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B Hohn

Publications and source records attributed to B Hohn.

At least 37 records · Page 2Linked to original sources
PubMed1999

Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium.

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Agrobacterium tumefaciens↗
PubMed1999

Bacterial conjugation protein MobA mediates integration of complex DNA structures into plant cells.

Agrobacterium tumefaciens transfers T-DNA to plant cells, where it integrates into the genome, a property that is ensured by bacterial proteins VirD2 and VirE2. Under natural conditions, the protein MobA mobilizes its encoding plasmid, RSF1010, between different bacteria. A detailed analysis of MobA-mediated DNA mobilization by Agrobacterium to plants was performed. We compared the ability of MobA to transfer DNA and integrate it into the plant genome to that of pilot protein VirD2. MobA was found to be about 100-fold less efficient than VirD2 in conducting the DNA from the pTi plasmid to the plant cell nucleus. However, interestingly, DNAs transferred by the two proteins were integrated into the plant cell genome with similar efficiencies. In contrast, most of the integrated DNA copies transferred from a MobA-containing strain were truncated at the 5' end. Isolation and analysis of the most conserved 5' ends revealed patterns which resulted from the illegitimate integration of one transferred DNA within another. These complex integration patterns indicate a specific deficiency in MobA. The data conform to a model according to which efficiency of T-DNA integration is determined by plant enzymes and integrity is determined by bacterial proteins.

Agrobacterium tumefaciens↗
PubMed1998

Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens: analysis by subcellular localization in mammalian cells.

Interaction between Agrobacterium tumefaciens and plants provides a unique example of interkingdom gene transfer. Agrobacterium, a plant pathogen, is capable to stably transform the plant cell with a segment of its own DNA called T-DNA (transferred DNA). This process depends, among others, on the specialized bacterial virulence proteins VirD1 and VirD2 that excise the T-DNA from its adjacent sequences. Subsequent to transfer to the plant cell, the virulence protein VirD2, through its nuclear localization signal (NLS), is believed to guide the T-DNA to the nucleus. The T-DNA then is integrated into the plant genome. Although both of these proteins are essential for bacterial virulence, physical interaction of them has not been analyzed so far. We studied associations between these proteins by expressing them in mammalian cells and by testing for intracellular localization and colocalization. When expressed in human cells [HeLa, human embryo kidney (HEK) 293], the VirD2 protein homogeneously distributed over the nucleoplasm. The presence of any of two NLSs, on the N and C termini of VirD2, was sufficient for its efficient nuclear localization whereas deletion of both NLSs rendered the protein cytoplasmic. However, this double NLS mutant was translocated to the nucleus in the presence of wild-type VirD2 protein, implying VirD2-VirD2 interaction. The VirD1 protein, by itself localized in the cytoplasm, moved to the nucleus when coexpressed with the VirD2 protein, suggesting VirD1-VirD2 interaction. This interaction was confirmed by coimmunoprecipitation tests. Of interest, both proteins coimported to the nucleus showed a similar, peculiar sublocalization. The data are discussed in terms of functions of the VirD proteins. In addition, coimport of proteins into nuclei is suggested as a useful system in studying individual protein-protein interactions.

Agrobacterium tumefaciens↗
PubMed1998

The Allium cepa chromosome aberration test reliably measures genotoxicity of soils of inhabited areas in the Ukraine contaminated by the Chernobyl accident.

