小剂量DNA毒剂诱导SV40转化的人细胞中的SV40DNA的增强复制

本文介绍了用~(32)P标记的pSV_2质粒为探针,通过DNA分子原位杂交研究小剂量DNA毒剂诱导被SV40转化的人细胞NB—E中SV40DNA的增强复制.最佳条件下,最大增强比可达4-5.而且,这种病毒DNA增强复制与病毒的增强复活与增强致突有相似的动力学过程和剂量响应关系.此结果为哺乳类细胞中可能存在SOS功能提供了进一步证据.被诱导细胞的Hirt沉淀与Hirt上清液中都存在SV40DNA片段,且Hirt上清液中SV40DNA片段大小均一,反映此过程与λ原噬菌体的诱导的起始过程有相似之处.小剂量紫外线可诱导被SV40转化的人XP细胞中的SV40 DNA的增强复制.说明这一诱导过程可能与切除修复功能有不同的遗传学过程.     

真核DNA复制机制的研究进展

人们对原核细胞ＤＮＡ复制机制早已有较深入的了解 ,但对真核细胞ＤＮＡ复制机制的认识 ,直到 90年代中期才比较清楚。ＳＶ40ＤＮＡ复制系统是研究真核染色体ＤＮＡ复制的理想体系。在ＳＶ40ＤＮＡ复制系统中由病毒编码的唯一的复制因子是Ｔ抗原 ,其余的复制因子全依赖于宿主细胞。利用纯化的蛋白质或人细胞抽提物在体外重新构建ＳＶ40ＤＮＡ复制体系 ,可以细致、深入地研究真核ＤＮＡ复制的起始、延伸和滞后链的成熟。1 .在ＤＮＡ复制的起始至延伸阶段发生ＤＮＡ聚合酶α/δ转换哺乳动物细胞有 5种ＤＮＡ聚合酶 (α、β、γ、δ、ε、)。由…     

复制酶体的瞻望和疑问

由若干酶组成的复合体参与双链DNA分子复制过程。这种活性蛋白质复合体称为复制酶体(replitase),而具有这些酶活性的蛋白质并不能独立起作用。这种复合体在DNA复制(S期)开始时才由其各种成分装配而成,而在细胞周期的Gl期则不复存在。在真核细胞的复制酶体中,参与dNTP生物合成的酶和参与DNA复制的蛋白质结合在一起。在此复合体被装配前,细胞内先合成了大量“粘合”蛋白,以便将这些有关的蛋白质粘合在一起。在真核细胞内是否有这样的复制酶体存在是多年来一个有争论的问题。作者认为真核细胞中的复制酶体可能由细胞核内、外的两个蛋白质复合体所组成,并由两者协同完成DNA复制。如果复制酶体的存在得到证实,将会对诸如细胞周期、DNA复制、癌症发生、抗癌化学疗法和抗代谢物的生物学作用等理论,产生相当大的影响。     

基因工程国内外进展和差距

基因工程是分子水平上的遗传工程。它主要运用重组DNA技术,在特殊酶的作用下,在体外人工连接来自不同生物体的目的基因于有自主复制能力的载体(质粒)DNA中,建成重组DNA的质粒:将此重组质粒送入受体生物细胞去复制和表达,达到遗传物质的转移,产生所需蛋白质。重组DNA技术的主要环节有:目的基因的分离或克隆、体外重组、载体传递或转染和复制、受体细胞繁殖和表达、蛋白质提纯和制备等。基因工程的最大特点是,打破了生物种间界限,使微生物、动植物、甚至人类之间的遗传物质可以互相转移和重组。     

蛋白质起始的DNA病毒复制机制

DNA聚合酶在DNA合成过程中需要的引物包括RNA引物、DNA自我引物和蛋白质引物3种类型。新DNA链(如冈崎片段)的复制多是在DNA模板上合成一段RNA引物,细小病毒利用其基因组末端的反向末端重复序列(ITRs)自我折叠成DNA引物,而一些DNA、RNA病毒及真菌质粒起始复制反应的引物则是蛋白质。以感染原核生物的噬菌体Phi29和真核DNA病毒腺病毒为例,从复制过程所涉及的蛋白质、对复制原点的识别、复制起始反应、新链的延伸、复制终止过程等方面详细阐述DNA病毒由蛋白质引发的复制机制,并对已商品化的Phi29 DNA聚合酶产品多重置换扩增及单细胞测序等的应用以及基于噬菌体Phi29蛋白质起始的最小复制系统体外扩增异源DNA等最新的应用研究作相关总结介绍。     

