Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

Plant transposable elements generate the DNA sequence diversity needed in evolution

Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild-type phenotype. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Enhanced frequency of transposition of the maize transposable elementActivator following excision from T-DNA inPetunia hybrida

Many of the systems currently employed for heterologous transposon tagging in plants rely on an excision assay to monitor transposon activity. We have used the streptomycin phosphotransferase (SPT) reporter system to assayAc activity inPetunia hybrida. In other species, such as tobacco orArabidopsis, excision ofAc from the SPT gene in sporogenous tissue gives rise to streptomycin-resistant seedlings in the following generation. The frequency of fully streptomycin-resistant seedlings in petunia was low (0.4%) but molecular analysis of these indicated that the actual excision frequency may be as low as 0.05%. This indicates that the SPT assay is not a reliable selection criterion for germinal excision in petunia. Extensive molecular screening for reinsertion ofAc was consistent with a low primary transposition frequency (0%–0.6%). In contrast to these findings, the progeny of confirmed germinal transpositions for three independent transformants showed frequent transposition to new sites (9.5%–17.0%). This suggests a high frequency of secondary transposition compared with primary transposition from the T-DNA. Segregation analysis indicates that the high transposition activity is closely associated with transposed copies ofAc. No evidence was found for an altered methylation state forAc following transposition. The implications of these results for heterologous transposon tagging in petunia are discussed in the context of the reliability of excision reporter systems in general.     

Assessment of utility of meiosis-associated promoters of lily for induction of germinal ds transposition in transgenic rice

In order to suppress the somatic excision of the Ds element and increase the independent transposition events of the Ac/Ds transposon tagging system in rice, we employed promoters of two meiosis-specific genes of lily, LIM10 and LIM18. The LIM10 promoter directed GUS expression specifically in anthers, with the LIM18 promoter doing the same in the anthers and somatic tissue. Both promoters induced independent germinal transposition with the frequency of approximately 1%. The LIM10 promoter, lacking induction of somatic transposition, is considered to be useful for improving transposon-tagging efficiencies in rice.     

Developmental regulation of excision timing of Mutator transposons of maize: Comparison of standard lines and an early excision bzl::Mu1 line

Three characteristics of standard Mutator lines reflect developmental regulation: new mutants usually involve single gametes, somatic excision is restricted to terminal cell divisions during tissue development, and germinal excision is rare. By selection for earlier (larger) somatic sectors in the aleurone, a Mutator line was identified that exhibits a dramatic elevation in somatic excision frequency during the first three nuclear divisions of the endosperm and more than a 10-fold increase in germinal reversion from the bzl::Mul reporter gene. The programming of early sectoring is dominant in crosses with Mutator lines containing diverse reporter alleles. Germinal reversion is biased 5- to 10-fold for events through the pollen compared to the ear. The timing of germinal excision in the tassel is late because somatic excision sectors in the anthers are small; however, 98% of the germinal revertants are concordant. These observations indicate that in the early sectoring line Mu excision usually occurs before the mitotic divisions that separate gametic nuclei and may be restricted to the early stages of microsporogenesis. © 1992 Wiley-Liss, Inc.     

Precise marker excision system using an animal‐derived piggyBac transposon in plants

Accurate and effective positive marker excision is indispensable for the introduction of desired mutations into the plant genome via gene targeting (GT) using a positive/negative counter selection system. In mammals, the moth‐derived piggyBac transposon system has been exploited successfully to eliminate a selectable marker from a GT locus without leaving a footprint. Here, we present evidence that the piggyBac transposon also functions in plant cells. To demonstrate the use of the piggyBac transposon for effective marker excision in plants, we designed a transposition assay system that allows the piggyBac transposition to be visualized as emerald luciferase (Eluc) luminescence in rice cells. The Eluc signal derived from piggyBac excision was observed in hyperactive piggyBac transposase‐expressing rice calli. Polymerase chain reaction, Southern blot analyses and sequencing revealed the efficient and precise transposition of piggyBac in these calli. Furthermore, we have demonstrated the excision of a selection marker from a reporter locus in T₀ plants without concomitant re‐integration of the transposon and at a high frequency (44.0% of excision events), even in the absence of negative selection.     

