Regulation of activator/dissociation transposition by replication and DNA methylation

In maize the transposable elements Activator/Dissociation (Ac/Ds) transpose shortly after replication from one of the two resulting chromatids ("chromatid selectivity"). A model has been suggested that explains this phenomenon as a consequence of different affinity for Ac transposase binding to holo-, hemi-, and unmethylated transposon ends. Here we demonstrate that in petunia cells a holomethylated Ds is unable to excise from a nonreplicating vector and that replication restores excision. A Ds element hemi-methylated on one DNA strand transposes in the absence of replication, whereas hemi-methylation of the complementary strand causes a >6.3-fold inhibition of Ds excision. Consistently in the active hemi-methylated state, the Ds ends have a high binding affinity for the transposase, whereas binding to inactive ends is strongly reduced. These results provide strong evidence for the above-mentioned model. Moreover, in the absence of DNA methylation, replication enhances Ds transposition in petunia protoplasts >8-fold and promotes formation of a predominant excision footprint. Accordingly, replication also has a methylation-independent regulatory effect on transposition.     

Gametophyte-specific transposition of the maize Ds element in transgenic tobacco

To assess the potential advantages of a transposon-tagging system based on gametophyte-specific transposition a fusion between the anther-specific Arabidopsis thaliana apg promoter and the maize Ac transposase gene was constructed and introduced into tobacco. The ability of this transposase source to activate Ds transposition in a developmentally controlled manner was monitored by crossing to plants harbouring the cell autonomous excision marker gene construct, Ds —SPT. A number of fully green, streptomycin-resistant seedlings resulting from germinal transposition events were observed in the progeny of apg -TPase x Ds —SPT F1 plants. Streptomycin-resistant sectors were not observed in either F₁ seedlings or F₂ progeny, indicating a complete lack of somatic excision. Further crosses of apg —TPase sources to plants containing Ds—bar herbicide selection excision marker constructs gave reproducible gametophytic excision frequencies of up to 0.3%. Sequencing of Ds excision sites from F₂ seedlings derived from single F₁ plants revealed various sequence alterations in the original Ds insertion 'footprint' indicative of independent Ds excision events. Independent re-insertion was confirmed by Southern analysis of F₂ siblings. It is concluded that apg -controlled Ac transposase expression activates male gametophyte-specific Ds transposition.     

Excision of a transposable element from a viral vector introduced into maize plants by agroinfection

The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants. MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection. Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone. The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus. The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize. From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.     

Origination of Ds elements from Ac elements in maize: evidence for rare repair synthesis at the site of Ac excision.

Although it has been known for some time that the maize transposon Ac can mutate to Ds by undergoing internal deletions, the mechanism by which these mutations arise has remained conjectural. To gain further insight into this mechanism in maize we have studied a series of Ds elements that originated de novo from Ac elements at known locations in the genome. We present evidence that new, internally deleted Ds elements can arise at the Ac donor site when Ac transposes to another site in the genome. However, internal deletions are rare relative to Ac excision footprints, the predominant products of Ac transposition. We have characterized the deletion junctions in five new Ds elements. Short direct repeats of variable length occur adjacent to the deletion junction in three of the five Ds derivatives. In the remaining two, extra sequences or filler DNA is inserted at the junction. The filler DNAs are identical to sequences found close to the junction in the Ac DNA, where they are flanked by the same sequences that flank the filler DNA in the deletion. These findings are explained most simply by a mechanism involving error-prone DNA replication as an occasional alternative to end-joining in the repair of Ac-generated double-strand breaks.     

