Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.     

Insect olfactory receptors are heteromeric ligand-gated ion channels

In insects, each olfactory sensory neuron expresses between one and three ligand-binding members of the olfactory receptor (OR) gene family, along with the highly conserved and broadly expressed Or83b co-receptor. The functional insect OR consists of a heteromeric complex of unknown stoichiometry but comprising at least one variable odorant-binding subunit and one constant Or83b family subunit. Insect ORs lack homology to G-protein-coupled chemosensory receptors in vertebrates and possess a distinct seven-transmembrane topology with the amino terminus located intracellularly. Here we provide evidence that heteromeric insect ORs comprise a new class of ligand-activated non-selective cation channels. Heterologous cells expressing silkmoth, fruitfly or mosquito heteromeric OR complexes showed extracellular Ca2+ influx and cation-non-selective ion conductance on stimulation with odorant. Odour-evoked OR currents are independent of known G-protein-coupled second messenger pathways. The fast response kinetics and OR-subunit-dependent K+ ion selectivity of the insect OR complex support the hypothesis that the complex between OR and Or83b itself confers channel activity. Direct evidence for odorant-gated channels was obtained by outside-out patch-clamp recording of Xenopus oocyte and HEK293T cell membranes expressing insect OR complexes. The ligand-gated ion channel formed by an insect OR complex seems to be the basis for a unique strategy that insects have acquired to respond to the olfactory environment.     

低温烧结3Y-TZP陶瓷的力学性能和耐磨性能

研究了低温烧结 3Y_TZP的烧结性能、力学性能以及耐磨性能 .经成型后的ZrO2(x(Y2 O3 ) =3% )在常压、12 5 0～ 145 0℃温度下 2h烧成 .由于该粉料有很高的烧结活性 ,在 130 0℃低温烧成下就获得了相对密度大于 99%的烧结体 ;在 140 0℃烧成温度下3Y_TZP获得最佳的力学性能和耐磨性能 ,其抗弯强度、断裂韧性和维氏硬度分别达到95 3MPa ,9.1MPa·m1/2 和 12 .7GPa .应力诱导相变是主要的增韧机理 .