R环的形成及对基因组稳定性的影响

R-环是由一个RNA:DNA杂交体和一条单链状态的DNA分子共同组成的三链核酸结构。其中, RNA:DNA杂交体的形成起因于基因转录所合成的RNA分子不能与模板分开, 或RNA分子重新与一段双链DNA分子中的一条链杂交。在基因转录过程中, 当转录泡遇到富含G碱基的非模板链区或位于某些与人类疾病有关的三核苷酸卫星DNA时, 转录泡后方累积的负超螺旋可促进R环形成。同时, 新生RNA分子未被及时加工、成熟或未被快速转运到细胞质等因素也会催生R环。研究表明, 细胞拥有多种管理R环的方法, 可以有效地管理R环的形成和处理已经形成的R环, 以尽量避免R环对DNA复制、基因突变和同源重组产生不利影响。文章重点分析了R-环的形成机制及R环对DNA复制、基因突变和同源重组的影响, 并针对R-环诱导的DNA复制在某些三核苷酸重复扩增有关的神经肌肉退行性疾病发生过程中的作用进行了分析和讨论。     

动物线粒体DNA的分子生物学研究进展

动物线粒体ＤＮＡ的分子生物学研究进展张方米志勇（中国科学院发育生物学研究所北京１０００８０）绝大多数的真核生物中都含有线粒体（ｍｉ－ｔｏｃｈｒｏｄｒｉａ,ｍｔ）这种细胞器,它自身携带ＤＮＡ,可自我复制、表达,并有核基因编码的蛋白质和酶从细胞质输入线粒体,共同完成生物氧化的生理功能。通过多年来对各种生物线粒体基因组结构和功能的研究,人们推测线粒体起源于紫色光合细菌,这种细菌入侵真核生物,与真核...     

High-level expression of a foreign gene by a recombinant baculovirus with an expanded host range

The usefulness of host range expanded viruses as an expressionvector system was investigated by following the expression ofthe E. coli lacZ gene. The host range expanded recombinantviruses were obtained from Sf-21 or BmN-4 cells coinfected withAutographa californica and Bombyx mori nuclearpolyhedrosis viruses. Among the host range expanded viruses,RecB-8 and RecS-B6 have similar enzyme digestion profiles butdifferent infection characteristics in cells. Therefore, tostudy the foreign gene expression efficiency of these twoviruses, we constructed recombinant viruses RecB8-LacZ andRecSB6-LacZ containing the lacZ gene instead of the polyhedringene. Also, the host range expanded recombinant AcNPV, Bac-BH,containing lacZ gene in the polyhedrin gene locus was constructedby substitution of the 0.6 kb region within the helicase gene ofBacPAK6 with that of BmNPV. -Galactosidase expressionefficiency by these viruses were determined and compared in Sf-21and BmN-4 cells. The result showed that Bac-BH has highexpression efficiency only in Sf-21 cells, whereas RecB8-LacZhas high expression efficiency both in Sf-21 and BmN-4 cells.Also, in BmN-4 cells, -galactosidase expressionefficiency of RecB8-LacZ was higher than that of recombinantBmNPV (BmK1-LacZ containing lacZ gene in polyhedrin gene locus).In addition, the expression efficiency was not correlated withvirus titer.     

基因组叠加生物型的基因表达特征

采用聚丙烯酰胺梯度凝胶电泳及特异性组织化学染色技术分析了鲤、鲫及其基因组叠加的不同组合个体的红细胞ＳＯＤ、ＥＳＴ和血红蛋白的电泳图谱。结果表明不同基因组合及不同倍性的个体间存在由基因组差异导致的生化多态性,为不同倍性的鱼类的同工酶（ＳＯＤ）表达存在基因剂量效应提供了依据,证实了不同组合的ＳＯＤ亚基间有协同表达、累积作用。研究表明每个基因有其独特的调控机制,因而在杂种三倍体内三分之一的基因组亦能够表     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV,and knockout of cysteinase gene for more efficient expression

AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insectbaculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect,A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm,Bombyx mori, which is wellknown for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for largescale industrial expression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an application example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in commonuse cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the production efficiency of the bFGF recombinant protein.     

