Dispensability of nanos mRNA localization for abdominal patterning but not for germ cell development.

The development of a functional germline is essential for species propagation. The nanos (nos) gene plays an evolutionarily conserved role in germline development and is also essential for abdominal patterning in Drosophila. A small fraction of nos mRNA is localized to the germ plasm at the posterior pole of the Drosophila embryo, where it becomes incorporated into the germ cells. Germ plasm associated nos mRNA is translated to produce a gradient of Nos protein that patterns the abdomen, whereas the remaining unlocalized RNA is translationally repressed to allow anterior development. Using transgenes that compromise nos mRNA localization and translational regulation, we show that wild-type body patterning can ensue without nos mRNA localization provided that nos translation is properly modulated. In contrast, localization of nos to the germ plasm, but not translational regulation, is essential for nos function in the developing germ cells. We propose that an imperative for nos localization in producing a functional germline has preserved an inefficient localization mechanism.

Protection of halogenated DNA from strand breakage and sister-chromatid exchange induced by the topoisomerase I inhibitor camptothecin.

The fundamental nuclear enzyme DNA topoisomerase I (topo I), cleaves the double-stranded DNA molecule at preferred sequences within its recognition/binding sites. We have recently reported that when cells incorporate halogenated nucleosides analogues of thymidine into DNA, it interferes with normal chromosome segregation, as shown by an extraordinarily high yield of endoreduplication, and results in a protection against DNA breakage induced by the topo II poison m-AMSA [F. Cortés, N. Pastor, S. Mateos, I. Domínguez, The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes, DNA Repair 2 (2003) 719-726; G. Cantero, S. Mateos, N. Pastor; F. Cortés, Halogen substitution of DNA protects from poisoning of topoisomerase II that results in DNA double-strand breaks (DSBs), DNA Repair 5 (2006) 667-674]. In the present investigation, we have assessed whether the presence of halogenated nucleosides in DNA diminishes the frequency of interaction of topo I with DNA and thus the frequency with which the stabilisation of cleavage complexes by the topo I poison camptothecin (CPT) takes place, in such a way that it protects from chromosome breakage and sister-chromatid exchange. This protective effect is shown to parallel a loss in halogen-substituted cells of the otherwise CPT-increased catalytic activity bound to DNA.

Spine morphogenesis in newborn granule cells is differentially regulated in the outer and middle molecular layers.

New neurons are continuously added to the hippocampus of adult mammals. Their survival and integration into the circuitry are highly dependent on experience. Here we show that mushroom spine formation in newborn granule cells was modulated by experience and that dendritic segments in different areas of the molecular layer were differentially regulated. Specifically, spines of new neurons in the outer molecular layer of the dentate gyrus were more readily influenced by nonspatial features in the living environment. Those in the middle molecular layer were more likely to be influenced by the size of the living environment. Therefore, the activity of cortical inputs into newborn granule cells may be reflected in the formation of mushroom spines in different dendritic segments in the molecular layer.

Effect of tissue-specific promoters and microRNA recognition elements on stability of transgene expression after hydrodynamic naked plasmid DNA delivery.

Intravenous hydrodynamic injections into the liver and skeletal muscle have increased the efficacy of naked DNA delivery to a level that makes therapeutically relevant gene transfer attainable. Although there are no concerns about the immunogenicity of the delivered DNA itself, transgene products that are foreign to the host can trigger an immune response and hamper the therapeutic effect. Our goal was to determine whether and to what extent some known preventive measures are applicable to these delivery methods in order to achieve longterm expression of foreign proteins in immunocompetent mice. We designed plasmid DNA vectors that expressed a marker gene under the control of either a ubiquitous or a tissue-specific promoter. We also included microRNA (miR) target sites in the transcripts in order to silence expression in antigen-presenting cells (APCs). The constructs were delivered either into muscle or liver, using outbred ICR and inbred C57BL=6 mice. The data suggest that firefly luciferase, a potent immunogen, triggered a uniform immune response only in outbred ICR mice, and only when expressed from a ubiquitous promoter. This response could not be prevented by including APC-specific miR target sites in the transcript. In contrast, the probability of immune rejection in ICR mice could be significantly diminished by using tissue-specific promoters, and under these circumstances, the silencing of transgene expression in APCs did confer some benefits. After a single hydrodynamic injection, inbred mice did not reject luciferase under any of the tested conditions for at least 8 weeks. To test whether they became tolerized, they were challenged with a second boost of a cytomegalovirus promoter-driven luciferase construct. This triggered a strong immune response, suggesting that luciferase-reactive cells from the animals' T and B cell repertoire had not been eliminated. This secondary reaction could not be prevented by silencing expression in APCs. In conclusion, for the clinical application of hydrodynamic naked DNA delivery the use of tissue-specific promoters in combination with silencing expression in APCs will increase the probability of long-term expression, but the most desirable outcome, the establishment of transgene tolerance, appears unlikely to be achieved by any of these measures.