High throughput sequencing of microRNAs in chicken somites

High throughput Solexa sequencing technology was applied to identify microRNAs in somites of developing chicken embryos. We obtained 651 273 reads, from which 340 415 were mapped to the chicken genome representing 1701 distinct sequences. Eighty-five of these were known microRNAs and 42 novel miRNA candidates were identified. Accumulation of 18 of 42 sequences was confirmed by Northern blot analysis. Ten of the 18 sequences are new variants of known miRNAs and eight short RNAs are novel miRNAs. Six of these eight have not been reported by other deep sequencing projects. One of the six new miRNAs is highly enriched in somite tissue suggesting that deep sequencing of other specific tissues has the potential to identify novel tissue specific miRNAs.     

Ageing is associated with a decline in peripheral blood CD56bright NK cells

Background  

Natural killer (NK) cells are cytotoxic lymphocytes that lack CD3 and express variable levels of CD16, CD56 and CD57. In recent years NK cells have been categorised into two major groups based on the level of CD56 expression. This phenotypic classification correlates with functional activity as CD56^bright NK cells are the major cytokine producing subset whereas CD56^dim NK cells exhibit greater cytotoxic activity. Previous studies have revealed a reduction in total NK cell numbers in association with ageing and this study sought to determine the potential influence of ageing on the number of NK cell subsets within peripheral blood.     

Targeted ablation of NrCAM or ankyrin-B results in disorganized lens fibers leading to cataract formation.

The NgCAM-related cell adhesion molecule (NrCAM) is an immunoglobulin superfamily member of the L1 subgroup that interacts intracellularly with ankyrins. We reveal that the absence of NrCAM causes the formation of mature cataracts in the mouse, whereas significant pathfinding errors of commissural axons at the midline of the spinal cord or of proprioceptive axon collaterals are not detected. Cataracts, the most common cause of visual impairment, are generated in NrCAM-deficient mice by a disorganization of lens fibers, followed by cellular disintegration and accumulation of cellular debris. The disorganization of fiber cells becomes histologically distinct during late embryonic development and includes abnormalities of the cytoskeleton and of connexin50-containing gap junctions. Furthermore, analysis of lenses of ankyrin-B mutant mice also reveals a disorganization of lens fibers at postnatal day 1, indistinguishable from that generated by the absence of NrCAM, indicating that NrCAM and ankyrin-B are required to maintain contact between lens fiber cells. Also, these studies provide genetic evidence of an interaction between NrCAM and ankyrin-B.     

High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance

Virus-induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto-mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto-mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx-mediated resistance against potato virus X and N-mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co-chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants.     

Directed differentiation of pluripotent cells to neural lineages: homogeneous formation and differentiation of a neurectoderm population

During embryogenesis the central and peripheral nervous systems arise from a neural precursor population, neurectoderm, formed during gastrulation. We demonstrate the differentiation of mouse embryonic stem cells to neurectoderm in culture, in a manner which recapitulates embryogenesis, with the sequential and homogeneous formation of primitive ectoderm, neural plate and neural tube. Formation of neurectoderm occurs in the absence of extraembryonic endoderm or mesoderm and results in a stratified epithelium of cells with morphology, gene expression and differentiation potential consistent with positionally unspecified neural tube. Differentiation of this population to homogeneous populations of neural crest or glia was also achieved. Neurectoderm formation in culture allows elucidation of signals involved in neural specification and generation of implantable cell populations for therapeutic use.     

Mapping loci associated with milling yield in wheat (Triticum aestivum L.)

A partial genetic linkage map constructed using 150 single seed descent (SSD) lines generated from a cross between the hexaploid wheat varieties Schomburgk and Yarralinka was used to identify loci controlling milling yield. Milling yield data were obtained using seed collected from field trials conducted at different sites over two seasons. The estimated broad-sense heritability of milling yield in this population was calculated as 0.48. In the preliminary analysis, two regions were identified on chromosomes 3A and 7D, which were significantly associated with milling yield and accounted for 22% and 19% of the genetic variation, respectively. Bulked segregant analysis in combination with AFLP identified other markers linked to these loci, as well as an additional region on chromosome 5A, which accounted for 19% of the genetic variation. The applicability of these markers as selection tools for breeding purposes is discussed.     

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Extension of neurites on axons is impaired by antibodies against specific neural cell surface glycoproteins

We have developed an in vitro assay which measures the ability of growth cones to extend on an axonal substrate. Neurite lengths were compared in the presence or absence of monovalent antibodies against specific neural cell surface glycoproteins. Fab fragments of antibodies against the neural cell adhesion molecule, NCAM, have an insignificant effect on the lengths of neurites elongating on either an axonal substrate or a laminin substrate. Fab fragments of polyclonal antibodies against two new neural cell surface antigens, defined by mAb G4 and mAb F11, decrease the lengths of neurites elongating on an axonal substrate, but have no effect on the lengths of neurites elongating on a laminin substrate. G4 antigen is related to mouse L1, while F11 antigen appears to be distinct from all known neural cell surface glycoproteins. Our results suggest that the G4 and F11 antigens help to promote the extension of growth cones on axons.