Construction of a host range-expanded hybrid baculovirus of BmNPV and AcNPV, and knockout of cysteinase gene for more efficient expression

The four main gene expression systems currently used to produce recombinant proteins are the prokary-otic, yeast, insect cell, and mammalian cell expression systems. The baculovirus expression vector system (BEVS) is a protein production system which uses a recombinant baculovirus harboring a foreign gene of interest to produce recombinant protein in an insect or its cultured cells. BEVS has many advantages: (i) BEVS requires less time to establish the production system than is needed in a…     

Genome Rearrangements Caused by Depletion of Essential DNA Replication Proteins in Saccharomyces cerevisiae

Genetic screens of the collection of ～4500 deletion mutants in Saccharomyces cerevisiae have identified the cohort of nonessential genes that promote maintenance of genome integrity. Here we probe the role of essential genes needed for genome stability. To this end, we screened 217 tetracycline-regulated promoter alleles of essential genes and identified 47 genes whose depletion results in spontaneous DNA damage. We further showed that 92 of these 217 essential genes have a role in suppressing chromosome rearrangements. We identified a core set of 15 genes involved in DNA replication that are critical in preventing both spontaneous DNA damage and genome rearrangements. Mapping, classification, and analysis of rearrangement breakpoints indicated that yeast fragile sites, Ty retrotransposons, tRNA genes, early origins of replication, and replication termination sites are common features at breakpoints when essential replication genes that suppress chromosome rearrangements are downregulated. We propose mechanisms by which depletion of essential replication proteins can lead to double-stranded DNA breaks near these features, which are subsequently repaired by homologous recombination at repeated elements.     

A chloroplast derived trnH gene is expressed in the mitochondrial genome of gramineous plants

We reported previously that the mitochondrial sequence that contains the chloroplast-derived trnH gene has been highly conserved in the region around one terminus of the junction between chloroplast-derived and mitochondrion-specific sequences in most of the gramineous plants analyzed [15]. The results of RT-PCR, northern hybridization, in vitro capping and ribonuclease protection experiments show that the chloroplast-derived trnH gene is transcribed from a putative promoter that is located in the mitochondrion-specific sequence. Gene expression in this region seems to be correlated with the conservation of the sequence at the junction between the chloroplast-derived fragment and the mitochondrion-specific sequence.     

Mitochondrial gene rearrangements: new paradigm in the evolutionary biology and systematics

Mitochondrial (mt) genomic study may reveal significant insight into the molecular evolution and several other aspects of genome evolution such as gene rearrangements evolution, gene regulation, and replication mechanisms. Other questions such as patterns of gene expression mechanism evolution, genomic variation and its correlation with physiology, and other molecular and biochemical mechanisms can be addressed by the mt genomics. Rare genomic changes have attracted evolutionary biology community for providing homoplasy free evidence of phylogenetic relationships. Gene rearrangements are considered to be rare evolutionary events and are being used to reconstruct the phylogeny of diverse group of organisms. Mt gene rearrangements have been established as a hotspot for the phylogenetic and evolutionary analysis of closely as well as distantly related organisms.     

DNA聚合酶在维持基因组稳定性中的多重功能及其相关疾病

遗传物质的稳定传递是生命繁衍的根本。基因组DNA的精确复制和分配是遗传物质传递的基础,也是细胞周期两大最核心的生物学事件。DNA聚合酶作为催化合成DNA双链的酶,是复制过程中最重要的因子之一。尽管对这类酶的研究已有将近60年的历史,但依然是生命科学基础研究的前沿之一。真核生物中已知的DNA聚合酶有十几种,它们不仅参与正常基因组DNA合成过程,也参与DNA损伤情况下多种修复过程。如此众多的具有不同特性的DNA聚合酶在细胞内是如何分工与合作的,在正常细胞传代与环境胁迫等情况下维护基因组稳定性中的关键作用及其分子机制又是什么。更有意思的是,最近的肿瘤细胞比较基因组数据表明,多种DNA聚合酶基因突变与某些肿瘤和遗传疾病相关,从而为这些疾病致病机理研究与诊治提供了新的思路和方法。对上述DNA聚合酶相关核心问题的最新研究进展进行了综述。     

