Rare-cutting Restriction Enzymes Research Articles

A series of bacterial co-expression vectors with rare-cutter recognition sequences

The bacterial co-expression method is a useful protein expression technique to reconstitute a hetero-oligomeric protein complex in vitro. However, during the plasmid subcloning for co-expression, unintended cleavage at the sequences of target cDNAs becomes more frequent as the number of DNA inserts increases. This problem also makes it difficult to change the combination of targeted proteins after preparing a certain co-expression construct. To avoid this problem, we have developed a series of bacterial co-expression vectors, in which each translation cassette can be subcloned at a set of rare-cutter restriction enzyme sites. We selected 9 different rare-cutter restriction enzymes that recognize a 7 or 8-base-pair sequence, and constructed 27 kinds of cloning vectors and 3 kinds of co-expression vectors, utilizing the rare-cutter recognition sequences as multi-cloning sites. Using this vector system, we co-expressed and co-purified a 7-subunit protein complex composed of the mammalian 26S proteasome regulatory subunits RPT1 to RPT6, and their associated factor, gankyrin. We verified the presence of all 7 subunits by western blotting, by taking advantage of the vector system in which the target proteins can be fused with a broad repertoire of epitope tags.

Ligation-mediated PCR, a fast and reliable technique for insertion sequence-based typing ofXanthomonas citripv.citri

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a major disease threatening citrus crops throughout the world. The most common methods for strain differentiation of this pathogen are repetitive element sequence-based PCR (rep-PCR) and pulsed field gel electrophoresis (PFGE), using rare-cutting restriction enzyme analysis. We developed a ligation-mediated PCR targeting three insertion sequences (IS-LM-PCR) present as several copies in the genome of the fully sequenced strain 306 of X. citri pv. citri. This technique amplifies DNA fragments between an insertion sequence element and an MspI restriction site. The analysis of strains can be conducted within 24 h, starting from very small amounts of bacterial DNA, which makes IS-LM-PCR much less labor-intensive than PFGE. We used IS-LM-PCR to analyze a collection of 66 strains of X. citri pv. citri from around the world. The overall reproducibility of IS-LM-PCR reached 98% in this data set and its discriminatory power was markedly superior than rep-PCR. We suggest that IS-LM-PCR could be used for the global surveillance of non-epidemiologically related strains of X. citri pv. citri.

Structures of the Rare-Cutting Restriction Endonuclease NotI Reveal a Unique Metal Binding Fold Involved in DNA Binding

The structure of the rare-cutting restriction endonuclease NotI, which recognizes the 8 bp target 5'-GCGGCCGC-3', has been solved with and without bound DNA. Because of its specificity (recognizing a site that occurs once per 65 kb), NotI is used to generate large genomic fragments and to map DNA methylation status. NotI contains a unique metal binding fold, found in a variety of putative endonucleases, occupied by an iron atom coordinated within a tetrahedral Cys4 motif. This domain positions nearby protein elements for DNA recognition, and serves a structural role. While recognition of the central six base pairs of the target is accomplished via a saturated hydrogen bond network typical of restriction enzymes, the most peripheral base pairs are engaged in a single direct contact in the major groove, reflecting reduced pressure to recognize those positions. NotI may represent an evolutionary intermediate between mobile endonucleases (which recognize longer target sites) and canonical restriction endonucleases.

Differences in Virulence and Genomic Features of Strains of ‘Candidatus Phytoplasma mali’, the Apple Proliferation Agent

ABSTRACT Root and shoot samples from 24 symptomatic or nonsymptomatic apple trees infected with 'Candidatus Phytoplasma mali' were collected at different locations in Germany and France and used to inoculate rootstock M11 top grafted with cv. Golden Delicious. Inoculated trees were monitored over a 12-year period for apple proliferation (AP) symptoms and categorized as not or slightly, moderately, or severely affected. Based on symptomatology, the phytoplasma strains were defined as being avirulent to mildly, moderately, or highly virulent. Determination of phytoplasma titers by quantitative polymerase chain reaction (PCR) with DNA from roots revealed similar phytoplasma concentrations in all virulence groups. Molecular characterization of the strains by differential PCR amplification with five sets of primers resulted in 13 profiles. Six strains that were maintained in periwinkle and tobacco were molecularly characterized in more detail. The genome sizes of these strains as determined by pulsed-field gel electrophoresis using yeast chromosomes as size references ranged between 640 and 680 kb. Cleavage of the chromosome with the rare cutting restriction enzymes ApaI, BamHI, BssHII, MluI, and SmaI resulted in macro fragment patterns distinctly different in all strains. Similar results were obtained by Southern blot hybridization with three probes derived from strain AT. Differential PCR amplification at an annealing temperature of 52 degrees C using eight primer pairs derived from strain AT revealed heterogeneity of target sequences among all strains. Based on these results, there is considerable variability in virulence and genomic traits in 'Ca. P. mali'. However, correlations between molecular markers and virulence or phytoplasma titer could not be identified.

