转座子标签法克隆分离植物基因的研究进展

转座子标签法是克隆与分离植物基因的一项十分有效的方法。概述了转座子标签技术克隆与分离植物基因的基本原理与方法 ,介绍了可用于转座子标签技术的转座子 ,对于转座子标签系统以及在克隆与分离异源植物基因方面的主要成就进行了综述 ,并对将来的研究方向进行了讨论。     

A system of transposon mutagenesis for bacteriophage T4

We have developed a system of transposon mutagenesis for bacteriophage T4. The transposon is a plasmid derivative of Tn5 which contains the essential T4 gene 24, permitting a direct selection for transposition events into a gene 24-deleted phage. The transposition occurred at a frequency of only 10(-7) per progeny phage, even though a dam- host was used to increase transposition frequency. Phage strains with a transposon insert were distinguished from most pseudorevertants of the gene 24 deletion by plaque hybridization using a transposon-specific probe. Mapping analysis showed that the transposon inserts into a large number of sites in the T4 genome, probably with a preference for certain regions. The transposon insertions in four strains were analysed by DNA sequencing using primers that hybridize to each end of the transposon and read out into the T4 genome. In each case, a 9 bp T4 target sequence had been duplicated and the insertions had occurred exactly at the IS50 ends of the transposon, demonstrating that bona fide transposition had occurred. Finally, the transposon insert strains were screened on the TabG Escherichia coli strain, which inhibits the growth of T4 motA mutants, and a motA transposon insert strain was found.     

The heat-stable toxin I gene from Escherichia coli 18D.

The heat-stable toxin I gene in the human Escherichia coli isolate 18D is the estA1 allele. The gene is not part of a composite transposon, but inspection of the flanking DNA sequences suggests that it was at one time part of a transposon. The hypothetical transposon originated from an event other than the occurrence that formed Tn1681.     

The phosphotriesterase gene opdA in Agrobacterium radiobacter P230 is transposable

We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10beta cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria.     

Counterselection and Co-Delivery of Transposon and Transposase Functions for <Emphasis Type="Italic">Sleeping Beauty</Emphasis>-Mediated Transposition in Cultured Mammalian Cells

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.     

Development of a mariner-Based Transposon for Use in Sorangium cellulosum

In order to generate marked insertions in the myxobacterium Sorangium cellulosum, a transposon based on the eukaryotic mariner transposon was developed. The transposition frequency was increased with the use of a mutated tnp gene. The transposon randomly inserts into the chromosome, as demonstrated by targeted mutagenesis of the epoK gene.     

Use of Transposons in Cloning Poorly Selectable Genes of Escherichia coli: Cloning of uvrA and Adjacent Genes

A transposon was introduced close to a poorly selectable gene. This gene could be cloned by using selection for the antibiotic resistance marker of the transposon.     

Transposon tagging of the sulfur gene of tobacco using engineered maize Ac/Ds elements

The Sulfur gene of tobacco is nuclearly encoded. A Su allele at this locus acts as a dominant semilethal mutation and causes reduced accumulation of chlorophyll, resulting in a yellow color in the plant. An engineered transposon tagging system, based upon the maize element Ac/Ds, was used to mutate the gene. High frequency of transposon excision from the Su locus produced variegated sectors. Plants regenerated from the variegated sector exhibited a similar variegated phenotype. Genetic analyses showed that the variegation was always associated with the transposase construct and the transposon was linked to the Su locus. Sequences surrounding the transposon were isolated, and five revertant sectors possessed typical direct repeats following Ds excisions. These genetic and molecular data are consistent with the tagging of the Su allele by the transposon.     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

