植物未知产物发育基因的分离方法

近年来,由于分子生物学和遗传学向发育生物学和生理学渗透,生长发育的遗传控制成为研究的热点,到目前为止,人们主要借助蛋白质的分离纯化分离了一些发育基因,但绝大部分发育基因的产物是未知的,其分离工作相当困难,近年来发展起来的基因克隆技术使未知产物的发育基因克隆取得了突破性的进展,本文对这些技术进行了简单的介绍和评述。     

植物基因分离的图位克隆技术

DNA是遗传的物质基础。遗传的基本单元是以基因的形式包含在 DNA序列内。要想从分子基础上来理解遗传的本质 ,基因的分离是最基本的前提。现在分离和克隆植物基因的方法很多 ,如传统的功能克隆及近年来发展十分迅速的表型克隆。但大多数情况下 ,我们并不知道基因的表达产物 ,在未知基因的功能信息又无适宜的相对表型用于表型克隆时 ,最常用的基因克隆技术有转座子示踪法、随机实变体筛选法和图位克隆法。其中 ,转座子示踪法中的转座子受其种类、活性和数量的制约 ,随机突变体筛选法随机性较大且不能控制其失活基因的种类和数量 ,也限制了…     

扩增未知序列DNA片段的PCR技术研究进展

1985年,Mullis等人发明了PCR技术,短短十余年间,这一技术得到迅速发展和应用,已由扩增已知基因发展到扩增未知基因。本文旨在介绍利用PCR技术扩增未知序列DNA片段的最新进展及这些技术在基因克隆研究中的应用。     

扩增未知序列DNA片段的PCR技术研究进展

1985年,Mullis等人发明了PCR技术,短短十余年间,这一技术得到迅速发展和应用,已由扩增已知基因发展到扩增未知基因,本文旨在介绍利用PCR技术扩增未知序列DNA片段的最新进展及这些技术在基因克隆研究中的应用。     

植物发育基因克隆技术的新进展

过去２０年,人们已经分离到了相当数量的与发育相关的基因,这主要借助于蛋白质产物的分离纯化,然后根据其氨基酸结构推算要应的核苷酸序列,并据此合成寡核苷酸探针,最终从ＣＤＮＡ文库或基因组文库中筛选出目的基因,而未知其编码产物的发育基因的分离克则是非常困难的工作,以前物主要方法有ＤＮＡ标签法,作图克隆法,差别筛选法和扣除发交法这些方法虽然都取得一定的成功,但由于各自的缺陷性,而限制了它们更广泛的应用,     

林木基因克隆研究进展

林木种质资源丰富,种质间遗传差异大,控制林木重要性状的基因克隆及转化对培育优良林木新品种具有很强的实用价值,但许多具有潜在应用价值的林木基因未得到充分发掘和有效分离。近年来,随着各种不同林木cDNA文库的建立,大规模随机EST测序技术的运用以及克隆技术的不断完善,特别是毛果杨（Populustrichocarpa）基因组测序计划的完成,大量与林木重要性状相关的基因被分离和鉴定。这些重要基因的获得为利用转基因技术培育高产、优质、抗逆、抗病虫害的林木新品种奠定了一定的基础。该文综述了20多年来国内外林木基因克隆的研究进展,对基因克隆及其应用过程中亟待解决的问题进行了讨论,并对其发展趋势进行展望。     

林木基因克隆研究进展

林木种质资源丰富, 种质间遗传差异大, 控制林木重要性状的基因克隆及转化对培育优良林木新品种具有很强的实用价值, 但许多具有潜在应用价值的林木基因未得到充分发掘和有效分离。近年来, 随着各种不同林木cDNA文库的建立, 大规模随机EST测序技术的运用以及克隆技术的不断完善, 特别是毛果杨(Populus trichocarpa)基因组测序计划的完成, 大量与林木重要性状相关的基因被分离和鉴定。这些重要基因的获得为利用转基因技术培育高产、优质、抗逆、抗病虫害的林木新品种奠定了一定的基础。该文综述了20多年来国内外林木基因克隆的研究进展, 对基因克隆及其应用过程中亟待解决的问题进行了讨论, 并对其发展趋势进行展望。     