Restriction landmark genomic scanning

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment. It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome. RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. Compared with the conventional gene-detection technologies, such as Southern blot analysis and PCR, RLGS has the following advantages even though it needs specially designed instruments: high-efficiency scanning capacity, scanning extensibility by using alternate restriction enzyme combinations, applicability to any organism, a spot intensity that reflects the copy number of restriction landmarks, and the ability, by using a methylation-sensitive enzyme, to screen the methylated state of genomic DNA. The RLGS protocol can be accomplished in 5 days to 2 weeks.

The Crystal Structure of the Rare-Cutting Restriction Enzyme SdaI Reveals Unexpected Domain Architecture

Rare-cutting restriction enzymes are important tools in genome analysis. We report here the crystal structure of SdaI restriction endonuclease, which is specific for the 8 bp sequence CCTGCA/GG ("/" designates the cleavage site). Unlike orthodox Type IIP enzymes, which are single domain proteins, the SdaI monomer is composed of two structural domains. The N domain contains a classical winged helix-turn-helix (wHTH) DNA binding motif, while the C domain shows a typical restriction endonuclease fold. The active site of SdaI is located within the C domain and represents a variant of the canonical PD-(D/E)XK motif. SdaI determinants of sequence specificity are clustered on the recognition helix of the wHTH motif at the N domain. The modular architecture of SdaI, wherein one domain mediates DNA binding while the other domain is predicted to catalyze hydrolysis, distinguishes SdaI from previously characterized restriction enzymes interacting with symmetric recognition sequences.

Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5′-GCGGCCGC-3′) has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5′-NCGGCCGN-3′ sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5′-GCTGCCGC-3′. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5′-GCTGCCGC-3′ with a specific activity of 8.2 × 104 U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.

3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.

CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.

Intragenomic recombination in the highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans

The highly leukotoxic JP2 clone of Actinobacillus actinomycetemcomitans is strongly associated with aggressive periodontitis in adolescents of African descent. DNA fingerprinting using the frequently cutting restriction enzyme MspI and multilocus sequence typing (MLST) showed that five strains of this clone were genetically virtually identical, although ribotyping of the six rrn genes and EcoRI RFLP analysis of the seven IS150-like elements revealed differences. PCR analyses demonstrated that these multi-copy sequences are subject to intragenomic homologous recombination, resulting in translocations or large inversions. The genome rearrangements were reflected in differences among 25 strains representing the JP2 clone in DNA fingerprinting using the rare-cutting restriction enzyme XhoI and resolved by PFGE. XhoI DNA fingerprinting provides a tool for studying local epidemiology, including transmission of this particularly pathogenic clone of A. actinomycetemcomitans.

Green light for gene targeting in plants

Precision engineering of eukaryotic organisms requires efficient techniques for homology-based replacement of an endogenous gene by an introduced gene, a process termed gene targeting (GT). Because foreign DNA is preferentially integrated by nonhomologous recombination in plants and animals, special strategies are needed to increase the frequency and facilitate the detection of GT. Developing these strategies has proved particularly difficult in plants, for which GT efficiencies are on the order of 10–3 to 10–4 targeted events per transformed plant (1, 2). This low frequency seems to be the result of a natural barrier to integration of homologous sequences in higher plants. By contrast, efficient GT has been demonstrated in lower plants, such as the moss Physcomitrella patens (3). The time appears ripe for a breakthrough in this field, and one has now been reported in this issue of PNAS. Avraham Levy and his group (4) describe a jump of between one and two orders of magnitude in GT frequency in Arabidopsis thaliana plants overexpressing the yeast chromatin remodeling protein Rad54. Their innovative approach was facilitated by the development of an ingenious assay that uses fluorescent seeds to identify targeted insertions. Previous attempts to improve GT efficiency include the introduction of double-stranded breaks (DSBs) at the target site and modification of proteins involved in homologous recombination (HR). DSBs are obligatory recombination intermediates, and dedicated endonucleases are recruited to introduce them during meiosis. The introduction of a target site for a rare cutter restriction enzyme increased the targeting efficiency to such a site by two orders of magnitude in the somatic tissues in which GT normally takes place (5). However, the DSB has to be introduced at or close to the gene to be changed, which has been achieved in mammalian cells by using reprogrammed endonucleases whose catalytic domain …

