Structural characterization of Set1 RNA recognition motifs and their role in histone H3 lysine 4 methylation

The yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, in addition to its catalytic SET domain, a conserved RNA recognition motif (RRM1). We present here the crystal structure and the secondary structure assignment in solution of the Set1 RRM1. Although RRM1 has the expected betaalphabetabetaalphabeta RRM-fold, it lacks the typical RNA-binding features of these modules. RRM1 is not able to bind RNA by itself in vitro, but a construct combining RRM1 with a newly identified downstream RRM2 specifically binds RNA. In vivo, H3K4 methylation is not affected by a point mutation in RRM2 that preserves Set1 stability but affects RNA binding in vitro. In contrast mutating RRM1 destabilizes Set1 and leads to an increase of dimethylation of H3K4 at the 5'-coding region of active genes at the expense of trimethylation, whereas both, dimethylation decreases at the 3'-coding region. Taken together, our results suggest that Set1 RRMs bind RNA, but Set1 RNA-binding activity is not linked to H3K4 methylation.     

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.     

Selective viral transduction of adult-born olfactory neurons for chronic in vivo optogenetic stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.     

Surface sensing triggers a broad-spectrum antimicrobial response in Pseudomonas aeruginosa

Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.     

Life and death decision in adult neurogenesis: in praise of napping

Among the thousands of new neurons that integrate into the adult olfactory bulb each day, 50% are eliminated through apoptosis. In this issue of Neuron, Yokoyama et?al. take steps toward deciphering the behavioral contexts that regulate newborn cell elimination.     

Differential correlations between changes to glutathione redox state,protein ubiquitination,and stress-inducible HSPA chaperone expression after different types of oxidative stress

In primary bovine fibroblasts with an hspa1b/luciferase transgene, we examined the intensity of heat-shock response (HSR) following four types of oxidative stress or heat stress (HS), and its putative relationship with changes to different cell parameters, including reactive oxygen species (ROS), the redox status of the key molecules glutathione (GSH), NADP(H) NAD(H), and the post-translational protein modifications carbonylation, S-glutathionylation, and ubiquitination. We determined the sub-lethal condition generating the maximal luciferase activity and inducible HSPA protein level for treatments with hydrogen peroxide (H₂O₂), UVA-induced oxygen photo-activation, the superoxide-generating agent menadione (MN), and diamide (DA), an electrophilic and sulfhydryl reagent. The level of HSR induced by oxidative stress was the highest after DA and MN, followed by UVA and H₂O₂ treatments, and was not correlated to the level of ROS production nor to the extent of protein S-glutathionylation or carbonylation observed immediately after stress. We found a correlation following oxidative treatments between HSR and the level of GSH/GSSG immediately after stress, and the increase in protein ubiquitination during the recovery period. Conversely, HS treatment, which led to the highest HSR level, did not generate ROS nor modified or depended on GSH redox state. Furthermore, the level of protein ubiquitination was maximum immediately after HS and lower than after MN and DA treatments thereafter. In these cells, heat-induced HSR was therefore clearly different from oxidative stress-induced HSR, in which conversely early redox changes of the major cellular thiol predicted the level of HSR and polyubiquinated proteins.     

The how and why of adult neurogenesis

Brain plasticity refers to the brain’s ability to change structure and/or function during maturation, learning, environmental challenges, or disease. Multiple and dissociable plastic changes in the adult brain involve many different levels of organization, ranging from molecules to systems, with changes in neural elements occurring hand-in-hand with changes in supportive tissue elements, such as glia cells and blood vessels. There is now substantial evidence indicating that new functional neurons are constitutively generated from endogenous pools of neural stem cells in restricted areas of the mammalian brain, throughout life. So, in addition to all the other known structural changes, entire new neurons can be added to the existing network circuitry. This addition of newborn neurons provides the brain with another tool for tinkering with the morphology of its own functional circuitry. Although the ongoing neurogenesis and migration have been extensively documented in non-mammalian species, its characteristics in mammals have just been revealed and thus several questions remain yet unanswered. Is adult neurogenesis an atavism, an empty-running leftover from evolution? What is adult neurogenesis good for and how does the brain ‘know’ that more neurons are needed? How is this functional demand translated into signals a precursor cell can detect? Adult neurogenesis may represent an adaptive response to challenges imposed by an environment and/or internal state of the animal. To ensure this function, the production, migration, and survival of newborn neurons must be tightly controlled. We attempt to address some of these questions here, using the olfactory bulb as a model system.     

Neuronal replacement in microcircuits of the adult olfactory system

The local-circuit inhibitory interneurons containing gamma-aminobutyric acid (GABA) are continuously replaced in the adult olfactory bulb. Here, we describe how the production of new GABAergic interneurons is adapted to experience-induced plasticity. In particular, we discuss how such an adaptation is sensitive to the level of sensory inputs and how, in turn, neurogenesis may adjust the neural network functioning to optimize processing of sensory information. Finally, this review brings together recently described properties of interneurons as well as emerging principles of their functions that indicate a much more complex role for these cells than just that of gatekeepers providing inhibition.     

Modulable Southern and Northern blot hybridization apparatus for routine screening of gene amplification and expression

An apparatus for Northern and Southern blot hybridization is described. It allows from one to twenty-four blots to be processed at the same time, with different probes. All the pre-, post- and hybridization steps are performed without handling the filters and the experimenter is totally protected from beta radiations.

The development of such modulable materials has become necessary since Southern and Northern techniques are becoming routine assays in hospitals, particularly in the field of oncology, in prognosis and for hereditary diseases, as an antenatal diagnostis procedure.