T-DNA介导的基因诱捕技术及其在植物功能基因组学研究中的应用

T-DNA介导的基因诱捕技术是近年来发展起来的鉴定和分离基因的方法。在拟南芥和水稻基因组测序已经完成的今天, 该技术将在两者基因功能的研究中扮演举足轻重的角色。本文就T-DNA介导的基因诱捕系统、 基因克隆和突变体库构建的研究进展及其在植物功能基因组学上的应用等内容进行了综述, 并讨论了该技术应用中的一些问题。     

植物发育基因克隆技术的新进展

过去２０年,人们已经分离到了相当数量的与发育相关的基因。这主要借助于蛋白质产物的分离纯化,然后根据其氨基酸结构推算其相应的核苷酸序列,并据此合成寡核苷酸探针,最终从ｃＤＮＡ文库或基因组文库中筛选出目的基因。而未知其编码产物的发育基因的分离克隆则是非常困难的工作,以前广泛应用的主要方法有ＤＮＡ标签法（ＤＮＡｔａｇｇｉｎｇ）［１,２,３］,作图克隆法（ｍａｐ－ｂａｓｅｄｃｌｏｎｉｎｇ）［４］,差别筛选法（ｄｉｆｅｒｅｎｔｉａｌｓｃｒｅｅｎｉｎｇ）［５］和扣除杂交法（ｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ）［６,７,８］。这些方法虽然都取得了一定的成功,但由于各自的缺陷性,而限制了它们更加广泛的应用。近年来在ＰＣＲ技术的基础上,人们已经建立了若干种分离克隆植物发育基因的新方法。１９９２年,ＬｉａｎｇＰ和ＰａｒｄｅｅＡ［９］首次提出并运用了ｍＲＮＡ差别显示技术（ｍＲＮＡｄｉｆ－ｆｅｒｅｎｔｉａｌｄｉｓｐｌａｙｒｅｖｅｒｓｅｔｒａｎｓｃｒｉｐｔｉｏｎ－ＰＣＲ,ＤＤＲＴ－ＰＣＲ）来进行基因的分离。实践表明,ｍＲＮＡ差别显示技术在分离、鉴定差别表达的新基因方面与以前的各种技术相比有其独特的优越性,但同时它也存在着重复性较差等缺点。因此,最近研究工作者又在其基础上,发展了诸如代表性差式分析（ｒｅｐｒｅｓｅｎｔａｔｉｏｎａｌｄｉｆｅｒｅｎｃｅａｎａｌｙｓｉｓ,ＲＤＡ）［１０,１１］和抑制性扣除（或减去）杂交（ｓｕｐｐｒｅｓｓｉｏｎｓｕｂｔｒａｃｔｉｖｅｈｙｂｒｉｄｉｚａｔｉｏｎ,ＳＳＨ）［１２］等一些更新的方法。下面就此作一简要概述。     

T—DNA介导的基因诱捕技术及其在植物功能基因组学研究中的应用

T-DNA介导的基因诱捕技术是近年来发展起来的鉴定和分离基因的方法。在拟南芥和水稻基因组测序已经完成的今天,该技术将在两者基因功能的研究中扮演举足轻重的角色。本文就T—DNA介导的基因诱捕系统、基因克隆和突变体库构建的研究进展及其在植物功能基因组学上的应用等内容进行了综述,并讨论了该技术应用中的一些问题。     

The role of homologous recombination processes in the repair of severe forms of DNA damage in mammalian cells

