Autonomous chaotic behaviour of the slime mould Dictyostelium discoideum predicted by a model for cyclic AMP signalling

How sustained oscillations lose their periodicity and thus give rise to chaos was first analysed in mathematical models, then observed in chemical systems such as the Belousov-Zhabotinsky reaction where chaos is autonomous because it originates from endogenous kinetic mechanisms. In contrast, chaos can also be obtained by periodically forcing an oscillatory system, as shown, for example, in cardiac cells and yeast glycolysis. Biochemical evidence for autonomous chaos has been obtained both in vitro for the peroxidase reaction and in enzymatic models not based directly on experimental systems. We report here the occurrence of autonomous chaos in a realistic model for the cyclic AMP signalling system of the slime mould Dictyostelium discoideum, based on receptor modification. This model is also capable of bursting, a phenomenon characteristic of some pacemaker neurones such as R15 in Aplysia. Whereas bursting has not been observed in D. discoideum, our model suggests that 'aperiodic signalling' in the mutant Fr17 provides the first example of autonomous chaos occurring spontaneously at the cellular level.     

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.     

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.     

Chemical structure of the morphogen differentiation inducing factor from Dictyostelium discoideum

Morphogens are signal molecules presumed to exist in embryos and to be involved in establishing the spatial pattern of cells during development. Differentiation inducing factor (DIF) has the properties of a morphogen required for producing the prestalk/prespore pattern in the aggregate formed by cells of the slime mould Dictyostelium in response to starvation. DIF-1, the major bioactive species after purification, has now been identified using a combined microchemical, spectroscopic and synthetic approach. The structure is defined as 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone, and represents a new class of effector molecule. The availability of relatively large quantities of synthetic and isotopically labelled materials should now allow progress towards a detailed understanding of the pattern-forming processes in Dictyostelium development.     

Different recombination site specificity of two developmentally regulated genome rearrangements

In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.     

Mechanism of sequential induction of cell-type specific mRNAs in Dictyostelium differentiation

Upon starvation, the cellular slime mould Dictyostelium discoideum initiates a 24-h programme of differentiation. Within 6 h, cells move towards aggregation centres in response to pulsatile synthesis and secretion of cyclic AMP. At about 12 h, aggregates of 10(5) cells are formed, held together by newly made surface adhesion molecules. The cells then differentiate into the two principal types found in the terminal stage of development, spores and stalks. Here we show that the chemotaxis and aggregation stages of this developmental programme can be described as a series of sequential events in which these extracellular signals--starvation, cyclic AMP and cell-cell contact--induce specific, sequential changes in the pattern of gene expression.     

Primary structure and functional expression of a developmentally regulated skeletal muscle chloride channel.

Skeletal muscle is unusual in that 70-85% of resting membrane conductance is carried by chloride ions. This conductance is essential for membrane-potential stability, as its block by 9-anthracene-carboxylic acid and other drugs causes myotonia. Fish electric organs are developmentally derived from skeletal muscle, suggesting that mammalian muscle may express a homologue of the Torpedo mamorata electroplax chloride channel. We have now cloned the complementary DNA encoding a rat skeletal muscle chloride channel by homology screening to the Cl- channel from Torpedo. It encodes a 994-amino-acid protein which is about 54% identical to the Torpedo channel and is predominantly expressed in skeletal muscle. Messenger RNA amounts in that tissue increase steeply in the first 3-4 weeks after birth, in parallel with the increase in muscle Cl- conductance. Expression from cRNA in Xenopus oocytes leads to 9-anthracene-carboxylic acid-sensitive currents with time and voltage dependence typical for macroscopic muscle Cl- conductance. This and the functional destruction of this channel in mouse myotonia suggests that we have cloned the major skeletal muscle chloride channel.     

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。     

A mutation altering the function of a carbohydrate binding protein blocks cell-cell cohesion in developing Dictyostelium discoideum.

In Dictyostelium discoideum, carbohydrate binding proteins (CBPs) or lectins have been implicated in the molecular basis of cellular cohesion. To determine the role of these CBPs, we have attempted to isolate structural gene mutants in which the CBPs have a defective affinity for carbohydrate ligands. We now report the isolation of a spontaneous, cross-reacting material (CRM) mutant which is non-cohesive and fails to develop. The mutant seems to have a defect in the structural gene for one of the two developmentally regulated carbohydrate binding proteins (CBP-26), which renders it unable to bind to galactose-containing ligands. The fact that wild-type cells interact with the mutant and carry it through development strongly supports a model of cell-cell interaction in which cohesion is mediated by complementary molecules.     

