Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.     

Activated signal transduction kinases frequently occupy target genes

Cellular signal transduction pathways modify gene expression programs in response to changes in the environment, but the mechanisms by which these pathways regulate populations of genes under their control are not entirely understood. We present evidence that most mitogen-activated protein kinases and protein kinase A subunits become physically associated with the genes that they regulate in the yeast (Saccharomyces cerevisiae) genome. The ability to detect this interaction of signaling kinases with target genes can be used to more precisely and comprehensively map the regulatory circuitry that eukaryotic cells use to respond to their environment.     

Cell biology. An age of instability

It is well established that as we age our cancer risk increases dramatically. As Sinclair explains in his Perspective, the link between cancer and aging is now solidified by new work in budding yeast (McMurray and Gottschling). As yeast cells age there is a marked increase in their genetic instability (a hallmark of cancer), which is independent of the mechanism that determines their life-span.     

Programmed DNA deletion as an RNA-guided system of genome defense

Genomewide DNA rearrangements occur in many eukaryotes during development, but their functions and mechanisms are poorly understood. Previous studies have implicated a sequence-recognition mechanism based on RNA-mediated interactions between nuclei in ciliated protozoa. In this study, we found that the process recognized and deleted a foreign gene integrated in a Tetrahymena chromosome, suggesting an unusual mechanism of genome surveillance. We further found that injection of double-stranded RNA into the cell at specific developmental stages triggers efficient deletion of the targeted genomic regions. Together the results indicate an RNA-based mechanism that directs genomewide DNA rearrangements and serves to disable invading genetic agents.     

植物线粒体结构基因组研究进展

植物线粒体基因组具有复杂的结构特征:基因组200~2 400 kb不等,变化大、重组频繁,存在水平基因迁移现象等。基因组中既含有非常保守的功能基因,也存在高度变异的基因间区序列。大量研究表明,线粒体基因组是植物细胞质雄性不育因子的载体。综述了近年来植物线粒基因组结构特征、基因组成和热点研究的进展,为进一步深入研究植物细胞质雄性不育的分子机理,并为胞质雄性不育三系杂交种选育、作物杂种优势利用提供理论指导。     

How viruses enter animal cells

Viruses replicate within living cells and use the cellular machinery for the synthesis of their genome and other components. To gain access, they have evolved a variety of elegant mechanisms to deliver their genes and accessory proteins into the host cell. Many animal viruses take advantage of endocytic pathways and rely on the cell to guide them through a complex entry and uncoating program. In the dialogue between the cell and the intruder, the cell provides critical cues that allow the virus to undergo molecular transformations that lead to successful internalization, intra-cellular transport, and uncoating.     

杆状病毒细胞凋亡抑制基因的研究进展

细胞凋亡是多细胞生物为维持自身的正常机能而产生的细胞自杀性行为，杆状病毒为维持自身的生长繁殖而产生了对抗细胞凋亡的基因p35和iap，二者分别作用于细胞凋亡途径的不同部位，以抑制细胞的凋亡。近年来人们对这两种基因的蛋白结构及作用机制等方面进行了大量的研究，对它们的作用机制有了进一步的了解，为其今后在医学及分子生物学等方面的应用打下了基础。     

逆转座子LINE-1在基因组中的调控作用

近年来研究发现真核生物基因组中的许多重复序列以及基因的内含子参与基因表达的调控。在这些重复序列中,有一种广泛分布于基因组中的逆转座子LINE-1,目前发现其对细胞的增殖与分化以及肿瘤的发生起着非常重要的作用,具体表现为影响基因的转录及整个基因组的稳定性、参与X染色体失活及基因组进化等。在正常细胞的分化调控中,基因的激活与沉默具有时间与空间的特异性,实现其“预定”的、有序的、不可逆转的分化过程。主要阐述了逆转座子LINE-1的结构特征和其在基因组中的调控作用及这些作用影响基因表达从而调节生物体的生命活动。研究LINE-1的调控机制对认识细胞的时空调控以及癌症的发生与发展具有重要的价值。     

Aging and genome maintenance: lessons from the mouse?

Recent progress in the science of aging is driven largely by the use of model systems, ranging from yeast and nematodes to mice. These models have revealed conservation in genetic pathways that balance energy production and its damaging by-products with pathways that preserve somatic maintenance. Maintaining genome integrity has emerged as a major factor in longevity and cell viability. Here we discuss the use of mouse models with defects in genome maintenance for understanding the molecular basis of aging in humans.     