Increased Ac excision (iae): Arabidopsis thaliana mutations affecting Ac transposition

The maize transposable element Ac is highly active in the heterologous hosts tobacco and tomato, but shows very much reduced levels of activity in Arabidopsis . A mutagenesis experiment was undertaken with the aim of identifying Arabidopsis host factors responsible for the observed low levels of Ac activity. Seed from a line carrying a single copy of the Ac element inserted into the streptomycin phosphotransferase (SPT) reporter fusion, and which displayed typically low levels of Ac activity, were mutagenized using gamma rays. Nineteen mutants displaying high levels of somatic Ac activity, as judged by their highly variegated phenotypes, were isolated after screening the M₂ generation on streptomycin-containing medium. The mutations fall into two complementation groups, iae1 and iae2 , are unlinked to the SPT::Ac locus and segregate in a Mendelian fashion. The iae1 mutation is recessive and the iae2 mutation is semi-dominant. The iae1 and iae2 mutants show 550- and 70-fold increases, respectively, in the average number of Ac excision sectors per cotyledon. The IAE1 locus maps to chromosome 2, whereas the SPT:: Ac reporter maps to chromosome 3. A molecular study of Ac activity in the iae1 mutant confirmed the very high levels of Ac excision predicted using the phenotypic assay, but revealed only low levels of Ac re-insertion. Analyses of germinal transposition in the iae1 mutant demonstrated an average germinal excision frequency of 3% and a frequency of independent Ac re-insertions following germinal excision of 22%. The iae mutants represent a possible means of improving the efficiency of Ac/Ds transposon tagging systems in Arabidopsis , and will enable the dissection of host involvement in Ac transposition and the mechanisms employed for controlling transposable element activity.     

Cytological visualization of DNA transposons and their transposition pattern in somatic cells of maize

Global genomic analysis of transposable element distributions of both natural (En/Spm, Ac-Ds, and MuDR/Mu) and modified (RescueMu) types was performed by fluorescence in situ hybridization (FISH) on somatic chromosomes coupled with karyotyping of each chromosome. In lines without an active transposable element, the locations of silent En/Spm, Ac-Ds, and MuDR/Mu elements were visualized, revealing variation in copy number and position among lines but no apparent locational bias. The ability to detect single elements was validated by using previously mapped active Ac elements. Somatic transpositions were documented in plants containing an engineered Mutator element, RescueMu, via use of the karyotyping system. By analyzing the RescueMu lines, we found that transposition of RescueMu in root-tip cells follows the cut-and-paste type of transposition. This work demonstrates the utility of FISH and karyotyping in the study of transposon activity and its consequences.     

Tn7 transposes proximal to DNA double-strand breaks and into regions where chromosomal DNA replication terminates

We report that the bacterial transposon Tn7 can preferentially transpose into regions where chromosomal DNA replication terminates. DNA double-strand breaks are associated with the termination of chromosomal replication; therefore, we directly tested the effect of DNA breaks on Tn7 transposition. When DNA double-strand breaks are induced at specific sites in the chromosome, Tn7 transposition is stimulated and insertions are directed proximal to the induced break. The targeting preference for the terminus of replication and DNA double-strand breaks is dependent on the Tn7-encoded protein TnsE. The results presented in this study could also explain the previous observation that Tn7 is attracted to events associated with conjugal DNA replication during plasmid DNA transfer.     