盘基网柄菌粘附分子gp150的定位及其与PKA活性关系的研究

细胞粘附分子gp150在盘基网柄菌细胞发育后期起着重要作用.用gp150抗体定位gp150在细胞发育重要阶段的分布,显示在细胞发育的不同时段,gp150的定位有着明显的区别.在聚集前的细胞流时期,gp150还均匀分布在细胞质内,到了细胞丘时期,gp150就移至多细胞聚集体的边缘部位.到了蛞蝓体时期,gp150在前柄细胞的表达量明显大于前孢子细胞,提示了gp150对柄细胞的作用.而当细胞发育至子实体阶段,gp150大量分布在成熟孢子的孢壁上,这是目前还没有报道的.高效液相色谱法检测野生型KAx-3细胞和gp150过表达细胞KAx-3:acct15/lagC的PKA活性,显示在细胞发育的早期(10 h之前),PKA在野生型细胞中的活性比KAx-3:act15/lagC细胞低.但是在细胞发育的后期,也就是野生型细胞中gp150开始快速积累之后,PKA在野生型细胞中的活性比KAx-3:act15/lagC细胞高.这些结果说明,gp150和PKA之间可能存在某种负反馈环的关系共同调节盘基网柄菌的生长发育.     

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.     

反相高效液相色谱法分析盘基网柄菌对氨基酸的利用

为观察盘基网柄菌(Dictyostelium discoideum)在SIH培养基上对氧基酸的利用情况,以2.4-二硝基氯苯为衍生化试剂,研究了用反相高效液相色谱(RP-HPLC)测定氨基酸的方法.在60 min内16种氨基酸达到基线分离,峰面积与氨基酸浓度的线性相关系数为0.992～0.999.对发酵液样品的分析结果表明,盘基网柄菌对赖氨酸的利用最为彻底,发酵后期赖氨酸已被消耗完全.蛋氯酸、色氨酸,精氨酸、组氧酸也较易被盘基网柄菌利用,而对天冬氨酸、谷氨酸、甘氨酸、苏氨酸、脯氨酸、缬氨酸的需求不大.这一代谢特点为改进盘基网柄菌培养基组成提供依据.     

Cyclic AMP/GMP-dependent modulation of Ca2+ channels sets the polarity of nerve growth-cone turning

Signalling by intracellular second messengers such as cyclic nucleotides and Ca2+ is known to regulate attractive and repulsive guidance of axons by extracellular factors. However, the mechanism of interaction among these second messengers in determining the polarity of the guidance response is largely unknown. Here, we report that the ratio of cyclic AMP to cyclic GMP activities sets the polarity of netrin-1-induced axon guidance: high ratios favour attraction, whereas low ratios favour repulsion. Whole-cell recordings of Ca2+ currents at Xenopus spinal neuron growth cones indicate that cyclic nucleotide signalling directly modulates the activity of L-type Ca2+ channels (LCCs) in axonal growth cones. Furthermore, cGMP signalling activated by an arachidonate 12-lipoxygenase metabolite suppresses LCC activity triggered by netrin-1, and is required for growth-cone repulsion mediated by the DCC-UNC5 receptor complex. By linking cAMP and cGMP signalling and modulation of Ca2+ channel activity in growth cones, these findings delineate an early membrane-associated event responsible for signal transduction during bi-directional axon guidance.     

Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination

Dendritic cells have a unique function in the immune response owing to their ability to stimulate immunologically naive T lymphocytes. In response to microbial and inflammatory stimuli, dendritic cells enhance their capacity for antigen presentation by a process of terminal differentiation, termed maturation. The conversion of immature to mature dendritic cells is accompanied by a marked cellular reorganization, including the redistribution of major histocompatibility complex class II molecules (MHC II) from late endosomal and lysosomal compartments to the plasma membrane and the downregulation of some forms of endocytosis, which has been thought to slow the clearance of MHC II from the surface. The relative extent to which these or other mechanisms contribute to the regulation of surface MHC II remains unclear, however. Here we find that the MHC II beta-chain cytoplasmic tail is ubiquitinated in mouse immature dendritic cells. Although only partly required for the sequestration of MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II ceased upon maturation, resulting in the accumulation of MHC II at the cell surface. Dendritic cells thus exhibit a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.