Structural dynamics of eukaryotic chromosome evolution

Large-scale genome sequencing is providing a comprehensive view of the complex evolutionary forces that have shaped the structure of eukaryotic chromosomes. Comparative sequence analyses reveal patterns of apparently random rearrangement interspersed with regions of extraordinarily rapid, localized genome evolution. Numerous subtle rearrangements near centromeres, telomeres, duplications, and interspersed repeats suggest hotspots for eukaryotic chromosome evolution. This localized chromosomal instability may play a role in rapidly evolving lineage-specific gene families and in fostering large-scale changes in gene order. Computational algorithms that take into account these dynamic forces along with traditional models of chromosomal rearrangement show promise for reconstructing the natural history of eukaryotic chromosomes.     

Comparative genomics of the eukaryotes

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.     

蜜蜂发育相关基因研究

蜜蜂是一种重要的授粉昆虫,也是研究人类疾病及社会行为的模式生物之一。由其基因组测序可知,蜜蜂基因组中(A+T)及CpG含量较高,其昼夜节律、RNAi和DNA甲基化基因更类似脊椎动物。先天免疫、表皮蛋白和味觉受体基因较少,气味受体基因较多。蜜蜂早期发育途径中的一些基因与果蝇的相似,功能却显著不同。本文综述了蜜蜂卵、幼虫、蛹和成虫期发育相关基因及其研究进展。     

耐辐射异常球菌DNA损伤修复重要功能蛋白的研究进展

耐辐射异常球菌(DR)具有对致死剂量的电离辐射极端的抗性,但对其超强辐射抗性的分子机制仍缺乏深入了解。耐辐射异常球菌基因组分析显示其拥有许多与修复相关的基因或蛋白,转录组学和蛋白组学分析等研究表明该菌能通过复杂的代谢途径控制的网络系统有效地调整并修复DNA。近年来已分析和鉴定了许多与DNA损伤修复,特别是辐射诱导的DNA双链断裂修复相关的蛋白,这些功能蛋白的研究有助于我们加深对其极端抗性机理的认识。主要针对DR双链断裂修复系统中重要功能蛋白的研究进展进行了概述,以期为DNA修复机理的基础研究及应用研究奠定基础。     

耐盐性Imt1基因表达载体的构建及其在酵母中的高效表达

 将Imt1（Inositol O-methyltransferase，肌醇甲基转移酶）构建成酵母表达载体pBD#-3，在酵母双突变体gpd#+（-）(glycerol 3-phosphate-dehydrogenase,3-磷酸甘油脱氢酶)中高效表达，使gpd#+（-）突变株对氯化钠的耐性由2.5%提高到5.0%。生化分析表明，在每mg干酵母细胞内积累高达2.2nmol的Ononitol(芒柄醇)含量。Western blot分析证明了Imt1基因的表达。这一研究为植物耐盐基因工程开辟了一条新途径。     

Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes

Budding yeast grown under continuous, nutrient-limited conditions exhibit robust, highly periodic cycles in the form of respiratory bursts. Microarray studies reveal that over half of the yeast genome is expressed periodically during these metabolic cycles. Genes encoding proteins having a common function exhibit similar temporal expression patterns, and genes specifying functions associated with energy and metabolism tend to be expressed with exceptionally robust periodicity. Essential cellular and metabolic events occur in synchrony with the metabolic cycle, demonstrating that key processes in a simple eukaryotic cell are compartmentalized in time.     

Human-specific changes of genome structure detected by genomic triangulation

Knowledge of the rhesus macaque genome sequence enables reconstruction of the ancestral state of the human genome before the divergence of chimpanzees. However, the draft quality of nonhuman primate genome assemblies challenges the ability of current methods to detect insertions, deletions, and copy-number variations between humans, chimpanzees, and rhesus macaques and hinders the identification of evolutionary changes between these species. Because of the abundance of segmental duplications, genome comparisons require the integration of genomic assemblies and data from large-insert clones, linkage maps, and radiation hybrid maps. With genomic triangulation, an integrative method that reconstructs ancestral states and the structural evolution of genomes, we identified 130 human-specific breakpoints in genome structure due to rearrangements at an intermediate scale (10 kilobases to 4 megabases), including 64 insertions affecting 58 genes. Comparison with a human structural polymorphism database indicates that many of the rearrangements are polymorphic.     

Isolation of single-copy human genes from a library of yeast artificial chromosome clones

A recently developed cloning system based on the propagation of large DNA molecules as linear, artificial chromosomes in the yeast Saccharomyces cerevisiae provides a potential method of cloning the entire human genome in segments of several hundred kilobase pairs. Most application of this system will require the ability to recover specific sequences from libraries of yeast artificial chromosome clones and to propagate these sequences in yeast without alterations. Two single-copy genes have now been cloned from a library of yeast artificial chromosome clones that was prepared from total human DNA. Multiple, independent isolates were obtained of the genes encoding factor IX and plasminogen activator inhibitor type 2. The clones, which ranged in size from 60 to 650 kilobases, were stable on prolonged propagation in yeast and appear to contain faithful replicas of human DNA.