Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Stable expression of a retrovirally transferred adhesion molecule in a human melanoma-specific cytotoxic T lymphocyte clone

 The adoptive transfer of in vitro generated tumor-specific cytotoxic T lymphocytes (CTL) is considered a promising perspective in cancer therapy. One possible drawback lies in the inappropriate homing of in vitro cultured lymphocytes, which could be circumvented by introducing the appropriate targeting molecules. Here we describe a protocol that allows a rapid and stable transfection of cytotoxic T cell clones. As a model system we used a CTL clone specific for the melanoma-associated antigen gp100 and a cDNA encoding for murine CD14 containing the variant exen v10 which is supposed to facilitate lymphocyte homing towards the skin. CD44v10 cDNA was ligated into the retroviral vector pMV-7, which was used to transfect the ecotropic GP-E-86 and the amphotropic PA317 cells. After several cycles of transduction to increase the viral titre, supernatants of the amphotropic PA317-CD44v10 line were used for transduction of CD44v10 into a human CTL clone. After three cycles of transduction at 12-h intervals, low but stable expression of CD44v10 was observed throughout the culture period of 10 weeks. The phenotype of the transduced CTL clone was unaltered and the cytotoxic potential was only slightly reduced as compared to the parental clone. The efficiency of stable transduction within a period of 1 week makes the protocol well suited for the in vivo transfer of transduced cells and, in the special case, should guarantee appropriate homing of the transduced CTL clone. Received: 14 August 1997 / Accepted: 20 November 1997     

State of art and limitations in genetic engineering to induce stable chondrogenic phenotype

Current protocols for chondrocyte expansion and chondrogenic differentiation of stem cells fail to reduce phenotypic loss and to mitigate hypertrophic tendency. To this end, cell genetic manipulation is gaining pace as a means of generating cells with stable chondrocyte phenotype. Herein, we provide an overview of candidate genes that either induce cartilage regeneration or inhibit cartilage degeneration. We further discuss in vitro, ex vivo and in vivo viral transduction and non-viral transfection strategies for targeted cells (chondrocytes, mesenchymal stem cells, induced pluripotent stem cells and synovial cells), along with the most representative results obtained in pre-clinical models and in clinical trials. We highlight current challenges and associated risks that slowdown clinical acceptance and commercialisation of gene transfer technologies.     

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.     

Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize

Summary The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.     

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.     

A reassessment of explanations for discordant introgressions of mitochondrial and nuclear genomes

Hybridization is increasingly recognized as a significant evolutionary process, in particular because it can lead to introgression of genes from one species to another. A striking pattern of discordance in the amount of introgression between mitochondrial and nuclear markers exists such that substantial mitochondrial introgression is often found in combination with no or little nuclear introgression. Multiple mechanisms have been proposed to explain this discordance, including positive selection for introgressing mitochondrial variants, several types of sex‐biases, drift, negative selection against introgression in the nuclear genome, and spatial expansion. Most of these hypotheses are verbal, and have not been quantitatively evaluated so far. We use individual‐based, multilocus, computer simulations of secondary contact under a wide range of demographic and genetic scenarios to evaluate the ability of the different mechanisms to produce discordant introgression. Sex‐biases and spatial expansions fail to produce substantial mito‐nuclear discordance. Drift and nuclear selection can produce strong discordance, but only under a limited range of conditions. In contrast, selection on the mitochondrial genome produces strong discordance, particularly when dispersal rates are low. However, commonly used statistical tests have little power to detect this selection. Altogether, these results dismiss several popular hypotheses, and provide support for adaptive mitochondrial introgression.     

Tomato contains two differentially expressed genes encoding B-type phytochromes,neither of which can be considered an ortholog of Arabidopsis phytochrome B

Tomato (Solanum lycopersicon L.) contains two B-type phytochrome genes (PHYB1 and PHYB2). Fragments of these two PHYB were cloned following amplification by the polymerase chain reaction of a portion of their relatively well conserved 5 coding regions. Polypeptides encoded by these gene fragments exhibit 90% sequence identity. These two PHYB are independently expressed in organ-specific fashion. In mature plants, PHYB2 mRNA is most abundant in fruit and PHYB1 mRNA in expanded leaves. A phylogenetic analysis fails to establish which tomato PHYB is orthologous to either Arabidopsis PHYB or PHYD, the latter being a second B-type phytochrome. Instead, this analysis indicates that following the divergence of the Solanaceae and Brassicaceae from one another, a PHYB gene duplicated independently in each lineage. Consequently, Arabidopsis PHYB mutants cannot be considered strictly equivalent to the tomato tri mutants, which appear to be mutated at the PHYB1 locus. Similarly, other putative PHYB mutants might not be equivalent to those described for Arabidopsis and tomato. This situation complicates efforts to determine PHYB function because there might be no one answer to this question.Abbreviations PCR polymerase chain reaction - PHY undesignated phytochrome gene - PHYA, PHYB, etc phytochrome gene(s) of the A, B, etc. type This research was supported by USDA NRICGP grant 93-00939 and by NATO travel grant CRG 931183. It was initiated when two of us (L.H.P., M.-M.C.-P.) spent a sabbatical year at the Institut National de la Recherche Agronomique in Versailles, France. L.H.P. gratefully acknowledges support provided by a senior guest fellowship from the Ministère de l'enseignement superieur et de la recherche during his stay in Versailles. L.H.P. and M.-M.C.-P thank all of their colleagues in Versailles for their warm hospitality and their willingness to share their expertise with us. We also thank Russell Malmberg, Richard Meagher and Robert Price for helpful discussions concerning the interpretation of molecular phylogenies.