A fourth complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DNA double-strand break repair.

The XR-V9B mutant of Chinese hamster V79 cells which exhibits hypersensitivity to ionizing radiation was isolated by the replica plating technique. The increased sensitivity of XR-V9B cells to X rays (approximately 4-fold, as judged by the D10) was accompanied by increased sensitivity to other DNA-damaging agents such as bleomycin (approximately 17-fold), VP16 (approximately 6-fold), and adriamycin (approximately 5-fold). Only a slightly increased sensitivity was observed after exposure to UV radiation, MMS, or mitomycin C (1.4-, 1.7-, and 2-fold, respectively). As measured by neutral elution after exposure to X rays, XR-V9B cells showed a defect in the rejoining of double-strand breaks (DSBs); after 4 h of repair more than 50% of DSBs remained in comparison to 5% in wild-type cells. No difference was observed in the kinetics of single-strand break rejoining between XR-V9B and wild-type cells, as measured by alkaline elution. To determine whether XR-V9B represents a new complementation group among ionizing radiation-sensitive Chinese hamster cell mutants defective in DSB repair, XR-V9B cells were fused with XR-V15B, XR-1, and V-3 cells, which have impaired DSB rejoining and belong to three different complementation groups. In all cases, the derived hybrids regained the sensitivity of wild-type cells when exposed to X rays, indicating that the XR-V9B mutant represents a new fourth complementation group among X-ray-sensitive Chinese hamster cell mutants defective in DSB repair.     

Single cell RNA‐seq in the sea urchin embryo show marked cell‐type specificity in the Delta/Notch pathway

Sea urchin embryos are excellent for in vivo functional studies because of their transparency and tractability in manipulation. They are also favorites for pharmacological approaches since they develop in an aquatic environment and addition of test substances is straightforward. A concern in many pharmacological tests though is the potential for pleiotropic effects that confound the conclusions drawn from the results. Precise cellular interpretations are often not feasible because the impact of the perturbant is not known. Here we use single‐cell mRNA (messenger RNA) sequencing as a metric of cell types in the embryo and to determine the selectivity of two commonly used inhibitors, one each for the Wnt and the Delta‐Notch pathways, on these nascent cell types. We identified 11 distinct cell types based on mRNA profiling, and that the cell lineages affected by Wnt and Delta/Notch inhibition were distinct from each other. These data support specificity and distinct effects of these signaling pathways in the embryo and illuminate how these conserved pathways selectively regulate cell lineages at a single cell level. Overall, we conclude that single cell RNA‐seq analysis in this embryo is revealing of the cell types present during development, of the changes in the gene regulatory network resulting from inhibition of various signaling pathways, and of the selectivity of these pathways in influencing developmental trajectories.     

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

The sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm-egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca(2+) signaling and homeostasis modules present in the egg and zygote. The sea urchin "calcium toolkit" as predicted by the genome is described. Emphasis is on the Ca(2+) signaling modules operating during egg activation, but the Ca(2+) signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca(2+) at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca(2+) serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies.     

Sparse Spatial Sampling for the Computation of Motion in Multiple Stages

The avian retino-tecto-rotundal pathway plays a central role in motion analysis and features complex connectivity. Yet, the relation between the pathway’s structural arrangement and motion computation has remained elusive. For an important type of tectal wide-field neuron, the stratum griseum centrale type I (SGC-I) neuron, we quantified its structure and found a spatially sparse but extensive sampling of the retinal projection. A computational investigation revealed that these structural properties enhance the neuron’s sensitivity to change, a behaviorally important stimulus attribute, while preserving information about the stimulus location in the SGC-I population activity. Furthermore, the SGC-I neurons project with an interdigitating topography to the nucleus rotundus, where the direction of motion is computed. We showed that, for accurate direction-of-motion estimation, the interdigitating projection of tectal wide-field neurons requires a two-stage rotundal algorithm, where the second rotundal stage estimates the direction of motion from the change in the relative stimulus position represented in the first stage     

Identification of genetic markers associated with Gyrodactylus salaris resistance in Atlantic salmon Salmo salar

Gyrodactylus salaris Malmberg, 1957 is a freshwater monogenean ectoparasite of salmonids, first recorded in Norway in 1975 and responsible for extensive epizootics in wild Atlantic salmon Salmo salar L. The susceptibility of different populations of Atlantic salmon to G. salaris infection differs markedly, with fish from the Baltic being characterised as relatively resistant whereas those from Norway or Scotland are known to be (extremely) susceptible. Resistance to Gyrodactylus infection in salmonids has been found to be heritable and a polygenic mechanism of control has been hypothesised. The current study utilises a 'Quantitative trait loci' (QTL) screening approach in order to identify molecular markers linked to QTL influencing G. salaris resistance in B1 backcrosses of Baltic and Scottish salmon. Infection patterns in these fish exhibited 3 distinct types; susceptible (exponential parasite growth), responding (parasite load builds before dropping) and resistant (parasite load never increases). B1 backcross fish were screened at 39 microsatellite markers and single marker-trait associations were examined using general linear modelling. We identified 10 genomic regions associated with heterogeneity in both innate and acquired resistance, explaining up to 27.3% of the total variation in parasite loads. We found that both innate and acquired parasite resistance in Atlantic salmon are under polygenic control, and that salmon would be well suited to a selection programme designed to quickly increase resistance to G. salaris in wild or farmed stocks.