植物DNA双链断裂修复的保守性和特异性

文章概述了植物DNA双链断裂（double-strand break，DSB）修复的研究进展。从酵母、脊椎动物、植物在此领域已取得的成果来看，真核生物DSB修复在过程和参与蛋白方面均有一定的进化保守性；另一方面，植物的DSB修复有其特异之处。     

DNA双链断裂与同源重组修复机理研究进展

双链断裂（double strand breaks,DSBs)是细胞染色体复制过程中经常出现的DNA损伤,它的修复过程顺真核生物中以同源重组（homology recombination,HR)修复为主。正常机体中有着一系列的基因和蛋白及时修复复这些损伤,这些蛋白归属于RAD52上位性集团（RAD52epistasis group)。它们对细胞发挥功能和维持生存意义重大,近来国外研究十分活跃。     

植物基因打靶技术

基因打靶是反向遗传学的基础工具,它通过同源重组置换染色体内的基因用于复杂基因组的基因功能分析。但是,在植物中,外源DNA的插入主要是非序列依赖的非同源末端连接方式,基因打靶频率很低,只有10-5～10-4的水平。综述了近年来为了提高植物基因打靶频率,研究人员的工作和最新进展 。     

植物基因同源重组研究现状

同源重组是普遍存在的生物学现象,从噬菌体、细菌到真核生物均有存在。它对生物的遗传与变异具有重大影响,一直是生物学家研究的热点。本文从染色体外同源重组、染色体内同源重组以及基因打靶三个方面综述了同源重组在植物方面的研究现状。从分子水平上较详尽的介绍了同源重组发生的机制以及同源重组在生物领域的应用、前景展望及其存在的局限性。     

DNA双链断裂损伤修复系统研究进展

多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内建立了复杂的修复系统来应对不同形式的损伤。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重的损伤形式,主要激活同源重组修复(Homologous recombination repair)和非同源末端连接(Non-homologous end joining)通路。这两条通路都是由多个修复元件参与、经过多步反应的复杂过程。两者各具特点、协同作用,共同维护细胞基因组的稳定性。对其分子机制的阐明为肿瘤放化疗的辅助治疗提供了潜在的作用靶点。     

Red同源重组技术研究进展

伴随着分子生物学的发展,一种基于λ噬菌体Red重组酶的同源重组系统已应用于大肠杆菌基因工程研究。Red重组系统由三种蛋白组成:Exo蛋白是一种核酸外切酶,结合在双链DNA的末端,从5′端向3′端降解DNA,产生3′突出端;Beta蛋白结合在单链DNA上,介导互补单链DNA退火;Gam蛋白可与RecBCD酶结合,抑制其降解外源DNA的活性。Red同源重组技术具有同源序列短(40～60bp)、重组效率高的特点。这种技术可在DNA靶标分子的任意位点进行基因敲除、敲入、点突变等操作,无需使用限制性内切酶和连接酶。此外,这种新型重组技术可直接将目的基因克隆于载体上,目的基因既可来源于细菌人工染色体也可是基因组DNA。Red同源重组技术使难度较大的基因工程实验顺利进行,大大推动功能基因组研究的发展。     

DNA双链断裂修复的选择性调控机制

在各种DNA损伤中,DNA双链断裂(double-strand break,DSB)是最为严重的一种,快速准确地修复DSB对维持基因组稳定性起着至关重要的作用。真核生物细胞通过一系列复杂的信号转导途径激活对DSB的修复,其中最为重要的是同源重组和非同源末端连接机制。最近的研究表明,这两种方式在DSB修复的早期是相互竞争的关系,其选择在很大程度上受到53BP1及同源蛋白质的调控。将讨论53BP1作为DSB修复途径的核心因子,在染色质水平整合BRCA1、Ct IP等修复因子和多种组蛋白修饰构成的信号途径,介导同源重组和非同源末端连接通路选择的分子机制。     

基因打靶技术的原理及操作

基因打靶是近几年发展起来的一种通过同源重组定点改变小鼠基因组特定位点的技术,其诞生是分子生物学与实验胚胎学方法相结合的产物,它的出现又导致了体内研究与体外研究、分子生物学与临床病理学的有机结合,为研究基因的体内功能和疾病的致病机理提供了一种有力的实验手段。本文以基因打靶的实验过程为主线,介绍该技术的原理、操作、进展和应用。     

基因打靶技术的研究进展

基因打靶技术是20世纪80年代发展起来的新技术,是一种利用DNA同源重组原理和胚胎干细胞(ES细胞)技术按定向组合的方式改变生物活体遗传信息的试验手段,具有广阔的应用前景。对基因打靶技术原理、步骤、条件性基因打靶以及应用进行综述。     