The role of homologous recombination processes in the repair of severe forms of DNA damage is reviewed, with particular attention to the functions of members of the recA/RAD51 family of genes. In the yeast Saccharomyces cerevisiae, several of the gene products involved in homologous recombination repair (HRR) have been studied in detail, and a picture is beginning to emerge of the repair mechanism for DNA double-strand breaks. Knowledge is fragmentary for other eukaryotic organisms and for other types of DNA damage. In mammalian cells, while it has been known for some years that HRR occurs, the relative importance of the process in repairing DNA damage is unknown and very few of the gene products involved have been identified. Very recently, a number of RAD51-like genes have been identified in mammals, either through cloning genes complementing cell lines sensitive to DNA-damaging agents (XRCC2, XRCC3), or through homology searches (RAD51L1, RAD51L2, RAD51L3). As yet the role of these genes and their possible functions are speculative, although the combination of sequence conservation and gene expression patterns suggest that they function in HRR pathways.     

Exploiting the yeast Saccharomyces cerevisiae for the study of the organization and evolution of complex genomes

Yeast artificial chromosome (YAC) cloning systems have advanced the analysis of complex genomes considerably. They permit the cloning of larger fragments than do bacterial artificial chromosome systems, and the cloned material is more easily modified. We recently developed a novel YAC cloning system called transformation-associated recombination (TAR) cloning. Using in vivo recombination in yeast, TAR cloning selectively isolates, as circular YACs, desired chromosome segments or entire genes from complex genomes. The ability to do that without constructing a representative genomic library of random clones greatly facilitates analysis of gene function and its role in disease. In this review, we summarize how recombinational cloning techniques have advanced the study of complex genome organization, gene expression, and comparative genomics.     

The "ups" and "downs" in Using Subtractive Cloning Techniques to Isolate Regulated Genes in Fish

Over the last decade, subtractive cloning approaches have beenused extensively to isolate genes that are up- or down-regulatedunder various conditions. These techniques have provided thefoundation for many subsequent studies concerning gene functionand regulation and, as such, have been valuable tools for manybiological fields. Over the past 10 years, we have used differentsubtractive cloning approaches to isolate genes in fish thatare regulated in relation to hormonal stimulation or the stageof ovarian maturation. These include conventional cDNA subtractionfollowed by library screening, differential display PCR, suppressionsubtraction hybridization, and more recently, iterative PCRsubtraction. We continue to use these techniques for the isolationof new genes involved in physiological processes in fish andbivalve molluscs. Examples that illustrate the use of thesedifferent subtractive cloning techniques are described, includingwhere possible the advantages and disadvantages of each. Inaddition, the use of ancillary methods (e.g., "Reverse Northerns")to facilitate the use of these subtractive approaches are discussed.     

Direct amplification of unknown genes and fragments by Uneven polymerase chain reaction

Gene cloning is a time-consuming task for molecular biologists, because it often takes weeks or months to construct, screen and finally clone a gene from a DNA library. Thus, more effective methods are needed for gene cloning. This paper describes a modified polymerase chain reaction (PCR) cycling condition, Uneven PCR, to generate specific unknown fragments or genes directly from total DNA instead of cloning fragments from DNA libraries. The essence of the method is to use two different annealing temperatures in consecutive PCR cycles to effectively amplify the target products while inhibiting the synthesis of non-specific products. Under favorable conditions, a desired DNA fragment or gene in the size range up to approximately 4 kb can be obtained and ready for cloning within a day or two.     

