Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.     

全基因组扩增策略在基因芯片分析SARS冠状病毒实验中的应用

针对SARS冠状病毒的分子生物学检测是控制SARS流行的关键环节。为评价全基因组扩增对SARS微量样本检测的影响 ,采用 6 mer随机引物反转录 ,用加接头的随机引物合成第二链 ,再以接头序列为引物扩增并掺入荧光标记 ,最后与带有 70 mer探针的基因芯片杂交。此非特异方法基本覆盖了样本中的全部DNA ,结果发现SARS冠状病毒全基因组的扩增效果对基因芯片杂交结果的均匀性有较大影响 ,PCR循环次数增多会导致扩增均匀性的降低。分析了不同的引物对全基因组扩增均匀性的影响 ,探讨了全基因组扩增策略的缺陷。     

Causes and consequences of centrosome abnormalities in cancer

Centrosome amplification is a hallmark of cancer. However, despite significant progress in recent years, we are still far from understanding how centrosome amplification affects tumorigenesis. Boveri''s hypothesis formulated more than 100 years ago was that aneuploidy induced by centrosome amplification promoted tumorigenesis. Although the hypothesis remains appealing 100 years later, it is also clear that the role of centrosome amplification in cancer is more complex than initially thought. Here, we review how centrosome abnormalities are generated in cancer and the mechanisms cells employ to adapt to centrosome amplification, in particular centrosome clustering. We discuss the different mechanisms by which centrosome amplification could contribute to tumour progression and the new advances in the development of therapies that target cells with extra centrosomes.     

The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.     

Oncogene amplification detected by in situ hybridization in radiation-induced skin cancers in rats.

Amplification of the c-myc oncogene has been detected by Southern blotting in the DNA of radiation-induced skin cancers in the rat. In the current work the localization of oncogene amplification within specific cells in the different cancers and in multiple biopsies of the same cancer was studied by in situ hybridization. The amount of amplification was measured by counting grains on tissue sections hybridized in situ to biotin-labeled human c-myc third exon, rat v-H-ras, and rat v-Ki-ras probes. The in situ estimates of c-myc amplification were generally correlated with previous findings using the Southern blot method, but within each cancer only a fraction of cells exhibited amplification. Multiple biopsies of a squamous carcinoma showed amplification of v-H-ras and c-myc but not v-Ki-ras during tumor growth, but none of these oncogenes were amplified during tumor regression. The c-myc-positive cells were distributed uniformly within the cancers and exhibited a more uniform nuclear structure in comparison to the more vacuolated c-myc-negative cells. A high [3H]thymidine labeling index was found in irradiated epidermal cells on Day 7 after exposure, and yet no evidence of c-myc oncogene amplification was found in situ. No c-myc amplification was found in unirradiated normal epidermis or in irradiated epidermal cells in the vicinity of radiation-induced cancers. The data indicate that c-myc amplification is cell-specific within radiation-induced carcinomas and does not occur in epidermal cells proliferating in response to radiation exposure.     

Gamma-ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair

To investigate the role of DNA double strand breaks (DSBs) and of their repair in gene amplification, we analyzed this process in the V3 Chinese hamster cell line and in the parental line AA8, after exposure to gamma-rays and to hydrogen peroxide (H2O2). V3 is defective in DSB repair because of a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene, a gene involved in the non-homologous end-joining pathway. As a measure of gene amplification we used the frequency of colonies resistant to N-(phosphonacetyl)-L-aspartate (PALA), since in rodent cells PALA resistance is mainly achieved through the amplification of the CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) gene. After treatment with different doses of gamma-rays and of H2O2, we found a dose related increase in the frequency of gene amplification and of chromosome aberrations. When the same doses of damaging agents were used, these increments were higher in V3 than in AA8. These results indicate that DSBs that are not efficiently repaired can be responsible for the induction of gene amplification. H2O2 stimulates gene amplification as well as gamma-rays, however, at similar levels of amplification induction, chromosome damage was about 50% lower. This suggests that gene amplification can be induced by H2O2 through pathways alternative to a direct DNA damage. Stimulation of gene amplification by H2O2, which is one of the products of the aerobic metabolism, supports the hypothesis that cellular metabolic products themselves can be a source of genome instability.     