SV40 T基因转化的山羊乳腺上皮细胞系及其生物学特性

目的建立能用于乳腺特异表达基因构件质量检验的山羊乳腺上皮细胞系.方法根据已发表的SV40病毒T基因序列设计引物,以整合有SV40 DNA早期基因区的COS-1细胞基因组DNA为模板,用高保真PCR扩增SV40 T基因.将获得的SV40 T基因克隆入真核表达载体,并用获得的重组表达质粒转染山羊原代乳腺上皮细胞.经有限稀释和反复传代后获得转化细胞克隆,对其生物学特性进行研究.结果扩增出序列正确的SV40T基因,重组质粒转染获得的转化细胞的对数生长期为接种后第4天,细胞群体倍增时间为23.5*!h,克隆形成率为26.7%.DNA斑点杂交试验证明转化细胞的基因组中整合有SV40 T基因,染色体核型分析试验表明转化细胞的核型无明显异常,裸鼠接种试验证明转化细胞不能形成肿瘤,软琼脂集落形成试验表明转化细胞在软琼脂中不能生长.部分细胞克隆已在体外传30代以上,保持正常乳腺上皮细胞的形态特征,在胶原基质上能形成腺泡样结构.结论本研究获得的SV40 T基因转化的山羊乳腺上皮细胞具有转化细胞系的生物学特性.     

HBV DNA环化结构在体外的复制表达水平优于HBV线性结构的表达质粒

为了研究乙型肝炎病毒（hepatitis B virus, HBV）DNA环化结构与HBV线性结构的表达质粒在体外复制表达水平的差异,采用3种含有HBV全长DNA的表达质粒与自连环化的HBV DNA分别转染Huh7细胞. 5 d后收集转染处理过的Huh7细胞和细胞上清,从感染细胞中抽提纯化HBV复制中间体进行Southern印迹分析,并将细胞培养上清进行ELISA分析.结果显示,HBV DNA通过环化后转染Huh7细胞,可高效地进行转录和复制表达,且优于质粒转染的效果.证明在细胞中,HBV DNA环状结构的复制表达能力优于HBV线性结构的表达质粒.     

细胞周期中DNA复制的控制

细胞周期中ＤＮＡ复制的控制陆长德（中国科学院上海生物化学研究所２０００３１）真核细胞ＤＮＡ复制发生在细胞周期的Ｓ期,细胞ＤＮＡ复制的控制从狭义上看发生在ＤＮＡ复制的起始上;从广义上看,也发生在参与ＤＮＡ复制的酶和蛋白质因子的基因表达调控上。关于这些调控机制虽然远还没搞清,但也取得了一些进展,与近来细胞生物学,以及肿瘤研究的一些进展结合起来,可以说已初露端倪。进一步研究真核细胞ＤＮＡ复制的控制进一步研究真核细胞DNA复制的控制无疑对于搞清细胞周期在G1\S 期的控制机制具有重要意义。 要意义。     

哺乳类线粒体基因组

作为真核细胞的一种重要细胞器的线粒体含有独立的并自主复制和转录的DNA基因组。虽然线粒体蛋白质的大部分系核DNA编码,但有一小部分是线粒体DNA(mt DNA)编码,并由线粒体的蛋白质合成系统合成。线粒体蛋白质合成系统中的rRNA和tRNA也是mt DNA编码。mt DNA的复制、转录以及蛋白质合成系统均有其本身特点,既与非线粒体真核系统有所不同,又有别于原核细胞中者。因此,线粒体基因组的研究在生物学上有重要意义。此外,线粒体的起源和进化是许多生物学家所感兴趣的和长期争论的问题,而mt DNA的进化比较     

哺乳类线粒体基因组

作为真核细胞的一种重要细胞器的线粒体含有独立的并自主复制和转录的DNA基因组。虽然线粒体蛋白质的大部分系核DNA编码,但有一小部分是线粒体DNA(mt DNA)编码,并由线粒体的蛋白质合成系统合成。线粒体蛋白质合成系统中的rRNA和tRNA也是mt DNA编码。mt DNA的复制、转录以及蛋白质合成系统均有其本身特点,既与非线粒体真核系统有所不同,又有别于原核细胞中者。因此,线粒体基因组的研究在生物学上有重要意义。此外,线粒体的起源和进化是许多生物学家所感兴趣的和长期争论的问题,而mt DNA的进化比较     

Carcinogen-induced DNA amplification in vitro: overreplication of the simian virus 40 origin region in extracts from carcinogen-treated CO60 cells.

