Specialized features of Rhynchosciara americana embryogenesis

Insect embryo development is a complex process which requires nuclear and cellular division, cell shape alteration, and cell movement. This process needs to be orchestrated in a specific spatial and temporal fashion. Different insect species, despite similarities, present distinct morphogenetic pathways. We used the dipteran R. americana as a comparative model for embryo morphogenesis studies, following embryo development with different histochemical and immunohistochemical procedures. Despite the phylogenetic proximity with D. melanogaster, R. americana presents a peculiar morphogenesis. We show that at the initial phases of development, from egg fertilization to blastoderm formation, R. americana is similar to Drosophila. The first cleavages are nuclear and cellularization only begins after nuclei spread throughout the egg’s cortex. However after this stage a series of cell movements establishes a short compact germ band anlage, which gastrulates in a pattern quite different from Drosophila. After gastrulation the germ band elongates anterior–posteriorly and segmentation occurs simultaneously along the embryo. Embryo development from egg fertilization to larva hatching takes about 12 days. Our results show that R. americana presents a different morphogenetic pathway which does not fit in the current short, intermediate or long germ band classification.     

Banding studies on the polytene chromosomes of Rhynchosciara hollaenderi

C-, Q-, H-, and Ag-banding have been carried out on the polytene chromosomes of Rhynchosciara hollaenderi. The results of these techniques are presented and are correlated with the molecular data which exists for Rhynchosciara polytene chromosomes. By systematic organization of both banding and molecular data, we have attempted to give as complete a picture as possible of the characteristics of the differentially banding regions. This presents an organized method of approaching mechanisms of banding in terms of structural and functional aspects of the intact chromosome. Polytene chromosomes are particularly suited for this type of analysis and with them, both developmental and evolutionary changes can be conveniently utilized for additional insights into the functions of banding regions.     

Feulgen-DNA absorption curves of polytene chromosome regions ofRhynchosciara americana

Summary Feulgen absorption spectra of polytene chromosome regions, differing in types of DNA composition and of chromatin compactness, were determined microspectrophotometrically in squashed salivary glands ofRhynchosciara americana. The absorption curves exhibited a secondary maximum at their ascending branch after the chromatins were maximally depurinated. The degree of shoulder prominence could not be specifically correlated with the packing state or even with richness in satellite repetitive DNA of the chromatins. It is assumed that other factors may exist which play a part in the phenomenon that gives rise to the Feulgen absorption shoulder characteristics. Differences in the extraction rate of the apurinic acid fragments due to their binding to specific non-histone proteins are suggested as contributing to the above-mentioned absorption patterns when comparing the various chromatin types.     

Embryonic expression of the engrailed homologue of Rhynchosciara americana

The segment polarity gene engrailed is involved in the determination of segment posterior identity in Drosophila. engrailed has been largely used for comparative developmental studies due to its evolutionary conservation from nematodes to humans. By in situ hybridization of an engrailed cDNA probe from Drosophila to polytene chromosomes of fourth instar larvae of Rhynchosciara americana we have shown that engrailed-like sequences must be localized in band 6 of chromosome A in this species. The pattern of engrailed protein expression during R. americana embryo development is diffuse at first evolving into a nuclear striped pattern after quite a length of time. In addition, our results suggest a possible developmentally regulated molecular modification of engrailed protein in R. americana embryos.     

A larval haemolymph protein in the eggs of Rhynchosciara americana

In Rhynchosciara americana larvae, a protein referred to as “protein 10” comprises one of the major plasma protein of the last instar. This protein was purified and an antiserum against it shows that an identical protein is present in the eggs from this fly. Protein 10 has an estimated molecular weight of 43,000 and an isoelectric pH of 6.6. Examination of protein 10 from eggs and from haemolymph by limited proteolysis indicates that the two are structurally identical. Protein 10 is synthesized in large amounts by the larval fat bodies up until the end of the feeding stage. Estimation by radial immunodiffusion shows that protein 10 is stored in the larval haemolymph, attaining a maximum level at the end of feeding stage and then decreasing until very little remains in the young adult. By the middle of the pupal stage the ovaries begin to sequester and acummulate the protein 10 which is deposited in the eggs.     

Characterisation of histones in the salivary glands of Rhynchosciara americana larvae

The histones from the salivary glands of Rhynchosciara americana larvae were identified. The electrophoretic patterns of the proteins studied resemble that of calf thymus histones, including the H₁ histone, which in Rhynchosciara has a lower electrophoretic mobility in urea/polyacrylamide gels but shows a molecular weight identical to the corresponding histone of calf thymus, as judged by SDS-polyacrylamide gel electrophoresis.     

The rate of DNA replication in the polytene chromosomes of Rhynchosciara angelae

The distribution pattern of 5-bromodeoxyuridine-labelled DNA from salivary glands of Rhynchosciara angelae upon caesium chloride gradient centrifugation was studied with DNA of different molecular weights. This pattern suggests a very low rate of DNA chain growth in polytene chromosomes. The rate of DNA chain growth was found to be 0.025 μm/min at 24 °C. The result was obtained through the development of a mathematical expression which took into account the distribution of the 5-bromodeoxyuridine-labelled DNA in CsCl gradients.DNA pulse-labelled for a short time sediments more slowly in alkaline sucrose gradients than DNA which has been labelled during a prolonged incubation. However, in neutral sucrose gradients the pattern of banding is the same for both DNAs. This indicates a discontinuity in the newly synthesized DNA strand, but not in the template strand. The transition of slow sedimenting to fast sedimenting DNA observed in alkaline sucrose gradients, occurs very slowly, as would be expected for a slow rate of DNA chain growth.The data obtained provided a means of comparing the number of replication points with the rate of DNA chain elongation and the length of S phase in Rhynchosciara.     

Acid lability of the hydrocarbon-deoxyribonucleoside linkages in 7,12-dimethylbenz[a]anthracene-modified deoxyribonucleic acid

DNA containing bound radioactive 7,12-dimethylbenz[a]anthracene was isolated from mouse fetal cell cultures exposed to this carcinogen. The carcinogen-deoxyriboside adducts within the DNA were found to be sensitive to acid-catalyzed hydrolysis. Adducts derived from reaction of a syn-dihydrodiol epoxide with deoxyadenosine residues in DNA were the most sensitive to acid and were hydrolyzed to yield a 1,2,3,4-tetrahydrotetraol of 7,12-dimethylbenz[a]anthracene under mild conditions. The structure of this tetraol was established by synthesis and mass spectrometry. Although definitive structures cannot be assigned at present to the nucleic acid adducts of this potent carcinogen, the present findings confirm and extend earlier work assigning partial structures to the major adducts.     

Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana

The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector gtB. Three different recombinants (gtRa1, gtRa23 and gtRa5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The gt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

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 -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 -amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in -amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with ³H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.This work was supported by a grant from the National Institutes of Health (GM 17590-03), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) of which one of us (G.M.M.S.) was a fellow during this research, and the Conselho Nacional de Pesquisas (CNPq).