DSB重组修复与肿瘤关系的研究进展

DNA损伤未及时有效地修复可导致基因组不稳定,增加肿瘤发生率。DSB是基因突变、染色体断裂的主要原因之一,并对肿瘤发生、发展具有一定影响,其修复主要是通过HR和NHEJ两条重组途径完成的。本综述了国外近来对DSB重组修复的HR和NHEJ途径,其与肿瘤抑制蛋白如P53、ATM、BRCA1和BRCA2之间的联系;DSB重组修复异常与某些肿瘤及具有肿瘤易感特征的共济失调性毛细血管扩张症和Nijmegen断裂综合征等疾病之间关系的研究进展。     

Homologous recombination: a basis for targeted genome optimization in crop species such as maize

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I- Sce I gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%–40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.     

Homologous Recombination in Zebrafish ES Cells

Targeted insertion of a plasmid by homologous recombination was demonstrated in zebrafish ES cell cultures. Two selection strategies were used to isolate ES cell colonies that contained targeted plasmid insertions in either the no tail or myostatin I gene. One selection strategy involved the manual isolation of targeted cell colonies that were identified by the loss of fluorescent protein gene expression. A second strategy used the diphtheria toxin A-chain gene in a positive-negative selection approach. Homologous recombination was confirmed by PCR, sequence and Southern blot analysis and colonies isolated using both selection methods were expanded and maintained for multiple passages. The results demonstrate that zebrafish ES cells have potential for use in a cell-mediated gene targeting approach.     

UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors

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Recombination in the Plant Genome and its Application in Biotechnology

Genetic variation within and between species is based on recombination of DNA molecules. Recombination also plays a very important role in the repair of damaged DNA. Clarity about the mechanism by which recombination occurs is of profound interest not only to understand how this process assures the maintenance of genome integrity and at the same time is the driving force of evolution, but also for its application in biotechnology. The isolation of genes involved in recombination and the elucidation of the role of many of the corresponding gene products in Escherichia coli and Saccharomyces cerevisiae has formed the basis for comparative analysis in other, more complex eukaryotic systems. The identification of homologous genes from different organisms, including plants, suggests a conservation of the general mechanisms of recombination. Transgenes introduced in an organism may be incorporated in the genome by either homologous or nonhomologous recombination (end joining). The preferred pathway differs strongly between organisms. In plants there is a preference for random integration of the introduced DNA by nonhomologous recombination, which might lead to the accidental inactivation of important genes and to variable and unpredictable expression of the transgene itself. Therefore, there is an urgent need for the development and improvement of techniques for the directed integration of transgenes at specific locations in the genome. The integration of transgenes by homologous recombination would allow specific modification or disruption of endogenous genes, providing a tool for more detailed analysis of gene function. In combination with the recent introduction of site-specific recombination systems from E. coli or yeast into plants, this may lead to the development of versatile systems for modification of the plant genome.     

基因打靶及其应用

用活细胞染色体DNA可与外源性DNA同源序列发生同源重组的性质,达到定点修饰改造染色体某基因的目的,此法称基因打靶．基因的同源重组是较普遍的生物现象,其分子机理尚未阐明,但活细胞内确有一酶系可使DNA的同源序列在细胞内发生重组,这一事实已无可争辨．此事实为基因打靶的理论基础．基因打靶技术操作的关键是建立一含筛选基因的重组载体,并有效地把它转入细胞核内．基因打靶命中的细胞可稳定遗传．基因打靶在改造生物品种,一些复杂生命现象（如发育的分子机制等）及临床理论研究均有广阔的前景．     

基因打靶技术的研究进展

基因打靶技术是一项新兴的分子生物学技术,是利用外源DNA与受体细胞染色体DNA上的同源序列之间发生重组,并整合在预定位点上,从而改变细胞遗传特性的方法。它的产生是遗传工程领域的一次革命,为发育生物学、分子遗传学、免疫学及医学等学科提供了一个全新的、强有力的研究手段。目前基因打靶技术在研究基因的结构和功能、表达与调控,转基因及基因治疗等方面均取得了进展。但基因打靶技术仍存在一些问题,主要是打靶的效率太低。本文综述了基因打靶技术的原理、操作程序并对提高基因打靶效率的可能途径进行了探讨。 Progress on Gene Targeting LIU Hong-quan1,DAI Ji-xun1,YU Wen-gong2,YANG Kun-feng1 1.Ocean University of Qingdao,College of Marine Life Sciences,Qingdao 266003,China; 2.Institute of Marine Drugs and Foods,Qingdao 266003,China Abstract:Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell.This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine.Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting.But there are some problems in gene targeting,especially for the low efficiency.This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting. Key words：gene targeting;homologous recombination;targeting vector;targeting efficiency