Induction of selective DNA amplification and morphological cell transformation in Syrian hamster embryo cells

DNA amplification is a frequently observed event in continuous cell lines and in tumors. It is likely that a common mechanism underlies the amplification of specific DNA sequences which confer drug resistance and genes which give a growth advantage to the tumor. To find a correlation between the induction of DNA amplification by chemicals and morphological cell transformation we treated Syrian hamster embryo (SHE) cells with diverse antineoplastic agents of different classes. Analysis of these agents seems to be important since they are potentially carcinogenic and resistance inducing. For the measurement of DNA amplification we established a new system using adeno-associated virus type 2 (AAV)-infected primary SHE cells as target cells and amplification of viral DNA as marker of DNA amplification. Simultaneously we determined morphological cell transformation in SHE cells. Our findings demonstrate that there is only a limited correlation between the induction of AAV DNA amplification and the morphological cell transformation in SHE cells. The newly established system of AAV DNA amplification appears to be a useful tool for the investigation of drug resistance in target cells of choice.     

基于PCR的染色体步移中单引物扩增的克服与非特异引物的利用

基于PCR的染色体步移(PCR-Walking)方法已有许多种,包括反向PCR、连接介导的PCR、随机引物PCR等.在众多的方法中,经常存在由通用引物引起的单引物非特异扩增现象.本文综述了连接介导的PCR-Walking中单引物扩增的形成原理及克服方法.克服单引物扩增主要是使接头引物在DNA两端的接头上只有1个结合位点,从而避开单引物扩增.常用的方法有3′端加氨基修饰的不对称接头、泡泡状接头或Y字型接头及单寡核苷酸接头等方法.还介绍了2种利用通用引物非特异扩增克隆目的序列的方法:引物错配法及基于RAPD原理的单引物PCR法.     

Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila.

Drosophila melanogaster follicle cells over-replicate the chromosomal domain containing the third chromosome chorion gene cluster. Multiple regions of this cluster are needed in cis for attainment of high levels of amplification. We have confirmed the importance of the proposed amplification control element (ACE3) and demonstrated that it can support low levels of follicular amplification in the absence of other elements, but that it lacks detectable activity as a DNA replication origin. We have also demonstrated the existence of additional amplification-enhancing regions (AERs), by analyzing the amplification levels of a series of in situ induced, nested deletions of the chorion cluster. These deletions were induced by P-transposase perturbation of a chorion transposon in a highly amplifying transformed line, and were not accompanied by re-transposition, making possible a quantitative analysis of amplification levels in the absence of chromosomal position effects. Analysis of endogenous replication intermediates in wild-type follicular DNA suggested that at least one of the AERs may be an origin of replication and that amplification uses at least one additional replication origin.     

Gene amplification in Chinese hamster DNA repair deficient mutants

In order to study the possible relationship between gene amplification and DNA repair we analyzed the amplification of the CAD gene in four mutants hypersensitive to UV light (CHO43RO, CHO7PV, UV5 and UV61) isolated in vitro from Chinese hamster cell lines (CHO-K1 and AA8). These mutants are characterized by different defects in the nucleotide excision repair mechanism and represent complementation groups 1, 9, 2, and 6 respectively. To evaluate the amplification ability of each cell line we measured the rate of appearance of PALA resistant clones with the Luria and Delbrück fluctuation test. Resistance to PALA is mainly due to amplification of the CAD gene. In the mutants CHO43RO, UV5 and CHO7PV we reproducibly found an amplification rate lower than in the parental cell lines (2–5 times), while in UV61 the amplification rate was about 4 times higher. This result indicates that each mutant is characterized by a specific amplification ability and that the unefficient removal of UV induced DNA damage can be associated with either a higher or a lower amplification rate. However, the analysis of randomly isolated CHO-K1 clones with normal UV sensitivity has shown variability in their amplification ability, making it difficult to relate the specific amplification ability of the mutants to the DNA repair defect and suggesting clonal heterogeneity of the parental population.     

核酸恒温扩增技术研究进展

核酸恒温扩增技术在生命科学研究及相关诸多领域已经得到了广泛应用。我们对核酸恒温扩增技术的最新进展作一简要综述,包括环介导恒温扩增、链替代扩增、依赖核酸序列的扩增、滚环扩增、切口酶核酸恒温扩增、依赖解旋酶的恒温扩增、转录依赖的扩增、杂交捕获法、转录介导的扩增等的原理、优缺点及应用。     

长片段融合PCR在构建烟曲霉rho1基因回补株中的应用

目的融合PCR是一种常用的构建重组片段或重组质粒的手段,但长片段融合PCR的难度较大。文中将探讨长片段融合PCR过程中引物设计及扩增条件对产物的影响。方法以构建烟曲霉rho 1基因回补株为例,采用融合PCR的方法扩增重组片段(长达6.5 kb),在引物设计时引入不同大小的同源区,并设置不同的扩增体系。结果当设计引物的同源区为35 bp,选用具有高扩增效率、高保真性的DNA聚合酶,以及各片段在融合PCR反应体系中的浓度为15 ng/μL时,实现了长达6.5 kb的片段扩增并完成了烟曲霉rho 1回补株的构建。结论在合适的PCR引物设计、片段浓度配比及聚合酶条件下,长片段融合PCR在丝状真菌的基因敲除及回补株的构建中是一种非常有效的工具。     

Global chromosomal structural instability in a subpopulation of starving Escherichia coli cells

Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.     

Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples

Analysis of genomic DNA derived from cells and fresh or fixed tissues often requires whole genome amplification prior to microarray screening. Technical hurdles to this process are the introduction of amplification bias and/or the inhibitory effects of formalin fixation on DNA amplification. Here we demonstrate a balanced-PCR procedure that allows unbiased amplification of genomic DNA from fresh or modestly degraded paraffin-embedded DNA samples. Following digestion and ligation of a target and a control genome with distinct linkers, the two are mixed and amplified in a single PCR, thereby avoiding biases associated with PCR saturation and impurities. We demonstrate genome-wide retention of allelic differences following balanced-PCR amplification of DNA from breast cancer and normal human cells and genomic profiling by array-CGH (cDNA arrays, 100 kb resolution) and by real-time PCR (single gene resolution). Comparison of balanced-PCR with multiple displacement amplification (MDA) demonstrates equivalent performance between the two when intact genomic DNA is used. When DNA from paraffin-embedded samples is used, balanced PCR overcomes problems associated with modest DNA degradation and produces unbiased amplification whereas MDA does not. Balanced-PCR allows amplification and recovery of modestly degraded genomic DNA for subsequent retrospective analysis of human tumors with known outcomes.     

Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number.In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C.The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.     

乳腺癌中EGFR蛋白表达和基因扩增的检测结果比较

目的:比较免疫组织化学技术检测乳腺癌中EGFR蛋白表达和荧光原位杂交检测EGFR基因扩增的结果的符合率,为EGFR靶向治疗病例的选择提供依据。方法:随机选取2005年1月到2011年12月冷水江市人民医院和湖南省肿瘤医院病理科的147例乳腺癌档案病例,采用免疫组织化学技术检测乳腺癌组织中EGFR蛋白表达,荧光原位杂交检测EGFR的基因扩增,比较两种方法阳性结果的符合率。结果:免疫组化染色结果显示EGFR在原发性和转移性乳腺癌中的阳性表达率分别为85%(105/123)和79%1(9/24),两组比较无显著差异(P0.05)。FISH检测结果显示原发性和转移性乳腺癌中分别有12%(15/123)和8%(2/24)存在EGFR基因扩增,两组比较结果无显著差异(P0.05)。所有存在EGFR基因扩增的原发性和转移性乳腺癌的EGFR免疫组织化学结果均为阳性。在原发性和转移性乳腺癌中,免疫组化阳性和基因扩增程度间呈显著正相关(P0.05),但免疫组化结果预测基因扩增的特异性较低。结论:免疫组织化学检测EGFR只能作为EGFR靶向治疗病例选择的初步筛选,进一步进行荧光原位杂交检测EGFR基因扩增是必须的。     

Analysis of PCR-diagnosis of brucellosis in human]

Polymerase chain reaction (PCR) was used for the diagnosis of brucellosis in humans with different forms of this disease. A high incidence (77.6%) of Brucella infection was revealed in the staff of cattle breeding centers with unfavorable situation with regard to brucellosis. Such a conclusion was made after PCR testing of native human sera. In acute brucellosis of humans amplification of the specific site of brucella DNA in PCR is possible only after extraction of DNA by a procedure adapted for DNA extraction from intact brucella cells. In chronic infection weak amplification of brucella genome DNA fragment was observed in investigation of native sera by the PCR. More expressed amplification product was recorded in PCR with a DNA precipitate from this serum obtained by ethanol precipitation. A still higher level of brucella DNA fragment amplification was observed after DNA extraction from sediment obtained by ethanol precipitation from this serum. These data confirmed the incomplete phagocytosis phenomenon at the early stage of infection, known in brucellosis pathogenesis, and allowed some hypotheses on the pathogenesis of chronic phase of brucellosis infection.     

Rapid re-amplification of PCR products purified in low melting point agarose gels

A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.     

Gene amplification in cancer

Gene amplification is a copy number increase of a restricted region of a chromosome arm. It is prevalent in some tumors and is associated with overexpression of the amplified gene(s). Amplified DNA can be organized as extrachromosomal elements, as repeated units at a single locus or scattered throughout the genome. Common chromosomal fragile sites, defects in DNA replication or telomere dysfunction might promote amplification. Some regions of amplification are complex, yet elements of the pattern are reproduced in different tumor types. A genetic basis for amplification is suggested by its relative frequency in some tumor subtypes, and its occurrence in "early" preneoplastic lesions. Clinically, amplification has prognostic and diagnostic usefulness, and is a mechanism of acquired drug resistance.