Comparative analysis of non-random DNA repair following Ac transposon excision in maize and Arabidopsis

Ac/Ds transposable elements often leave short DNA rearrangements, or 'footprints,' at the sites where they excise. Previous studies at the maize waxy ( wx ) gene suggest that the DNA repair that forms transposon footprints is not random. Each excision site consistently displays a different, predominant repair product suggesting flanking DNA may influence footprint formation. We have expanded these studies to show that predominant end-joining products also form in association with Ac/Ds excision in Arabidopsis and that chromosomal location of the Ac -containing construct does not appear to influence this repair. The predominant repair product is identical in both maize and Arabidopsis for Ac elements with the same adjacent DNA sequences. However, a broader range of minor footprint types is observed in Arabidopsis , including footprints that are rare in maize, suggesting potential differences in the host proteins involved in either transposition, repair or both. The data also suggest that the sequences influencing footprint formation are within 39 bp 5' and 18 bp 3' of the transposon. These studies demonstrate that transgenic Ac/Ds -containing plants will be useful tools in dissecting plant DNA repair processes.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

Excision of Ds1 from the genome of maize streak virus in response to different transposase-encoding genes

We have previously established a reverse genetic system for studying excision of the transposable element Ds1 in maize plants. Ds1 carried by the genome of maize streak virus (MSV) is introduced into maize plants by agroinfection. Excision of Ds1 from the MSV genome depends on the presence of an active Ac element in the recipient maize plants. With the purpose of exploiting MSV-Ds1 as vector for maize transformation, we studied different genes encoding the transposase (TPase) for their efficiency of activating Ds1 excision. These genes were inserted in the same T-DNA carrying MSV-Ds1 and introduced into maize plants by Agrobacterium-mediated transformation. We showed that the wild-type TPase transcribed by the 2 promoter produced much higher efficiency of Ds1 excision than that transcribed by the Ac promoter. In contrast to what had been observed in tobacco and petunia, the truncated TPase (103–807) lacking the amino-terminal 102 amino acids gave a much more reduced Ds1 excision efficiency than the wild-type TPase when both genes were transcribed by the 2 promoter.     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.     

The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

One strand of ars189 from the maxicircle of Crithidia fasciculata, transforms Saccharomyces cerevisiae more efficiently than its complementary strand as a single stranded DNA

An autonomously replicating element (ars 189) has been isolated from the maxicircle DNA of an insect trypanosomatid Crithidia fasciculata. This 189-bp fragment contains two copies of the yeast consensus ARS sequence of (A/T)TTTATPuTTT(T/A), has an A + T composition of 79.4%, and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. The complementary strands of ars 189 have been cloned into an M 13 vector containing the URA3 gene of Saccharomyces cerevisiae. When these circular single-stranded (ss) DNAs were used to transform yeast spheroplasts, the M 13 chimeric DNA carrying the strand of ars189 rich in adenine generated approximately four times more yeast Ura⁺ transformants than the construct containing the thymine-rich strand. In contrast, both strands of yeast ARS1 cloned into an M 13 vector transformed yeast at an equivalent level. The conversion of ARS -containing ss DNAs to duplex forms in vivo and their subsequent autonomous replication have been verified by Southern hybridization analysis of extracts from yeast transformants.     

Instability of the L6 gene for rust resistance in flax is correlated with the presence of a linked Ac element

In a programme aimed at tagging rust-resistance genes in flax with the maize transposable element Ac , a primary transformant of a line called 'Forge' that is homozygous for four rust-resistance genes, L ⁶, M, N and P ², was identified that possessed 10 copies of the Ac element, one of which was linked (29 map units) to L ⁶. Descendants of this plant, which had from 8 to 15 copies of Ac , were crossed to a rust-susceptible line and the progeny screened for rust-susceptible mutants. When the Ac linked to L ⁶ was present in the parent, a high frequency of L ⁶ mutants was observed (29 mutants in 30 575). By contrast, when this Ac was absent, no such mutants were observed in 9258 progeny. The background frequency of L ⁶ mutants was low (five in 124 088). A detailed analysis was made of the first 11 L ⁶ mutants recovered from parents carrying the L ⁶-linked Ac element. While none of the mutants possessed a tagged resistance gene, all lacked an RFLP marker closely linked to L ⁶, suggesting that deletions were responsible for loss of the L ⁶ specificity. In many of the mutants, one or more RFLP markers in the vicinity of the linked Ac were also absent. These findings suggest that the linked Ac may be inducing chromosome breakage.     

