Genetic analysis of the pAD1 pheromone response in Streptococcus faecalis, using transposon Tn917 as an insertional mutagen

The conjugative plasmid pAD1 (56.7 kilobases) in Streptococcus faecalis has been shown to confer a mating response to the sex pheromone cAD1 excreted by recipient strains. The response is characterized by the synthesis of a proteinaceous adhesin which coats the surface of the pAD1 -containing donor cell and facilitates the formation of mating aggregates. Donors exposed to cAD1 -containing filtrates of recipients undergo self-aggregation (clumping), an event believed to be associated with an interaction between the adhesin and a binding substance always present on the surface of both recipients and donors. To analyze the molecular processes involved in the mating response, mutants were generated by the erythromycin resistance transposon Tn917 . Transpositions to pAD1 in S. faecalis DS16 gave rise to a number of derivatives that exhibited "constitutive clumping" and the ability to transfer at high frequencies in short (10-min) matings. These mutants fell into two subclasses, which exhibited colony morphologies that were "dry" or "normal". The Tn917 insertions were mapped by restriction enzyme analysis to two separate clusters, designated traA and traB. The dry colony subclass corresponded to traA and represented a span of 1.5 kilobases, whereas the normal subclass corresponded to traB and spanned 1.3 kilobases. The two clusters were separated by 1.7 kilobases in which insertions of Tn917 did not affect the ability to respond normally to cAD1 . Neither type of constitutive clumper produced cAD1 . Another series of insertions exhibited reduced donor potential. In two cases, the reduction in transfer was three to four orders of magnitude; these mapped in traA . In two other cases, the reduction was one to two orders of magnitude. These mapped outside of traA and traB, and one was associated with an increase in plasmid copy number.     

The rice miniature inverted repeat transposable element mPing is an effective insertional mutagen in soybean

Insertional mutagenesis of legume genomes such as soybean (Glycine max) should aid in identifying genes responsible for key traits such as nitrogen fixation and seed quality. The relatively low throughput of soybean transformation necessitates the use of a transposon-tagging strategy where a single transformation event will produce many mutations over a number of generations. However, existing transposon-tagging tools being used in legumes are of limited utility because of restricted transposition (Ac/Ds: soybean) or the requirement for tissue culture activation (Tnt1: Medicago truncatula). A recently discovered transposable element from rice (Oryza sativa), mPing, and the genes required for its mobilization, were transferred to soybean to determine if it will be an improvement over the other available transposon-tagging tools. Stable transformation events in soybean were tested for mPing transposition. Analysis of mPing excision at early and late embryo developmental stages revealed increased excision during late development in most transgenic lines, suggesting that transposition is developmentally regulated. Transgenic lines that produced heritable mPing insertions were identified, with the plants from the highest activity line producing at least one new insertion per generation. Analysis of the mPing insertion sites in the soybean genome revealed that features displayed in rice were retained including transposition to unlinked sites and a preference for insertion within 2.5 kb of a gene. Taken together these findings indicate that mPing has the characteristics necessary for an effective transposon-tagging resource.     

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15–50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.     

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.     

Resources for targeted insertional and deletional mutagenesis in Arabidopsis

The maize transposons Activator (Ac) and Dissociation (Ds) are active in many monocots and dicots, including Arabidopsis. We describe a new Ac-derived transposon construct, designated the Ds-loxP T-DNA, which can be used for both insertional and deletional mutagenesis. There are loxP sites in both orientations on both the transposon and the donor site T-DNA and an arrangement of marker genes that permits selection of transposition events, as well as deletions and inversions extending from the donor site to a transposon reinserted on either side of it. We show that Cre-mediated deletions and inversions occur at a high frequency. The tendency of Ac-Ds transposons to reinsert near the donor site can be used to target both insertional and deletional mutagenesis, but efficient exploitation of this property requires a library of mapped marked donor sites distributed in the genome. We have created a population of independent Ds T-DNA transformants and we have mapped an initial set of 75 Ds T-DNA integration sites. We assessed the potential efficiency of targeted mutagenesis by detecting Ds reinsertion events at several loci over a 400 kb interval from each of two donor sites with different Ds T-DNA constructs. The distribution of reinsertion sites is similar around the two tested loci, with roughly 10, 4, and ca. 1% of reinsertions detected within 1-2 kb of sites 10, 100, and 200-400 kb from the donor site, respectively. To facilitate the use of this targeted mutagenesis system. we have constructed a searchable database of the mapped Ds T-DNA integration sites.     