A Modified Autonomous En Transposon in Maize (Zea Mays L.) Elicits a Differential Response of Reporter Alleles

Transposable elements in maize are composed of a defined molecular structure that includes coding sequences, determiners of functionality and ordered terminal motifs that provide binding sites for transposase proteins. Alterations in these components change the phenotypic expression of unstable genes with transposon inserts. The molecular basis for the altered timing and frequency of transposition as determined by the size and number of spots on kernels or stripes on leaves has generally been described for defective inserts in genes. Most differential patterns can be ascribed to alterations in the terminal motifs of the reporter allele structure that supplies a substrate (terminal inverted repeat motifs) for transposase activity. For autonomously functioning alleles, the explanations for changes in phenotype are not so clear. In this report, an En-related element identified as F-En is described that shares with En the recognition of a specific defective element c1(mr)888104 but differs from En in that this F-En element does not recognize the canonical c1(mr) elements that are recognized by En. Evidence is provided suggesting that F-En does not recognize other En/Spm-related defective elements, some of whose sequences are known. This modified En arose from a c1-m autonomously mutating En allele.     

Transfer of plastid DNA to the nucleus is elevated during male gametogenesis in tobacco

In eukaryotes, many genes were transferred to the nucleus from prokaryotic ancestors of the cytoplasmic organelles during endosymbiotic evolution. In plants, the transfer of genetic material from the plastid (chloroplast) and mitochondrion to the nucleus is a continuing process. The cellular location of a kanamycin resistance gene tailored for nuclear expression (35SneoSTLS2) was monitored in the progeny of reciprocal crosses of tobacco (Nicotiana tabacum) in which, at the start of the experiments, the reporter gene was confined either to the male or the female parental plastid genome. Among 146,000 progeny from crosses where the transplastomic parent was male, 13 transposition events were identified, whereas only one atypical transposition was identified in a screen of 273,000 transplastomic ovules. In a second experiment, a transplastomic beta-glucuronidase reporter gene, tailored to be expressed only in the nucleus, showed frequent stochastic expression that was confined to the cytoplasm in the somatic cells of several plant tissues. This gene was stably transferred in two out of 98,000 seedlings derived from a male transplastomic line crossed with a female wild type. These data demonstrate relocation of plastid DNA to the nucleus in both somatic and gametophytic tissue and reveal a large elevation of the frequency of transposition in the male germline. The results suggest a new explanation for the occurrence of uniparental inheritance in eukaryotes.     

Gene conversion and end-joining-repair double-strand breaks in the Caenorhabditis elegans germline

Excision of a Mos1 transposon in the germline of Caenorhabditis elegans generates a double-strand break in the chromosome. We demonstrate that breaks are most prominently repaired by gene conversion from the homolog, but also rarely by nonhomologous end-joining. In some cases, gene conversion events are resolved by crossing over. Surprisingly, expression of the transposase using an intestine-specific promoter can induce repair, raising the possibility that activation of transposase expression in somatic cells can lead to transposition of Mos1 in the germline.     

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.     

piggyBac转座系统的功能改进及在哺乳动物中的应用

piggyBac (PB)转座系统具有转座效率高、删除精确、半随机插入和携带片段较大等优点。但是作为一种转基因实验的工具,特别是在哺乳动物个体水平的转基因方面,还需要提高其转基因效率,并降低外源基因随机插入对内源基因破坏的风险。近年来的研究结果显示,PB转座系统得到了进一步改进：采用PB转座酶与DNA特异性结合蛋白融合而构成的融合型转座酶,表现出外源片段有插入到染色体靶向位点的倾向;采用突变体筛选的方法提高了PB转座酶的活性,获得了只具有切除活性而没有插入活性的新型PB转座酶;采用PB转座系统与细菌人工染色体(Bacterial artificial chromosomes, BAC)载体联合使携带的外源片段长度提高到了207 kb。改进后的PB转座系统在基因组研究、基因治疗、诱导多能干细胞(Induced pluripotent stem cells, iPSCs)诱导及其分化方面发挥了较大的作用。文章对PB转座系统的最新研究进展和应用前景进行了综述。