time the latter begin amplification/transcription through their regression, it appears that Ec and EcR are necessary as a sustained stimulus at these regions.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

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.

The chromosomes of Rhynchosciara baschanti (Diptera: Sciaridae): molecular cytogenetic comparisons with taxa in the americana-like group.

Polytene chromosome analysis is presented for Rhynchosciara baschanti, a species belonging to the americana-like group of Rhynchosciara. R. baschanti chromosomes show morphological differences in centromeric and telomeric regions compared to two other members within the group, R. americana and R. hollaenderi. In addition, fixed band and autosomal inversion differences were noted. Physical mapping data showed synteny among the taxa under study for DNA puffs and single-copy or histone gene probes, whereas rDNA and poly-(r)A probes showed different diagnostic patterns. The activity of developmentally active genes and the pattern of thymidine incorporation into DNA puff sites of R. baschanti are consistent with those found in the two previously studied species, except for lower levels of expression at some of these sites. These results suggest that differential duplication of specific DNA sequences, in particular repetitive and homopolymeric DNA, has played a role in the chromosomal evolution of these Rhynchosciara species. Inversions and band dimorphisms have also occurred, but the processes leading to their maintenance and fixation appear to have been slow, since these three species are in general chromosomally monomorphic.

The localization of ribosomal DNA in Sciaridae (Diptera: Nematocera) reassessed

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.

Chromatin structure of ribosomal genes in Chironomus thummi (Diptera: Chironomidae): tissue specificity and behaviour under drug treatment

In eukaryotes the ribosomal gene population shows two different states in terms of chromatin structure. One subset is organized as nucleosomes (silent copies) while the other has a non-nucleosomal configuration (active copies). Insect cells are not the exception and this bimodal distribution of ribosomal chromatin also occurs in salivary gland cells, and cells of other larval tissues, of the midge Chironomus thummi. In run-on experiments on salivary glands cells we confirmed that transcribed rRNA genes show a non-nucleosomal configuration. The proportion of rRNA genes adopting an open, non-nucleosomal configuration was found to be tissue-dependent, suggesting that the population of unfolded ribosomal chromatin in C. thummi was established during cell differentiation. We propose that cell differentiation determines the fraction of non-nucleosomal rRNA gene copies and thus defines the range of possible rRNA synthesis rates in a particular cell type. In the salivary gland the fraction of unfolded chromatin was not significantly affected when transcription was repressed. However, transcription activation by pilocarpine led to a moderate increase in this fraction. These findings indicate that, in addition to a possible increase in the number of RNA-polymerases per transcribing rDNA unit, the proportion of transcribed ribosomal genes in differentiated cells can be modulated in response to an exceptional rRNA synthesis requirement.

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.

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.

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.

In memoriam—Crodowaldo Pavan

Crodowaldo Pavan (Fig. 1) was born in Campinas, in the state of São Paulo, Brazil, on December 1, 1919. In 1941, he obtained his Bachelor’s degree in Natural History at the Faculty of Philosophy, Sciences and Letters (FFCL), University of São Paulo (USP). He concluded his Ph.D. in 1944, working in the Department of General Biology of the FFCL under the supervision of Prof. André Dreyfus, director of the FFCL (1943–1947). In 1943, Pavan started collaboration with Theodosius Dobzhansky and a school of Animal Evolutionary Genetics was born in Brazil. In that year, they published two papers, the first on Drosophila species reared in the laboratory (Dobzhansky and Pavan 1943a) and a second on chromosomal evolution of Drosophila species (Dobzhansky and Pavan 1943b). Other results obtained by Pavan, Dobzhansky and colleagues were published in later years. In 1951, Pavan collected the lower dipteran Rhynchosciara angelae (Diptera: Sciaridae; renamed Rhynchosciara americana) in coastal regions of the state of São Paulo. This fly was particularly suitable for cytogenetic studies, given its oversized polytene chromosomes. After identifying local decondensation patterns in polytene chromosomes from different tissues of Rhynchosciara, he and Marta MMEE Breuer concluded that all the cells of a multicellular organism have the same “disc patterns” (namely the same gene set). However, there were differences in the degree of condensation of “discs” (polytene bands) in certain chromosome sections from distinct cell types (Pavan and Breuer 1952). Although this paper is considerably simpler than Beermann’s (1952) outstanding treatise made with the lower dipteran Chironomus, both were in agreement and the view of differential gene expression as the central process in cell differentiation and development has since then been firmly established. Perhaps the most remarkable achievement of Pavan’s scientific career would come one year later when, together with Marta Breuer, he observed an increase in the DNA content within certain polytene bands, uncoupled from polyteny, during larval development of R. angelae (Fig. 2). According to the authors, “Another point suggested by our data in Rhynchosciara is related to the theory of the constancy of the amount of DNA in all cells of a species.... The observations on bulb formation suggest to us that this theory may not hold in all cases. We observed an increase of the DNA content in certain bands (Feulgen reaction), which reaches an amount several times greater than that which the band had before going through the process. This increase of DNA is however not uniform along the length of the chromosome. There are some bands where the increase of DNA is very great while in some other bands there is no evidence of increase or decrease at all. The increases of DNA in the regions which we have studied in detail.... are independent of the process of polytenization”. The paper containing this observation (Breuer and Pavan 1955) presents the first description of developmentally controlled gene amplification in specific genomic regions undergoing puffing, later called “DNA puffs”. Pavan said on several occasions that, in discussions at scientific meetings, Prof. Beermann as well as other researchers expressed their doubts about that finding. This was not surprising, since the observations challenged the invariability of the amount of genetic material in the cells of an organism, a dogma of that time. Also contributing to the Chromosoma (2009) 118:401–403 DOI 10.1007/s00412-009-0217-5