Mapping of single-copy genes by TSA-FISH in the codling moth, <Emphasis Type="Italic">Cydia pomonella</Emphasis>

Background

We work on the development of transgenic sexing strains in the codling moth, Cydia pomonella (Tortricidae), which would enable to produce male-only progeny for the population control of this pest using sterile insect technique (SIT). To facilitate this research, we have developed a number of cytogenetic and molecular tools, including a physical map of the codling moth Z chromosome using BAC-FISH (fluorescence in situ hybridization with bacterial artificial chromosome probes). However, chromosomal localization of unique, single-copy sequences such as a transgene cassette by conventional FISH remains challenging. In this study, we adapted a FISH protocol with tyramide signal amplification (TSA-FISH) for detection of single-copy genes in Lepidoptera. We tested the protocol with probes prepared from partial sequences of Z-linked genes in the codling moth.

Results

Using a modified TSA-FISH protocol we successfully mapped a partial sequence of the Acetylcholinesterase 1 (Ace-1) gene to the Z chromosome and confirmed thus its Z-linkage. A subsequent combination of BAC-FISH with BAC probes containing anticipated neighbouring Z-linked genes and TSA-FISH with the Ace-1 probe allowed the integration of Ace-1 in the physical map of the codling moth Z chromosome. We also developed a two-colour TSA-FISH protocol which enabled us simultaneous localization of two Z-linked genes, Ace-1 and Notch, to the expected regions of the Z chromosome.

Conclusions

We showed that TSA-FISH represents a reliable technique for physical mapping of genes on chromosomes of moths and butterflies. Our results suggest that this technique can be combined with BAC-FISH and in the future used for physical localization of transgene cassettes on chromosomes of transgenic lines in the codling moth or other lepidopteran species. Furthermore, the developed protocol for two-colour TSA-FISH might become a powerful tool for synteny mapping in non-model organisms.

     

Improvement of Pro-Oxidant Capacity of Protocatechuic Acid by Esterification

Pro-oxidant effects of phenolic compounds are usually correlated to the one-electron redox potential of the phenoxyl radicals. Here we demonstrated that, besides their oxidizability, hydrophobicity can also be a decisive factor. We found that esterification of protocatechuic acid (P0) provoked a profound influence in its pro-oxidant capacity. The esters bearing alkyl chains containing two (P2), four (P4) and seven (P7) carbons, but not the acid precursor (P0), were able to exacerbate the oxidation of trolox, α-tocopherol and rifampicin. This effect was also dependent on the catechol moiety, since neither gallic acid nor butyl gallate showed any pro-oxidant effects. A comparison was also made with apocynin, which is well-characterized regarding its pro-oxidant properties. P7 was more efficient than apocynin regarding co-oxidation of trolox. However, P7 was not able to co-oxidize glutathione and NADH, which are targets of the apocynin radical. A correlation was found between pro-oxidant capacity and the stability of the radicals, as suggested by the intensity of the peak current in the differential pulse voltammetry experiments. In conclusion, taking into account that hydroquinone and related moieties are frequently found in biomolecules and quinone-based chemotherapeutics, our demonstration that esters of protocatechuic acid are specific and potent co-catalysts in their oxidations may be very relevant as a pathway to exacerbate redox cycling reactions, which are usually involved in their biological and pharmacological mechanisms of action.     

Seasonal Photochemical Transformations of Nitrogen Species in a Forest Stream and Lake

The photochemical release of inorganic nitrogen from dissolved organic matter is an important source of bio-available nitrogen (N) in N-limited aquatic ecosystems. We conducted photochemical experiments and used mathematical models based on pseudo-first-order reaction kinetics to quantify the photochemical transformations of individual N species and their seasonal effects on N cycling in a mountain forest stream and lake (Plešné Lake, Czech Republic). Results from laboratory experiments on photochemical changes in N speciation were compared to measured lake N budgets. Concentrations of organic nitrogen (N_org; 40–58 µmol L⁻¹) decreased from 3 to 26% during 48-hour laboratory irradiation (an equivalent of 4–5 days of natural solar insolation) due to photochemical mineralization to ammonium (NH₄ ⁺) and other N forms (N_x; possibly N oxides and N₂). In addition to N_org mineralization, N_x also originated from photochemical nitrate (NO₃ ⁻) reduction. Laboratory exposure of a first-order forest stream water samples showed a high amount of seasonality, with the maximum rates of N_org mineralization and NH₄ ⁺ production in winter and spring, and the maximum NO₃ ⁻ reduction occurring in summer. These photochemical changes could have an ecologically significant effect on NH₄ ⁺ concentrations in streams (doubling their terrestrial fluxes from soils) and on concentrations of dissolved N_org in the lake. In contrast, photochemical reactions reduced NO₃ ⁻ fluxes by a negligible (<1%) amount and had a negligible effect on the aquatic cycle of this N form.     

How laminin-1 can be recognized by the protozoan parasite Tritrichomonas foetus: possible role played by the extracellular matrix glycoprotein in both cytoadhesion and cytotoxicity exerted by the parasite

The isoform 1 of the extracellular matrix glycoprotein Laminin is known to be an important ligand for some parasitic protozoa including Trichomonas vaginalis. The bovine parasite Tritrichomonas foetus seems to display a similar recognition process to laminin-1, as some amino acid sequences found in the LNS module of laminin-1 can also be recognized by this parasite. Which of the laminin-1 residing adhesion sequences are recognized by T. foetus, and the role played by such a protein-cell recognition process in both cytoadhesion and cytotoxicity exerted by the parasite are the subjects briefly reviewed and discussed here.     

Three-Dimensional Architecture and Biogenesis of Membrane Structures Associated with Hepatitis C Virus Replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.     

Stretching the rules: monocentric chromosomes with multiple centromere domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.