An in vitro system to study carcinogen-induced amplification in simian virus 40 (SV40)-transformed Chinese hamster (CO60) cells is described. SV40 amplification in this system resembled in many aspects the viral overreplication observed in drug-treated CO60 cells. Cytosolic extracts from N-methyl-N'-nitro-N-nitrosoguanidine-treated cells supported de novo DNA synthesis in the presence of excess exogenous T antigen and the SV40-containing plasmid pSVK1. The pattern of viral replication in these extracts was unique, since only the 2.4-kilobase-pair region spanning the origin was overreplicated, whereas distal sequences were not replicated significantly. Extracts from control cells supported only marginal levels of replication. In HeLa extracts, complete SV40 DNA molecules were replicated efficiently. The overreplication of the origin region in CO60 cell extracts was bidirectional and symmetrical. A fraction of the newly synthesized DNA molecules underwent a second round of replication, yielding MboI-sensitive fragments representing the 2.4-kilobase-pair region around the origin. The mechanisms controlling the amplification of the viral origin region, the nature of the cellular factors induced in the carcinogen-treated cells, and their putative association with general drug-induced SOS-like responses are discussed.     

Purification and functional characterization of bovine RP-A in an<Emphasis Type="Italic">in vitro</Emphasis> SV40 DNA replication system

The single-stranded DNA binding protein RP-A is required in SV40 DNAin vitro replication. The RP-A purified from calf thymus contains 4 polypeptides with molecular weights 70kDa, 53kDa, 32kDa, and 14kDa. The p70 subunit and its proteolysed form p53 are recognized by the monoclonal antibody 70C (Kenny et al. (1990)) and bind to ssDNA. The p70 and p32 subunits of bovine RP-A are phosphorylated by CDC2-cyclin B kinase. Bovine RP-A supports the origin dependent unwinding of SV40 DNA by T antigen. Furthermore, bovine RP-A can efficiently substitute for human RP-A in SV40 DNA replicationin vitro. A modified blotting technique revealed that RP-A interacts specifically and directly with the p48 subunit of DNA polymerase α-primase complex.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts

Cellular DNA is under constant attack from numerous exogenous and endogenous agents. The resulting DNA lesions, if not repaired timely, could stall DNA replication, leading to genome instability. To better understand the mechanism of DNA lesion replication at the biochemical level, we have attempted to reconstitute this process in Xenopus egg extracts, the only eukaryotic in vitro system that relies solely on cellular proteins for DNA replication. By using a plasmid DNA that carries a site-specific apurinic/apyrimidinic (AP) lesion as template, we have found that DNA replication is stalled one nucleotide before the lesion. The stalling is temporary and the lesion is eventually replicated by both an error-prone mechanism and an error-free mechanism. This is the first biochemical system that recapitulates efficiently and faithfully all major aspects of DNA lesion replication. It has provided the first direct evidence for the existence of an error-free lesion replication mechanism and also demonstrated that the error-prone mechanism is a major contributor to lesion replication.     

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.     

In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus

Summary Linear, extrachromosomal DNA's of the filamentous fungus Ascobolus immersus are localized within the mitochondria. These linear plasmids have no homology to the high molecular weight mtDNA (hmw mtDNA). For analysis of plasmid replication an in organello DNA synthesis system was developed, in which radionucleotides were incorporated into intact mitochondria. Plasmid DNA is labelled preferentially in this system. From replication analysis of a specific plasmid there is evidence of a virus-like protein-primed replication. Sequence analysis of this plasmid reveals that a viral DNA polymerase is encoded. Thus, these genetic elements presumably are viral remnants rather than true plasmids.     

In vitro initiation of DNA replication in simian virus 40 chromosomes

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.     

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.     

Inactivation of topoisomerase I or II may lead to recombination or to aberrant replication termination on both SV40 and yeast 2 μm DNA

Topoisomerase I is believed to be sufficient for early replication of circular viral genomes such as those of SV40 and of yeast plasmids. Topoisomerase II is required for the decatenation of the daughter genomes and probably also for fork elongation during the later stages of SV40 replication. Using the neutral-neutral two-dimensional gel system, we have followed the progression of replication of both SV40 and the yeast 2μm plasmid under various conditions of topoisomerase inhibition. During SV40 replication, inhibition of topoisomerase II by VP16, VM26 or hypertonic shock (but not by merbarone), and inhibition of topoisomerase I by camptothecin all led to the accumulation of aberrant DNA structures containing two almost completely replicated genomes. These aberrant structures resembled either recombination intermediates or late Cairns structures in which the site of replication termination had shifted and now mapped to a continuum of sites throughout the genome. Replication of the 2 μm plasmid in a topoisomerase II- but not a topoisomerase I-deficient yeast gave rise to very similar structures. The data suggest that inactivation of topoisomerase I or II either stimulates recombination or, by differentially affecting replication fork progression, leads to aberrant replication termination. Edited by: J. Huberman