Saving insertional recessive lethal mutants of Arabidopsis thaliana

A group of 13 recessive lethal mutants was selected on the basis of the collection of Arabidopsis thaliana transgenic plants with insertions of T-DNA vector plasmid pLD3 or pPCVRN4, which was produced by agrobacterial transformation of germinating seeds. The use of media containing exogenous hormones made it possible to compensate the lethal effect, identify phenotypes, and characterize six lines of recessive lethal germlings using genetic and molecular-genetic methods.     

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.     

Use of the transposon Ac as a gene-searching engine in the maize genome

We show here that, although genes constitute only a small percentage of the maize genome, it is possible to identify them phenotypically as Ac receptor sites. Simple and efficient Ac transposition assays based on the well-studied endosperm markers bz and wx were used to generate a collection of >1300 independent Ac transposants. The majority of transposed Ac elements are linked to either the bz or the wx donor loci on chromosome 9. A few of the insertions produce obvious visible phenotypes, but most of them do not, suggesting that these populations will be more useful for reverse genetics than for forward transposon mutagenesis. An inverse polymerase chain reaction method was adapted for the isolation of DNA adjacent to the transposed Ac elements (tac sites). Most Ac insertions were into unique DNA. By sequencing tac sites and comparing the sequences to existing databases, insertions were identified in a number of putative maize genes. The expression of most of these genes was confirmed by RNA gel blot analysis. We report here the isolation and characterization of the first 46 tac sites from the two insertion libraries.     

Behaviour of the maize transposable element Ac in Arabidopsis thaliana

The somatic and germinal activity of the maize transposable element, Ac, has been analysed in progeny of 43 transformants of A. thaliana using a streptomycin resistance assay to monitor Ac excision. The ability to assay somatic activity enabled, for the first time, a detailed analysis of Ac activity in individual A. thaliana seedlings to be made. The effects of T-DNA copy number, generation, dosage at each locus, flanking sequences and orientation of the element were compared. The most striking observation was the variability in Ac activity in genotypically identical individuals and the poor penetrance of the variegated phenotype. In general, increasing Ac dosage increased both somatic and germinal excision frequencies. The majority of families from individuals selected as inheriting an excision event carried transposed Ac elements re-integrated in different positions in the genome.     

Creation of a collection of morphological insertional mutants of Arabidopsis thaliana

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

Selection for enhanced germinal excision of Ac in transgenic Arabidopsis thaliana

Gene tagging in Arabidopsis thaliana using the autonomous Ac (Activator) transposable element has so far been hampered by low frequencies of germinal transposition events. Here we describe a procedure by which the frequency of independent germinal reinsertions has been much improved by a process of long-term selection on kanamycin for the continued growth of tissues in which somatic excisions have occurred. Growth on artificial media increased the somatic excision frequency, and the long-term selection procedure channelled somatic transposition events into the germline. This resulted in an overall germinal excision frequency in the progeny of longterm selected plants of 15%, as confirmed by Southern blotting, with 63% of the plants bearing excision events having detectable reinsertions of the Ac element. This compares with a germinal excision frequency of approximately 1% when no long-term selection is employed. However, offspring from individual plants tended to have identical germinal Ac reinsertion patterns, thus the critical parameter for evaluating the system for tagging purposes is the frequency of individual plants yielding offspring with reinsertions, which was 64%. This high frequency, when coupled to the enhanced germinal transposition rate overall, easily allows the generation of a large population of plants with independent reinsertions.