Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modifiedDs transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.Equal contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this work     

Reduction of calcium currents in identified neurons of Helix pomatia: intracellular injection of D890

The actions of intracellularly applied D890 on membrane currents of the identified neurons B1, B2 and B3 of Helix pomatia were investigated. The TTX-resistant component of the inward current, the inward currents in Na+-free sucrose solution and in Ca2+-free Ba2+ solution were reduced. In Ca2+-free Co2+ solution the inward current was not affected. The late outward currents were strongly reduced. In solutions containing 20 mmol/l NiCl2 the remaining parts of these currents were blocked only to a lesser extent. The early outward current remained unchanged. It is concluded that intracellularly applied D890 mainly exerts its effects on the calcium current.     

Acetylcholine responses of identified neurons in Helix pomatia--II. Pharmacological properties of acetylcholine responses

A pharmacological separation of depolarizing and hyperpolarizing mechanisms involved in the generation of acetylcholine (ACh) depolarizations was attempted in the identified neurons B1 and B3 of the buccal ganglia of Helix pomatia. The selectivity of the drugs employed was assayed in non-identified buccal neurons in which ACh increased a hyperpolarizing Cl- conductance. Voltage clamp techniques were used. Under control conditions the depolarizing ACh currents increased non-linearly with more negative membrane potentials. The hyperpolarizing ACh currents showed a linear potential dependence. The buffer substance Tris (5 mmol/l) depressed the depolarizing ACh currents. The effect was accentuated with more negative membrane potentials. Tris failed to affect hyperpolarizing ACh responses. HEPES (5 mmol/l) did not change depolarizing or hyperpolarizing ACh responses. d-Tubocurarine (0.02-0.2 mmol/l), hexamethonium (0.5-5.0 mmol/l) and atropine (0.1 mmol/l) blocked the depolarizing and hyperpolarizing ACh responses. Arecoline (0.1 mmol/l) had neither an agonistic nor an antagonistic effect on the identified and on the non-identified neurons. It displayed an anticholinesterase activity. Anthracene-9-carbonic acid (0.5 mmol/l) depressed selectively the hyperpolarizing ACh responses. In the neurons B1 and B3 no pharmacologically separable hyperpolarizing ACh responses were detected to be superimposed on the ACh depolarizations.     

Acetylcholine responses of identified neurons in Helix pomatia--I. Interactions between acetylcholine-induced and potential-dependent membrane conductances

The influence of potential-dependent membrane conductances on amplitude and time course of acetylcholine (ACh) responses was studied. The investigations were performed on the identified neurons B1 and B3 of the buccal ganglion of Helix pomatia. The neurons B1 and B3 were depolarized by ACh. The depolarization was accompanied by a decrease of membrane resistance. An inward rectification occurring negative to the resting membrane potential (RMP) reduced the amplitude of the ACh depolarizations. An outward rectification occurring positive to the RMP consisted of two parts and ceiled the ACh responses. The early outward current reduced the amplitude and modified the time course of ACh responses. Local responses or axonal action potentials increased the amplitude of the ACh depolarizations.     

Neuronal PAS Domain Protein 4 Suppression of Oxygen Sensing Optimizes Metabolism during Excitation of Neuroendocrine Cells

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Archaeal guide RNAs function in rRNA modification in the eukaryotic nucleus

In eukaryotes, many Box C/D small nucleolar RNAs base pair with ribosomal RNA through short complementary guide sequences, thereby marking up to 100 individual nucleotides of ribosomal RNA for 2'-O-methylation. Function of the eukaryotic Box C/D RNAs depends upon interaction with at least six known proteins. Box C/D RNAs are not known to exist in Bacteria but were recently identified in Archaea by biochemical analysis and computational genomic screens and have likely evolved independently in Archaea and Eukarya for more than 2000 million years. We have microinjected Box C/D RNAs from Pyrococcus furiosus, a hyperthermophilic archaeon, into the nuclei of oocytes from the aquatic frog Xenopus laevis. Our results show that Box C/D RNAs derived from this prokaryote are retained in the nucleus, localize to nucleoli, and interact with the X. laevis Box C/D RNA binding proteins fibrillarin, Nop56, and Nop58. Furthermore, we have demonstrated the ability of archaeal Box C/D RNAs to direct site-specific 2'-O-methylation of ribosomal RNA. Our studies have revealed the remarkable ability of archaeal Box C/D RNAs to assemble into functional RNA-protein complexes in the eukaryotic nucleus.     

Analysis and visualization of animal movement

The interdisciplinary workshop 'Analysis and Visualization of Moving Objects' was held at the Lorentz Centre in Leiden, The Netherlands, from 27 June to 1 July 2011. It brought together international specialists from ecology, computer science and geographical information science actively involved in the exploration, visualization and analysis of moving objects, such as marine reptiles, mammals, birds, storms, ships, cars and pedestrians. The aim was to share expertise, methodologies, data and common questions between different fields, and to work towards making significant advances in movement research. A data challenge based on GPS tracking of lesser black-backed gulls (Larus fuscus) was used to stimulate initial discussions, cross-fertilization between research groups and to serve as an initial focus for activities during the workshop.     

Time courses of lidocaine effects on sodium membrane currents in small and large neurons

Time courses of effects of lidocaine on sodium currents and sodium dependent action potentials were studied in somata of small and large neurons. Cultured rat sensory spinal ganglion cells (diameter: 30 microns) and neurons of the buccal ganglion of Helix pomatia (diameter: 150 microns) served as the test cells. The latency of the suppressive action of lidocaine was the longer the larger the of the cells was. Maximal blocking effects occurred within 10 min in sensory spinal ganglion cells and within 40 min in snail neurons. Model calculations based on the assumptions (i) that lidocaine is distributed in the extra- and intracellular space by simple diffusion and (ii) that the drug concentration at the outer surface of the cells is elevated stepwisely, revealed a strong dependency of intracellular concentration changes on the size of the cells. From these findings it is concluded that lidocaine blocks sodium channels primarily from the intracellular side.     

The BEACH protein LRBA is required for hair bundle maintenance in cochlear hair cells and for hearing

Lipopolysaccharide‐responsive beige‐like anchor protein (LRBA) belongs to the enigmatic class of BEACH domain‐containing proteins, which have been attributed various cellular functions, typically involving intracellular protein and membrane transport processes. Here, we show that LRBA deficiency in mice leads to progressive sensorineural hearing loss. In LRBA knockout mice, inner and outer hair cell stereociliary bundles initially develop normally, but then partially degenerate during the second postnatal week. LRBA deficiency is associated with a reduced abundance of radixin and Nherf2, two adaptor proteins, which are important for the mechanical stability of the basal taper region of stereocilia. Our data suggest that due to the loss of structural integrity of the central parts of the hair bundle, the hair cell receptor potential is reduced, resulting in a loss of cochlear sensitivity and functional loss of the fraction of spiral ganglion neurons with low spontaneous firing rates. Clinical data obtained from two human patients with protein‐truncating nonsense or frameshift mutations suggest that LRBA deficiency may likewise cause syndromic sensorineural hearing impairment in humans, albeit less severe than in our mouse model.