Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.
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PMID:Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism. 155 47

The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that product(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.
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PMID:The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA. 347 33

Seven Tn904 insertion mutants of pTi Ach5 affecting Agrobacterium tumefaciens virulence were studied. The mutant character was shown to be plasmid borne. Four of these mutants were avirulent and carried an insertion in restriction endonuclease HpaI fragment 12, a 3.3-megadalton fragment, which therefore appears to be a Ti plasmid region essential for virulence. Two mutants were attenuated in virulence. The inserts mapped close to HpaI fragment 12. One mutant giving rise to small tumors with excessive adventitious root formation on Kalanchoe daigremontiana carried an insertion in the right side of the common sequence in the deoxyribonucleic acid of the Ti plasmid detected in crown gall tumors. The insertion behavior of Tn904 was studied by analyzing 11 independently isolated and randomly chosen mutants. The Tn904 inserts did not affect oncogenicity, tumor morphology, bacterial transfer functions, octopine catabolism functions, or vital parts of the Ti plasmid, such as the origin of replication. Most of the Tn904 inserts were concentrated in a small part of the map. The size of additional deoxyribonucleic acid as a result of Tn904 inserts varied between 5 and 15 megadaltons. In two cases a Ti plasmid was found with two Tn904 insertions at different positions.
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PMID:Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens. 625 98

This paper presents a method for insertion of genetic material into a specific site in T-DNA, the portion of Agrobacterium tumor-inducing (Ti) plasmid that becomes incorporated into the nuclear DNA of transformed plant cells when crown gall tumors are incited by this plant pathogen. The three stages of our procedure are as follows: 1. A T-DNA subfragment cloned in pBR322 is cleaved by a restriction endonuclease at a unique central site and target DNA (a kanamycin resistance marker) is ligated into this site. 2. The resulting recombinant plasmid is purified and cleaved with EcoRI; the resulting fragment bearing the kanamycin resistance marker is ligated into the unique EcoRI site of pRK290, a wide-host-range plasmid. 3. The pRK290 recombinant plasmid is transformed into an agrobacterium tumefaciens strain containing a wild type Ti plasmid. Double recombination between the altered T-DNA fragment of the clone and its wild-type counterpart in the Ti plasmid is selected for by introduction of R751-pMG2, a plasmid incompatible with pRK290. The approach described here can be adapted for introducing genes into higher plant cells with the Ti plasmid as vector. It can likewise be used for site- or fragment-specific mutagenesis of the Ti plasmid as a means of detailed functional analysis.
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PMID:Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells. 695 19

During the initial stages of crown gall tumorigenesis, the T-DNA region of the Agrobacterium tumefaciens Ti-plasmid is processed, resulting in the production of T-DNA molecules that are subsequently transferred to the plant cell. Processing of the T-DNA in the bacterium involves the nicking of T-DNA border sequences by an endonuclease encoded by the virD locus, and the subsequent tight (possibly covalent) association of the VirD2 protein with the 5' end of the processed single-stranded or double-stranded T-DNA molecule. To investigate the interaction of the VirD1,D2 endonuclease with a right T-DNA border, a set of plasmids containing both the border and virD sequences on the same high-copy-number replicon has been constructed and introduced into Escherichia coli. In this model system a tight nucleoprotein complex is formed between the relaxed double-stranded substrate plasmid and the VirD2 protein. This putative T-DNA processing complex may be analogous to the covalent relaxation complex formed between the pilot protein and plasmid DNA during bacterial conjugation. VirD2 attachment to the relaxed substrate plasmid was resistant to denaturing agents but sensitive to S1 nuclease digestion, indicating a single-stranded region near the site of protein attachment. We speculate that this structure may be an intermediate formed prior to T-strand unwinding from the substrate plasmid in a host bacterium.
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PMID:Formation of a putative relaxation intermediate during T-DNA processing directed by the Agrobacterium tumefaciens VirD1,D2 endonuclease. 835 16

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5' end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2-cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.
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PMID:Agrobacterium VirD2 protein interacts with plant host cyclophilins. 961 35

The crown gall teratoma tumor line BT37, incited by Agrobacterium tumefaciens strain T37, has been found to contain part of the tumor-inducing plasmid, pTi T37, of the inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diploid tumor cell. We have examined subcellular DNA fractions from this tumor line in an effort to determine whether T-DNA is in chloroplasts, mitochondria, or nuclei. Tumor cell chloroplast DNA exhibited EcoRI and Bst I endonuclease cleavage patterns identical to those of normal tobacco chloroplast DNA. Tumor cell mitochondrial DNA exhibited a complex Bst I cleavage pattern that did not differ detectably from that of normal tobacco mitochondrial DNA. Southern blots of tumor chloroplast and mitochondrial cleavage products did not hybridize with labeled pTi T37 DNA, whereas blots of tumor cell nuclear DNA cleavage products hybridized strongly. We conclude that T-DNA is located not in chloroplasts or mitochondria but rather in the nuclear fraction of this tumor line.
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PMID:T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. 1659 50

Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.
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PMID:Studies on the structure of cointegrates between octopine and nopaline Ti-plasmids and their tumour-inducing properties. 2431 33

Agrobacterium was identified as the agent causing the plant tumor, crown gall over 100 years ago. Since then, studies have resulted in many surprising observations. Armin Braun demonstrated that Agrobacterium infected cells had unusual nutritional properties, and that the bacterium was necessary to start the infection but not for continued tumor development. He developed the concept of a tumor inducing principle (TIP), the factor that actually caused the disease. Thirty years later the TIP was shown to be a piece of a tumor inducing (Ti) plasmid excised by an endonuclease. In the next 20 years, most of the key features of the disease were described. The single-strand DNA (T-DNA) with the endonuclease attached is transferred through a type IV secretion system into the host cell where it is likely coated and protected from nucleases by a bacterial secreted protein to form the T-complex. A nuclear localization signal in the endonuclease guides the transferred strand (T-strand), into the nucleus where it is integrated randomly into the host chromosome. Other secreted proteins likely aid in uncoating the T-complex. The T-DNA encodes enzymes of auxin, cytokinin, and opine synthesis, the latter a food source for Agrobacterium. The genes associated with T-strand formation and transfer (vir) map to the Ti plasmid and are only expressed when the bacteria are in close association with a plant. Plant signals are recognized by a two-component regulatory system which activates vir genes. Chromosomal genes with pleiotropic functions also play important roles in plant transformation. The data now explain Braun's old observations and also explain why Agrobacterium is nature's genetic engineer. Any DNA inserted between the border sequences which define the T-DNA will be transferred and integrated into host cells. Thus, Agrobacterium has become the major vector in plant genetic engineering.
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PMID:Agrobacterium: nature's genetic engineer. 2561 Apr 42