KLP-7 acts through the Ndc80 complex to limit pole number in C. elegans oocyte meiotic spindle assembly

During oocyte meiotic cell division in many animals, bipolar spindles assemble in the absence of centrosomes, but the mechanisms that restrict pole assembly to a bipolar state are unknown. We show that KLP-7, the single mitotic centromere–associated kinesin (MCAK)/kinesin-13 in Caenorhabditis elegans, is required for bipolar oocyte meiotic spindle assembly. In klp-7(−) mutants, extra microtubules accumulated, extra functional spindle poles assembled, and chromosomes frequently segregated as three distinct masses during meiosis I anaphase. Moreover, reducing KLP-7 function in monopolar klp-18(−) mutants often restored spindle bipolarity and chromosome segregation. MCAKs act at kinetochores to correct improper kinetochore–microtubule (k–MT) attachments, and depletion of the Ndc-80 kinetochore complex, which binds microtubules to mediate kinetochore attachment, restored bipolarity in klp-7(−) mutant oocytes. We propose a model in which KLP-7/MCAK regulates k–MT attachment and spindle tension to promote the coalescence of early spindle pole foci that produces a bipolar structure during the acentrosomal process of oocyte meiotic spindle assembly.     

人肺腺癌细胞分化相关基因cDNAs的克隆

在用10^-5 mol/L全反式维甲酸(RA)诱导人肺腺癌细胞系GLC-82分化的基础上,以M13噬菌粒pSPORT1为载体,应用定向克隆技术,分别构建了未经RA诱导和RA诱导1d及4d细胞的3个cDNA文库.以含重组子的诱导文库单链DNA为靶标(Target)同未诱导文库的cDNA驱除子(Driver)进行消减杂交,富集RA特异性单链DNA,将富集的单链DNA回复为双链后转化感受态菌,建立细胞诱导分化过程中活化表达基因的cDNA消减文库,得到124个cDNA消减克隆.经同源性分析和与文库总cDNA作Southern印迹杂交,进而与RA诱导前后细胞的RNA作Northern印迹杂交,筛选出2个(RA5,RA28)诱导后呈早期瞬时表达和1个(RA42)呈早期并持续表达的cDNA克隆,cDNA全长分别为1.8,1.5和0.7kb.序列测定及初步功能分析结果表明,RA5,RA28和RA42这3个首次报道的序列,可能是人肺腺癌细胞分化相关基因的cDNA克隆.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.     

Sequence- and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.

Oligonucleotides containing a 3'-thiothymidine residue (T3's) at the cleavage site for the EcoRV restriction endonuclease (between the central T and A residues of the sequence GATATC) have been prepared on an automated DNA synthesizer using 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl N,N-diisopropylphosphorothioamidite). The self-complementary sequence GACGAT3'sATCGTC was completely resistant to cleavage by EcoRV, while the heteroduplex composed of 5'-TCTGAT3'sATCCTC and 5'-GAGGATATCAGA (duplex 4) was cleaved only in the unmodified strand (5'-GAGGATATCAGA). In contrast, strands containing a 3'-S-phosphorothiolate linkage could be chemically cleaved specifically at this site with Ag+. A T3's residue has also been incorporated in the (-) strand of double-stranded closed circular (RF IV) M13mp18 DNA at the cleavage site of a unique EcoRV recognition sequence by using 5'-pCGAGCTCGAT3'sATCGTAAT as a primer for polymerization on the template (+) strand of M13mp18 DNA. On treatment of this substrate with EcoRV, only one strand was cleaved to produce the RF II or nicked DNA. Taken in conjunction with the cleavage studies on the oligonucleotides, this result demonstrates that the 3'-S-phosphorothiolate linkage is resistant to scission by EcoRV. Additionally, the phosphorothiolate-containing strand of the M13mp18 DNA could be cleaved specifically at the point of modification using iodine in aqueous pyridine. The combination of enzymatic and chemical techniques provides, for the first time, a demonstrated method for the sequence-specific cleavage of either the (+) or (-) strand.     

36 Identification and characterization of small subunit ribosomal intermediates

Bacterial ribosome biogenesis is poorly understood especially in terms of the role of ribosomal RNA (rRNA) maturation in vivo. A major problem in addressing these questions are asynchronous biogenesis, a large population of mature particles and the lack of techniques to isolated in vivo formed ribosome biogenesis intermediates. Our group has taken multiple approaches to allow study of ribosome biogenesis in Escherichia coli. We have used genetic manipulation to discover that for specific biogenesis factors, there is a delicate balance that is necessary for viability. Additionally, we have pioneered an affinity purification approach to allow for isolation of in vivo formed intermediates. Data will be present on our findings for the role of rRNA maturation in biogenesis, subsequent ribosome function, and cell viability. Our findings may result in identification of novel targets for antimicrobial development.     

Faunal breaks and species composition of Indo-Pacific corals: the role of plate tectonics,environment and habitat distribution

Species richness gradients are ubiquitous in nature, but the mechanisms that generate and maintain these patterns at macroecological scales remain unresolved. We use a new approach that focuses on overlapping geographical ranges of species to reveal that Indo-Pacific corals are assembled within 11 distinct faunal provinces. Province limits are characterized by co-occurrence of multiple species range boundaries. Unexpectedly, these faunal breaks are poorly predicted by contemporary environmental conditions and the present-day distribution of habitat. Instead, faunal breaks show striking concordance with geological features (tectonic plates and mantle plume tracks). The depth range over which a species occurs, its larval development rate and genus age are important determinants of the likelihood that species will straddle faunal breaks. Our findings indicate that historical processes, habitat heterogeneity and species colonization ability account for more of the present-day biogeographical patterns of corals than explanations based on the contemporary distribution of reefs or environmental conditions.     

Using a Strategic Environmental Assessment framework to quantify the environmental impact of bioenergy plans

Renewable energy and greenhouse gas (GHG) reduction targets are driving an acceleration in the use of bioenergy resources. The environmental impact of national and regional development plans must be assessed in compliance with the EU Strategic Environmental Assessment (SEA) Directive (2001/42/EC). Here, we quantify the environmental impact of an Irish Government bioenergy plan to replace 30% of peat used in three peat‐burning power stations, located within the midlands region, with biomass. Four plan alternatives for supplying biomass to the power plant were considered in this study: (1) importation of palm kernel shell from south‐east Asia, (2) importation of olive cake pellets from Spain and (3) growing either willow or (4) Miscanthus in the vicinity of the power stations. The impact of each alternative on each of the environmental receptors proposed in the SEA Directive was first quantified before the data were normalized on either an Irish, regional or global scale. Positive environmental impacts were very small compared to the negative environmental impacts for each of the plan alternatives considered. Comparison of normalized indicator values confirmed that the adverse environmental consequences of each plan alternative are concentrated at the location where the biomass is produced. The analysis showed that the adverse environmental consequences of biomass importation are substantially greater than those associated with the use of willow and Miscanthus grown on former grassland. The use of olive cake pellets had a greater adverse environmental effect compared to the use of peat whereas replacement of peat with either willow or Miscanthus feedstocks led to a substantial reduction in environmental pressure. The proposed assessment framework combines the scope of SEA with the quantitative benefits of life cycle assessment and can be used to evaluate the environmental consequences of bioenergy plans.