"睡美人"转座子的研究进展

“睡美人( Sleeping Beauty, SB) ”转座系统是Tc1/mariner 转座子超家族中的一员,已经失活了一千多万年。1997年,Ivics 等根据积累的系统发生数据,利用生物信息学的方法, 对其进行分子重建, 终于唤醒了其转座活性。近年来对“睡美人”转座系统的转座效率和转座机理进行的研究,已证明SB转座子在基因筛选,转基因及基因治疗等领域具有广阔的应用前景。文章重点论述了SB转座子在结构及其优化、转座机制和应用等方面的进展,同时对其研究中出现的各种问题进行了总结并提出了一些解决方案。     

Sleeping Beauty transposition: from biology to applications

Sleeping Beauty (SB) is the first synthetic DNA transposon that was shown to be active in a wide variety of species. Here, we review studies from the last two decades addressing both basic biology and applications of this transposon. We discuss how host–transposon interaction modulates transposition at different steps of the transposition reaction. We also discuss how the transposon was translated for gene delivery and gene discovery purposes. We critically review the system in clinical, pre-clinical and non-clinical settings as a non-viral gene delivery tool in comparison with viral technologies. We also discuss emerging SB-based hybrid vectors aimed at combining the attractive safety features of the transposon with effective viral delivery. The success of the SB-based technology can be fundamentally attributed to being able to insert fairly randomly into genomic regions that allow stable long-term expression of the delivered transgene cassette. SB has emerged as an efficient and economical toolkit for safe and efficient gene delivery for medical applications.     

Enhancement of Sleeping Beauty transposition by CpG methylation: possible role of heterochromatin formation

The Sleeping Beauty (SB) transposase is the most active transposase in vertebrate cells, and the SB transposon system has been used as a tool for insertional mutagenesis and gene delivery. Previous studies have indicated that the frequency of chromosomal transposition is considerably higher in mouse germ cells than in mouse embryonic stem cells, suggesting the existence of unknown mechanisms that regulate SB transposition. Here, we demonstrated that CpG methylation of the transposon region enhances SB transposition. The transposition efficiencies of a methylated transposon and an unmethylated transposon which had been targeted in the same genomic loci by recombination-mediated cassette exchange in mouse erythroleukemia cells were compared, and at least a 100-fold increase was observed in the methylated transposon. CpG methylation also enhanced transposition from plasmids into the genome. Chromatin immunoprecipitation assays revealed that histone H3 methylated at lysine-9, a hallmark of condensed heterochromatin, was enriched at the methylated transposon, whereas the unmethylated transposon formed a relaxed euchromatin structure, as evidenced by enrichment of acetylated histone H3 and reporter gene expression. Possible roles of heterochromatin formation in the transposition reaction are discussed. Our findings indicate a novel relationship between CpG methylation and transposon mobilization.     

Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome

Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.     

转座子挽救法对转座子突变菌株中插入位点的定位分析

为寻找谷氨酸棒杆菌转座子插入突变菌株中的转座子插入位点,采用了转座子挽救法对转座子及其插入位点附近的序列进行分离,并测定插入位点相邻DNA序列,获得了三个转座子插入位点DNA序列,其中一个是柠檬酸合成酶基因,另两个为目前未知基因,暂命名为orfA和orfB。该方法简便易行,是分析转座子插入位点的理想方法。     

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.     

PiggyBac transposon-mediated gene transfer in Cashmere goat fetal fibroblast cells

PiggyBac (PB) has recently been found to be functional in various organisms. To verify and exploit its application in the cashmere goat, a PB transposon system including donor and helper vector of was developed, in which the EGFP gene in donor of vector was used as reporter. Cashmere goat fetal fibroblasts cells (GFFs) were transfected with the PB transposon system and the efficiency of gene transfer was determined. Compared with random integration, PB-mediated EGFP expression levels increased 7.78-fold in the GFFs, confirming that the PB transposon system constructed successfully mediated efficient foreign gene integration in the GFFs. To further investigate the characteristics of PB-mediated integration instance, PB integration site distribution in the goat genome was examined. The results showed that PB had a preference for AT rich regions of the goat genome. Thus this study confirms the function of PB transposon in GFFs and provides a potential genetic tool for producing transgenic goats.