Sequence and analysis of chromosome 2 of Dictyostelium discoideum

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.     

发酵罐高密度培养盘基网柄菌

利用SIH合成培养基,在7 L发酵罐培养盘基网柄菌.培养147 h后,细胞密度达到4.0×107 mL-1,为在复杂培养基上所能达到的细胞密度的2～4倍.培养过程中葡萄糖的消耗量为6.7 g/L,产氨浓度达0.88 g/L.对培养基中的氨基酸分析表明,赖氨酸、色氨酸、甲硫氨酸和苯丙氨酸消耗较快, 显示SIH培养基的氨基酸成分还可进一步优化.采用基于Monod生长动力学的半经验模型可很好模拟细胞生长和底物消耗,并估计出动力学参数μmax = 0.115 h-1, Nmax = 6.0×107 mL-1.本研究为进一步优化合成培养基和为利用这一新型真核表达系统大规模生产重组异源蛋白奠定了基础.     

Altruism and social cheating in the social amoeba Dictyostelium discoideum

The social amoeba, Dictyostelium discoideum, is widely used as a simple model organism for multicellular development, but its multicellular fruiting stage is really a society. Most of the time, D. discoideum lives as haploid, free-living, amoeboid cells that divide asexually. When starved, 10(4)-10(5) of these cells aggregate into a slug. The anterior 20% of the slug altruistically differentiates into a non-viable stalk, supporting the remaining cells, most of which become viable spores. If aggregating cells come from multiple clones, there should be selection for clones to exploit other clones by contributing less than their proportional share to the sterile stalk. Here we use microsatellite markers to show that different clones collected from a field population readily mix to form chimaeras. Half of the chimaeric mixtures show a clear cheater and victim. Thus, unlike the clonal and highly cooperative development of most multicellular organisms, the development of D. discoideum is partly competitive, with conflicts of interests among cells. These conflicts complicate the use of D. discoideum as a model for some aspects of development, but they make it highly attractive as a model system for social evolution.     

ATR-Chk1-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控

显微镜观察盘基网柄菌野生型KAx--3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍.为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1Cdc25信号通路在其中的可能作用.实验结果表明:RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞.突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞.这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.     

Cellular fluorescent high-throughput screening assays of the ATP-gated P2X7 receptor

The P2X 7 receptor (P2X7R) is an important member of the P2X family of ligand-gated ion channels that respond to ATP as the endogenous agonist. Studies suggest that P2X7R plays a potentially pivotal role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. Thus, P2X7R may be a potential target for drug development. Here, we used a FlexStation to examine the function of recombinant P2X7R stably expressed in human embryonic kidney 293 cells and to compare three high-throughput screening assays: a membrane potential assay, an ethidium bromide uptake assay, and a calcium influx assay. We found that all three assays were suitable for the analysis of P2X7R, but the calcium influx assay was the most robust and is the best choice as a first high-throughput screening assay when embarking on a P2X7R drug discovery project.     

Extracellular sodium regulates airway ciliary motility by inhibiting a P2X receptor.

The mucociliary system is responsible for clearing inhaled particles and pathogens from the airways. This important task is performed by the beating of cilia and the consequent movement of mucus from the lungs to the upper airways. Because ciliary motility is enhanced by elevated intracellular calcium concentrations, inhibition of calcium influx could lead to disease by jeopardizing mucociliary clearance. Several hormones and neurotransmitters stimulate ciliary motility, one of the most potent of which is extracellular ATP (ATP0), which acts by releasing calcium ions from internal stores and by activating calcium influx. Here we show that, in airway ciliated cells, extracellular sodium ions (Na+(0)) specifically and competitively inhibit an ATP0-gated channel that is permeable to calcium ions, and thereby attenuate ATP0-induced ciliary motility. Our finding points to a physiological role for Na+(0) in ciliary function, and indicates that mucociliary clearance might be improved in respiratory disorders such as chronic bronchitis and cystic fibrosis by decreasing the sodium concentration of the airway surface fluid in which the cilia are bathed.     

ATR-Chkl-Cdc25信号通路参与盘基网柄菌G2/M转变期的调控简

显微镜观察盘基网柄菌野生型KAx-3细胞和突变型RNAi-allC细胞,计数结果表明后者的单细胞繁殖速度约为前者的8倍. 为探究该突变型盘基网柄菌细胞周期缩短的原因,用荧光定量PCR和western blot研究了ATR-Chk1-Cdc25信号通路在其中的可能作用. 实验结果表明：RNAi-allC细胞中cdc25基因相对表达量约为KAx-3细胞的8倍,而其Chk1与ATR基因的相对表达量却明显低于KAx-3细胞. 突变细胞中Cdc25蛋白含量高于KAx-3细胞,但其Chk1蛋白含量却显著低于KAx-3细胞. 这些数据表明,两种类型细胞之间的ATR、Chk1、Cdc25在mRNA水平和蛋白表达上均存在差异,特别是ATR基因表达量的不同明显影响ChK1和Cdc25的表达量,提示ATR-Chk1-Cdc25信号通路应该在一定程度上参与了盘基网柄菌细胞周期G2/M期的调控.     

不同介质中分化逆转对细胞粘菌mRNA稳定性影响

细胞粘菌(Dictyostelium discoideum)发育后期细胞被重新分散于营养培养基或缓冲液后,mRNAs稳定性在这二种介质中的变化相似。一些mRNAs稳定性下降,快速降解,另一些mRNAs不受影响。环状AMP选择性地保护其中一些受快速降解的mRNAs。放线菌酮有稳定mRNA作用。受检mRNAs在不同分化逆转介质中的相同效应表明,营养条件不是引起分化逆转细胞mRNA稳定性变化的因素。