Sustained transgene expression using MAR elements.

Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.

RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

Whole-cell living biosensors--are they ready for environmental application?

Since the development of the first whole-cell living biosensor or bioreporter about 15 years ago, construction and testing of new genetically modified microorganisms for environmental sensing and reporting has proceeded at an ever increasing rate. One and a half decades appear as a reasonable time span for a new technology to reach the maturity needed for application and commercial success. It seems, however, that the research into cellular biosensors is still mostly in a proof-of-principle or demonstration phase and not close to extensive or commercial use outside of academia. In this review, we consider the motivations for bioreporter developments and discuss the suitability of extant bioreporters for the proposed applications to stimulate complementary research and to help researchers to develop realistic objectives. This includes the identification of some popular misconceptions about the qualities and shortcomings of bioreporters.

Molecular biology of baculovirus and its use in biological control in Brazil

Baculoviruses are insect viruses found mainly in Lepidoptera. The family Baculoviridae is taxonomically divided in two genera, Nucleopolyhedrovirus and Granulovirus, which differ by occlusion body morphology. NPVs (Nucleopolyhedroviruses) have polyhedrical inclusion bodies (PIBs) containing multiple viral particles, while GVs (Granuloviruses) appear to be generally single particles occluded in oval shaped occlusion bodies. During the life cycle, two different viral progenies are produced: BV (Budded Virus) and PDV (Polyhedra Derived Virus), which are essential for the infectious process and virus propagation in host cells. Baculoviruses are being used for pest control and they are especially safe due to their specificity and invertebrate-restricted host range. Baculoviruses have been used as vectors for high level protein expression ofheterologous genes from prokaryotic and eukaryotic organisms. Also, recombinant DNA techniques have allowed the production of genetically modified viral insecticides. This study is a review on the taxonomy, structure, replication and molecular biology of baculoviruses, as well as their use as bioinsecticides in Brazil.

A downstream mediator in the growth repression limb of the jasmonate pathway.

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

The α1-adrenergic receptors: diversity of signaling networks and regulation.

The α(1)-adrenergic receptor (AR) subtypes (α(1a), α(1b), and α(1d)) mediate several physiological effects of epinephrine and norepinephrine. Despite several studies in recombinant systems and insight from genetically modified mice, our understanding of the physiological relevance and specificity of the α(1)-AR subtypes is still limited. Constitutive activity and receptor oligomerization have emerged as potential features regulating receptor function. Another recent paradigm is that β arrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effects. The aim of this review is to summarize our current knowledge on some recently identified functional paradigms and signaling networks that might help to elucidate the functional diversity of the α(1)-AR subtypes in various organs.

A simple and reliable method for the screening of transgenic tobacco plants

Even though much improvement has been made in plant transformation methods, the screening of transgenic plants is often a laborious work. Most approaches for detecting the transgene in transformed plants are still timeconsuming, and can be quite expensive. The objective of this study was to search for a simpler method to screen for transgenic plants. The infiltration of kanamycin (100 mg/mL) into tobacco leaves resulted in conspicuous chlorotic spots on the non-transgenic plant leaves, while no spots were seen on the leaves of transformed plants. This reaction occurred regardless of age of the tested plants, and the method has proven to be simple, fast, non-destructive, relatively cheap, and reliable. These results were comparable to those obtained by the polymerase chain reaction (PCR) amplification of the transgene using specific primers.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation.

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that in Arabidopsis thaliana, two paralogous auxin response transcription factors, ARF6 and ARF8, regulate both stamen and gynoecium maturation. arf6 arf8 double-null mutant flowers arrested as infertile closed buds with short petals, short stamen filaments, undehisced anthers that did not release pollen and immature gynoecia. Numerous developmentally regulated genes failed to be induced. ARF6 and ARF8 thus coordinate the transition from immature to mature fertile flowers. Jasmonic acid (JA) measurements and JA feeding experiments showed that decreased jasmonate production caused the block in pollen release, but not the gynoecium arrest. The double mutant had altered auxin responsive gene expression. However, whole flower auxin levels did not change during flower maturation, suggesting that auxin might regulate flower maturation only under specific environmental conditions, or in localized organs or tissues of flowers. arf6 and arf8 single mutants and sesquimutants (homozygous for one mutation and heterozygous for the other) had delayed stamen development and decreased fecundity, indicating that ARF6 and ARF8 gene dosage affects timing of flower maturation quantitatively.