Analysis of extrachromosomal Ac/Ds transposable elements

The mechanism of transposition of the maize Ac/Ds elements is not well understood. The true transposition intermediates are not known and it has not been possible to distinguish between excision models involving 8-bp staggered cuts or 1-bp staggered cuts followed by hairpin formation. In this work, we have analyzed extrachromosomal excision products to gain insight into the excision mechanism. Plasmid rescue was used to demonstrate that Ds excision is associated with the formation of circular molecules. In addition, we present evidence for the formation of linear extrachromosomal species during Ds excision. Sequences found at the termini of circular and linear elements showed a broad range of nucleotide additions or deletions, suggesting that these species are not true intermediates. Additional nucleotides adjacent to the termini in extrachromosomal elements were compared to the sequence of the original donor site. This analysis showed that: (1) the first nucleotide adjacent to the transposon end was significantly more similar to the first nucleotide flanking the element in the donor site than to a random sequence and (2) the second and farther nucleotides did not resemble the donor site. The implications of these findings for excision models are discussed.     

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.     

Replication of a geminivirus derived shuttle vector in maize endosperm cells.

A maize (Zea mays L.) endosperm cell culture has been shown to efficiently replicate DNA sequences derived from wheat dwarf virus (WDV), a monopartite monocot geminivirus. To analyze sequences necessary for viral replication and to verify their application for a plant gene expression vector, we have developed a 3.7 kilobase pairs Escherichia coli--plant cell shuttle vector, pWI-11. The p15A origin of replication, functional in E. coli, was introduced into the viral sequences. We have replaced the coding region of the coat protein gene by that of bacterial neomycin phosphotransferase II (NPT II) gene. The resulting NPT II gene fusion can serve as a selectable marker in both plant and E. coli systems. Into a unique cloning site in this pWI-11 vector, we introduced a gene fusion carrying the bacterial beta-glucuronidase (GUS) coding region under control of the cauliflower mosaic virus 35S (CaMV35S) gene promoter and terminator. By transferring these viral sequences into protoplasts derived from maize endosperm cell cultures, we have demonstrated that the plasmid pWI-11 can replicate in maize endosperm cells, that the GUS reporter gene introduced into pWI-11 can be expressed at high level in the transformed cells, and that the replicating viral DNA can be rescued from endosperm cells by transforming E. coli in the presence of kanamycin. The level of GUS gene expression increased progressively in transformed endosperm cells during a prolonged culture period, coinciding with replication of the viral sequences in these cells.     

Adsorption of DNA on clay minerals: protection against DNaseI and influence on gene transfer

Abstract Numerous authors have investigated DNA relationships with sandy soil. A model composed of various DNAs adsorbed on montmorillonite clay was developed to assay enzyme (DNaseI) activity on clay-adsorbed nucleic acids. The extent of DNA adsorption was affected by the concentration and valency of the cations used (Mg²⁺, Ca²⁺, Na⁺), indicating a charge-dependent process. Calf thymus DNA was found to be highly adsorbed by smectite (up to 30 mg g⁻¹ of dry clay). Adsorbed DNA was shown to be more resistant to degradation by DNaseI than free DNA. Experimental data with plasmid and short linear amplified (through polymerase chain reaction) DNA showed that protection against nucleases was only partial. Nevertheless, clay-adsorbed DNA was found to be still able, even after a strong DNaseI treatment, to artificially transform competent Escherichia coli cells. The results show that persistance of DNA and gene transfer by genetic transformation may occur in soil.     

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.     

Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development

Abstract Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA ⁻ background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA ⁻ C. pseudotuberculosis was 10–12-fold lower than that in the recA ⁺ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA ⁻ background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suc ⁰ and N. crassa inv strains transformed with p NC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suc ⁰ ( p NC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa , although S. cerevisiae suc ⁺ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI -restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv^- to Inv⁺ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.