基因捕获技术及其最新进展

基因捕获技术是目前最具应用前景的基因克隆方法之一。利用该技术建立的随机插入突变的突变体文库,可用于寻找、鉴定和研究大量末知功能和已知功能的活化基因,它是继自然突变、物理突变和化学突变之后发展起来的新的分子生物学方法。基因捕获载体是带有报告基因和/或选择标记基因的不完整的基因表达载体;这些载体所带的基因只有在整合到宿主功能基因内部且与融合的宿主基因编码框一致时才能得以表达。经典的基因捕获载体有：增强子捕获载体、基因捕获载体和启动子捕获载体。增强子载体只有一个最小化的启动子控制下游报告基因的表达活性。只有当启动子上游存在一个增强子时才能启动其下游基因的转录：而单独依靠这个启动了则不能转录。狭义的基因捕获载体是指插入到结构基因内部从而捕获该基因的载体,分为内含子捕获载体和外显了捕获载体。前者插入内含子,因此需要在无启动子的报告基因前面添加一个splice acceptor（SA）位点：后者插入外显子,因此不需SA位点就能产生融合蛋白mRNA。启动子捕获载体由一个无启动子的报告基因和选择标记基因组成,只有在捕状载体捕入到内源基因的外显子中,且两阅读框一致的时候才会有报告基因和被捕获的内源基因上游编码区的融合蛋白的表达。利用某些遗传元件的遗传特性也可以构建非常有用的捕获载体。常用的有：逆转录病毒介导的捕抉载体和转座子介导的捕荻载体等等。该文较为全面地概括了转座子介导的捕获载体的用途和研究现状。比如：在拟南芥中应用Ac／Ds转座子元件,存果蝇中应用P-element和piggyBac转座元件,在斑马鱼中应用T012转座了元件,在脊椎动物研究中应用Tc1/meriner转座了超家族,以及存哺乳动物和小鼠ES细胞中应用piggyBac转座子元件。还列举了设计新颖的载体优化策略,比如：发展新的选择标记基因,利用内源核糖体识别／结合位点（IRES）,去掉起始密码子和加一小段接头（1inker）等方法。最后介绍了几种针特定实验目的而设计的捕获载体。附录部分还列出了关于基因捕获技术的网络资源。     

Cloning and mutational analysis of SRY.

A candidate for the male sex-determining gene has recently been isolated. This sex-determining gene (SRY) has been found to be mutated in some individuals with failed testis development, and, in mouse transgenesis, the SRY murine homologue (Sry) causes female-to-male sex reversal. The cloning of SRY should facilitate the characterisation of other genes in the testis-determining pathway and provide information on the mechanism of mammalian developmental decisions.     

Rapid one-step recombinational cloning

As an increasing number of genes and open reading frames of unknown function are discovered, expression of the encoded proteins is critical toward establishing function. Accordingly, there is an increased need for highly efficient, high-fidelity methods for directional cloning. Among the available methods, site-specific recombination-based cloning techniques, which eliminate the use of restriction endonucleases and ligase, have been widely used for high-throughput (HTP) procedures. We have developed a recombination cloning method, which uses truncated recombination sites to clone PCR products directly into destination/expression vectors, thereby bypassing the requirement for first producing an entry clone. Cloning efficiencies in excess of 80% are obtained providing a highly efficient method for directional HTP cloning.     

pKILLIN: a versatile positive-selection cloning vector based on the toxicity of Killin in Escherichia coli

The invention of DNA cloning over 40 years ago marked the advent of molecular biology. The technique has now become a routine practice in any modern biomedical laboratory. Although positive-selection of recombinants in DNA cloning seems to be superior to blue/white selection based on the disruption of the lacZ gene, it is rarely practiced due to its high background, lack of multiple cloning sites, and inability to express the genes of interest or purify the protein products. Here we report the creation of a new positive-selection cloning vector dubbed pKILLIN, which overcomes all of the above pitfalls. The essence behind its high cloning efficiency is the extreme toxicity and small size of the toxic domain of killin, a recently discovered p53 target gene. Insertion inactivation of killin within the multiple cloning site via either blunt- or sticky-end ligation not only serves as a highly efficient cloning trap, but also may allow any cloned genes to be expressed as His-tagged fusion proteins for subsequent purification. Thus, pKILLIN is a versatile positive-selection vector ideal for cloning PCR products, making DNA libraries, as well as routine cloning and bacterial expression of genes.     

Genetics and Molecular Biology of Mouse Pigmentation

The formation of mouse coat color is a relatively complex developmental process that is affected by a large number of mutations, both naturally occurring and induced. The cloning of the genes in which these mutations occur and the elucidation of the mechanisms by which these mutations disrupt the normal pigmentation pattern is leading to an understanding of the way interactions between gene products lead to a final phenotype.