Genome dynamics in major bacterial pathogens

Pathogenic bacteria continuously encounter multiple forms of stress in their hostile environments, which leads to DNA damage. With the new insight into biology offered by genome sequences, the elucidation of the gene content encoding proteins provides clues toward understanding the microbial lifestyle related to habitat and niche. Campylobacter jejuni, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis , the pathogenic Neisseria, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are major human pathogens causing detrimental morbidity and mortality at a global scale. An algorithm for the clustering of orthologs was established in order to identify whether orthologs of selected genes were present or absent in the genomes of the pathogenic bacteria under study. Based on the known genes for the various functions and their orthologs in selected pathogenic bacteria, an overview of the presence of the different types of genes was created. In this context, we focus on selected processes enabling genome dynamics in these particular pathogens, namely DNA repair, recombination and horizontal gene transfer. An understanding of the precise molecular functions of the enzymes participating in DNA metabolism and their importance in the maintenance of bacterial genome integrity has also, in recent years, indicated a future role for these enzymes as targets for therapeutic intervention.     

比较基因组学和人类基因组研究

在人类基因组计划突飞猛进地开展的同时,模式生物基因组计划也在轰轰烈烈地进行,并取得许多实质性的进展,是人类对于模式生物基因组有了更广泛更深刻的认识。这些知识的积累,从根本上推动了人类基因组计划的进行。     

Genome affinities and epitope profiles of whitefly-transmitted geminiviruses from the Americas

The relationships among fifteen isolates of whitefly-transmitted geminiviruses (WTGs) from North, Central and South America and six from other continents were assessed (a) in nucleic acid hybridisation tests with sulphonated DNA probes for eight of the viruses, and/or (b) in triple-antibody-sandwich ELISA with panels of monoclonal antibodies (MAbs) to particles of African cassava mosaic virus (ACMV) and Indian cassava mosaic virus (ICMV). Probes specific for DNA-A of four American viruses, abutilon mosaic (AbMV), bean golden mosaic (BGMV), squash leaf curl (SLCV) and tomato golden mosaic (TGMV), detected virtually all the American viruses but reacted weakly if at all with ICMV, ACMV or tomato yellow leaf curl virus from Thailand (TYLCV-T). Conversely, the probe for ACMV DNA-A did not detect any of the American viruses, and that for TYLCV-T DNA-A reacted weakly with SLCV and TGMV0020but did not detect the others. In contrast, probes specific for DNA-B of the four American viruses or ACMV detected only the homologous virus, except for slight reactions between the AbMV DNA-B probe and both chino del tomate virus (CdTV)-DNA and SLCV-DNA. However, a probe for DNA-B of bean calico mosaic virus (BCMoV) reacted weakly with BGMV-PR DNA, and a probe for DNA-B of CdTV from Mexico detected several American viruses. Six out of 17 MAbs specific for ACMV and six out of 10 MAbs specific for ICMV reacted with one or other of the 14 American virus isolates tested. Two and-ACMV MAbs reacted with all, and one anti-ACMV MAb and two anti-ICMV MAbs reacted with nearly all the American viruses, one anti-ACMV MAb reacted with about half the American viruses and six other MAbs reacted with only one or two of them. Of the American viruses, CdTV and AbMV were the least closely related to the others. The epitope profiles of BCMoV, BGMV, cotton leaf crumple virus, serrano golden mosaic virus and SLCV were virtually indistinguishable. TGMV, potato yellow mosaic virus (PYMV) and an euphorbia virus had profiles intermediate between those of the BGMV cluster and AbMV-CdTV. In general, the epitope profiles and the results of hybridisation tests with DNA-A probes show that the similarities among the American viruses are greater than those between the American viruses and the viruses from other continents; the hybridisation tests with DNA-B probes show that substantial differences exist between individual American viruses. In America, geminivirus evolution seems to have proceeded convergently from different progenitor viruses, or divergently from one ancestral form, with DNA-B diverging to a greater extent than DNA-A and its particle-protein gene.     

GeneAnnot: interfacing GeneCards with high-throughput gene expression compendia

The interpretation of microarray expression results often includes extensive efforts to identify and annotate the gene representatives immobilised on the arrays. In this paper we describe the usage of our automatic GeneAnnot system, which links between Affymetrix arrays and the rich human gene annotations available in GeneCards. We explain GeneCards search options and results display; elaborate on the presentation of expression information in GeneCards, including both our whole-genome GeneNote project and external expression resources; describe the various parameters and displays used by GeneAnnot to assess the annotation quality and probeset specificity; and show how to search GeneAnnot and GeneNote websites directly.