Pulsed field gel electrophoresis to rapidly detect the presence of IncJ conjugative transposon-like elements

To develop a screening method to detect the presence of the IncJ group of integrating conjugative transposon-like elements upon transfer to Escherichia coli. The unique insertion site of known IncJ elements, the prfC gene, is located in a region of the E. coli chromosome between 98.5 and 100 min on the E. coli genetic map. Using pulsed field gel electrophoresis and the rare cutting restriction enzymes SfiI and XbaI insertions of IncJ elements and an estimate of their size could be determined physically. This method allows initial screening of putative IncJ conjugative transposon-like elements by physical determination of their integration. IncJ-like elements, which appear to be highly homologous to the prototype IncJ element R391, have been found associated with recent epidemic outbreaks of cholera in a number of locations worldwide. Because of their integrative biology this method provides the first initial screening method to physically determine their presence upon transfer to E. coli.

Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis

Brucella ovis is recognized worldwide as an important pathogen of sheep, and has also been identified in farmed deer in New Zealand. Previously, only one strain type of B. ovis has been identified. The objective of this paper was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. ovis to determine whether strain variations exist, whether sheep and deer are affected by the same strains, and to compare the performance of the rare-cutting restriction enzymes XbaI and SwaI. Ten B. ovis isolates from sheep and two from deer in New Zealand, as well as the type strain, were subjected to PFGE analysis using both XbaI and SwaI. PFGE of XbaI restriction fragments produced two banding patterns consisting of 27-28 bands, which were found to be 98% similar by cluster analysis, and were named X1 and X1a. PFGE of SwaI restriction fragments resulted in three banding patterns consisting of 13-15 bands each. Ten of the isolates had identical banding patterns and were named S1. One isolate differed by one band, representing a subtype named S1a. Two isolates differed by six bands, representing a different strain type of B. ovis and this was named S2. Cluster analysis showed S2 to be 78% similar to the S1/S1a cluster. Both strain types were isolated from both sheep and deer. Thus, two distinct strain types of B. ovis were identified in New Zealand, which is the first report of more than one strain type being identified worldwide. Neither strain was species-specific for sheep or deer. The restriction endonuclease SwaI was found to be more discriminatory than the enzyme XbaI, which has been used in previous studies.

Diversity of Proteolytic Clostridium botulinum Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.

Construction of a combined physical and genetic map of the chromosome of Lactobacillus acidophilus ATCC 4356 and characterization of the rRNA operons

The combination of PFGE and hybridization approaches was used to study the genome of Lactobacillus acidophilus neotype strain ATCC 4356. PFGE analysis of chromosomal DNA after digestion with each of the rare-cutting restriction enzymes I-CeuI, NotI, CspI, SmaI, ApaI and SgrAI allowed the size of the circular chromosome of L. acidophilus to be estimated at 2.061 Mbp. The physical map contained 86 restriction sites for the six enzymes employed, with intervals between the sites varying from 1 to 88 kbp (approximately 0.05-4.3 % of the chromosome). Based on the physical map, a genetic map was constructed via Southern blot analyses of L. acidophilus DNA using specific gene probes. A total of 73 probes representing key genes, including 12 rRNA (rrn) genes, were positioned on the latter map. Mapping analysis also indicated the presence of four rrn operons (rrnA-D) on the chromosome, each containing a single copy of each of the three rrn genes 16S (rrl), 23S (rrs) and 5S (rrf). Operon rrnD was inverted in orientation with respect to the others and contained a long 16S-23S intergenic spacer region with tRNAIle and tRNAAla genes, whereas the other operons contained a short spacer lacking any tRNA genes. The high-resolution physical/genetic map constructed in this study provides a platform for genomic and genetic studies of Lactobacillus species and for improving industrial and probiotic strains.