Two-kidney, one clip and one-kidney, one clip hypertension in mice.

The mouse remains the animal of choice in transgenic experiments, creating a need for methods of evaluating the physiology of genetically modified animals. We have established and characterized two murine models of renovascular hypertension known as the two-kidney, one clip and one-kidney, one clip models. The appropriate size of the clip lumen needed to induce high blood pressure was determined to be 0.12 mm. Clips with a lumen of 0.11 mm induced a high percentage of renal infarction, and clips with a 0.13-mm opening did not produce hypertension. Four weeks after clipping, two-kidney, one clip hypertensive mice exhibited blood pressure approximately 20 mm Hg higher than their sham-operated controls. After a similar period, this increase reached almost 35 mm Hg in the one-kidney, one clip model. Depending on the model, mice develop either renin-dependent or renin-independent hypertension. Both models are characterized by the development of cardiovascular hypertrophy.

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Functional expression of PHO1 to the Golgi and trans-Golgi network and its role in export of inorganic phosphate.

Arabidopsis thaliana PHO1 is primarily expressed in the root vascular cylinder and is involved in the transfer of inorganic phosphate (Pi) from roots to shoots. To analyze the role of PHO1 in transport of Pi, we have generated transgenic plants expressing PHO1 in ectopic A. thaliana tissues using an estradiol-inducible promoter. Leaves treated with estradiol showed strong PHO1 expression, leading to detectable accumulation of PHO1 protein. Estradiol-mediated induction of PHO1 in leaves from soil-grown plants, in leaves and roots of plants grown in liquid culture, or in leaf mesophyll protoplasts, was all accompanied by the specific release of Pi to the extracellular medium as early as 2-3 h after addition of estradiol. Net Pi export triggered by PHO1 induction was enhanced by high extracellular Pi and weakly inhibited by the proton-ionophore carbonyl cyanide m-chlorophenylhydrazone. Expression of a PHO1-GFP construct complementing the pho1 mutant revealed GFP expression in punctate structures in the pericycle cells but no fluorescence at the plasma membrane. When expressed in onion epidermal cells or in tobacco mesophyll cells, PHO1-GFP was associated with similar punctate structures that co-localized with the Golgi/trans-Golgi network and uncharacterized vesicles. However, PHO1-GFP could be partially relocated to the plasma membrane in leaves infiltrated with a high-phosphate solution. Together, these results show that PHO1 can trigger Pi export in ectopic plant cells, strongly indicating that PHO1 is itself a Pi exporter. Interestingly, PHO1-mediated Pi export was associated with its localization to the Golgi and trans-Golgi networks, revealing a role for these organelles in Pi transport.

Spider mites associated to soybean in Rio Grande do Sul, Brazil

The objective of this work was to study the occurrence and geographic distribution of phytophagous mites associated to soybean in Rio Grande do Sul, Brazil. Samplings were performed from January to May 2005 on genetically modified soybean (glyphosate-tolerant) crops, in 27 municipalities of six regions - Alto Vale do Uruguai, Campanha, Depressão Central, Planalto Médio, Missões and Serra do Sudeste. Five phytophagous mite species belonging to the family Tetranychidae - Mononychellus planki, Tetranychus desertorum, T. gigas, T. ludeni and T. urticae - occurred in 21, 12, 5, 3 and 14 municipalities, respectively. A map of Rio Grande do Sul with the geographic distribution of each species is presented, as well as an illustrated dichotomous key to help the identification of the spider mites found.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.