A General Method to Modify BACs to Generate Large Recombinant DNA Fragments

Bacterial artificial chromosome (BAC) has the capacity to clone DNA fragments in excess of 300 kb. It also has the considerable advantages of stable propagation and ease of purification. These features make BAC suitable in genetic research, such as library construction, transgenic mice production, and gene targeting constructs. Homologous recombination in Escherichia coli, a process named recombineering, has made the modification of BACs easy and reliable. We report here a modified recombineering method that can efficiently mediate the fusion of large DNA fragments from two or more different BACs. With the introduction of kanamycin-resistant gene and proposed rare-cutting restriction endonuclease (RCRE) sites into two BACs, a 82.6-kb DNA fragment containing the inverted human alpha-globin genes (theta, alpha1, alpha2, and zeta) from BAC191K2 and the locus control region (LCR) of human beta-globin gene locus (from the BAC186D7) was reconstructed. This approach for combining different BAC DNA fragments should facilitate many kinds of genomic experiments.

Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont

The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic α-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.

Intrachromosomal homologous recombination in Arabidopsis thaliana.

Because of the availability of the complete sequence of the genome of the model plant Arabidopsis and of insertion mutants for most genes in public mutant collections, the elucidation of the particular role of different factors involved in DNA recombination and repair processes, an important task for plant biology, is becoming feasible. An assay system based on transgenes harboring homologous overlaps of the beta-glucuronidase (uidA) gene is available to determine recombination behavior in various mutant backgrounds. Restoration of the marker gene by homologous recombination can be detected by histochemical staining in planta. Inclusion of a site of the rare cutting restriction enzyme I-SceI in the transgene construct enables the determination of recombination frequencies after induction of double-strand breaks. In this chapter we describe how the respective transgene is transferred by transformation or crossing into the mutant background, how recombination frequencies are determined, and, if necessary, how cells carrying a restored uidA gene can be isolated and propagated for molecular analysis of the particular recombination event.

NotI passporting to identify species composition of complex microbial systems.

We describe here a new method for large-scale scanning of microbial genomes on a quantitative and qualitative basis. To achieve this aim we propose to create NotI passports: databases containing NotI tags. We demonstrated that these tags comprising 19 bp of sequence information could be successfully generated using DNA isolated from intestinal or fecal samples. Such NotI passports allow the discrimination between closely related bacterial species and even strains. This procedure for generating restriction site tagged sequences (RSTS) is called passporting and can be adapted to any other rare cutting restriction enzyme. A comparison of 1312 tags from available sequenced Escherichia coli genomes, generated with the NotI, PmeI and SbfI restriction enzymes, revealed only 219 tags that were not unique. None of these tags matched human or rodent sequences. Therefore the approach allows analysis of complex microbial mixtures such as in human gut and identification with high accuracy of a particular bacterial strain on a quantitative and qualitative basis.

Knockout of 'metal-responsive transcription factor' MTF-1 in Drosophila by homologous recombination reveals its central role in heavy metal homeostasis.

'Metal-responsive transcription factor-1' (MTF-1), a zinc finger protein, is conserved from mammals to insects. In the mouse, it activates metallothionein genes and other target genes in response to several cell stress conditions, notably heavy metal load. The knockout of MTF-1 in the mouse has an embryonic lethal phenotype accompanied by liver degeneration. Here we describe the targeted disruption of the MTF-1 gene in Drosophila by homologous recombination. Unlike the situation in the mouse, knockout of MTF-1 in Drosophila is not lethal. Flies survive well under laboratory conditions but are sensitive to elevated concentrations of copper, cadmium and zinc. Basal and metal-induced expression of Drosophila metallothionein genes MtnA (Mtn) and MtnB (Mto), and of two new metallothionein genes described here, MtnC and MtnD, is abolished in MTF-1 mutants. Unexpectedly, MTF-1 mutant larvae are sensitive not only to copper load but also to copper depletion. In MTF-1 mutants, copper depletion prevents metamorphosis and dramatically extends larval development/lifespan from normally 4-5 days to as many as 32 days, possibly reflecting the effects of impaired oxygen metabolism. These findings expand the roles of MTF-1 in the control of heavy metal homeostasis.

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome.

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.