The accident on the Chernobyl Nuclear Power Plant reactor IV in April 1986 led to the release of an enormous amount of radioactive material into the biosphere and to the formation of a complex pattern of nuclear contamination over a large area. As a consequence more than 5 million km2 of the soil in the Ukraine became contaminated with more than 1 Ci/km2 [1,2]. An assessment of the genetic consequences of the nuclear pollution is one of the most important problems. We applied the Allium cepa test to estimate the impact on plant chromosomes of nuclear pollution in the inhabited zones of the Ukraine. We tested soil from the obligatory resettlement zone (zone 2), where the mean density of pollution is 15-40 Ci/km2; zones of enhanced radiological control-zone 3, 5-15 Ci/km2 and zone 4, 1-5 Ci/km2. We found a dose-dependent increase in the fraction of aberrant mitoses from control values of 1.6 +/- 0.9% up to 23.8 +/- 5.0%, and a corresponding monotonous decrease of the mitotic index from 49.4 +/- 4.8% to a limiting value of 22.5 +/- 4.0% at pollution levels exceeding 35 Ci/km2 (activity of the soil samples exceeding 6000 Bq/kg, respectively). We observed a strong, significant correlation of 137Cs activity of soil samples with the percentage of chromosomal abnormalities, r = 0.97 (P < 0.05), and with the mitotic index, r = -0.93 (P < 0.05), in the roots of A. cepa, respectively. The results showed high toxicity and genotoxicity of radioactively polluted soils and confirmed the efficiency of the A. cepa test as a quick and inexpensive biological test for ecological and genetic risk assessment in the 'Chernobyl' zones.

Chromosome Aberrations↗
PubMed1998

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.

DNA Transposable Elements↗
PubMed1998

Transgenic plants are sensitive bioindicators of nuclear pollution caused by the Chernobyl accident.

To evaluate the genetic consequences of radioactive contamination originating from the nuclear reactor accident of Chernobyl on indigenous populations of plants and animals, it is essential to determine the rates of accumulating genetic changes in chronically irradiated populations. An increase in germline mutation rates in humans living close to the Chernobyl Nuclear Power Plant site, and a two- to tenfold increase in germline mutations in barn swallows breeding in Chernobyl have been reported. Little is known, however, about the effects of chronic irradiation on plant genomes. Ionizing radiation causes double-strand breaks in DNA, which are repaired via illegitimate or homologous recombination. We make use of Arabidopsis thaliana plants carrying a beta-glucuronidase marker gene as a recombination substrate to monitor genetic alterations in plant populations, which are caused by nuclear pollution of the environment around Chernobyl. A significant (p<0.05) increase in somatic intrachromosomal recombination frequencies was observed at nuclear pollution levels from 0.1-900 Ci/km2, consistent with an increase in chromosomal aberrations. This bioindicator may serve as a convenient and ethically acceptable alternative to animal systems.

Animals↗
PubMed1998

The omega sequence of VirD2 is important but not essential for efficient transfer of T-DNA by Agrobacterium tumefaciens.

The VirD2 protein of Agrobacterium tumefaciens contains defined sequences necessary for processing and transferring the T-DNA during transformation of plant cells. We performed a mutational analysis of the conserved omega sequence of VirD2, whose role has proven to be difficult to elucidate so far. In this report, we show that a deletion of these 5 amino acids or their replacement by 5 glycines reduced T-DNA transfer considerably, compared with wild type, demonstrating that the omega sequence is important for the efficient transfer of T-DNAs. However, the efficiency and pattern of integration of the T-DNAs were not affected by any modifications of the omega sequence. The importance of the C terminus of VirD2 for T-DNA transfer is discussed.

Agrobacterium tumefaciens↗
PubMed1996

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

Base Sequence↗
PubMed1996

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Agrobacterium tumefaciens↗
PubMed1995

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize.

A reverse genetic system for studying excision of the transposable element Ds1 in maize plants has been established previously. In this system, the Ds1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of the Ac element, excision of Ds1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences required in cis for excision of Ds1. The Ds1 element contains the Ac transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5' subterminal region). We showed that mutation of these motifs abolished completely the excision capacity of Ds1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3') subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.

Base Sequence↗
PubMed1995

The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome.

The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T-DNA by using a virD2 mutant. This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration.

Agrobacterium tumefaciens↗
PubMed1995

Intracellular Agrobacterium can transfer DNA to the cell nucleus of the host plant.

Agrobacterium tumefaciens is a Gram-negative, soil-borne bacterium responsible for the crown gall disease of plants. The galls result from genetic transformation of plant cells by the bacteria. Genes located on the transferred DNA (T-DNA), which is part of the large tumor-inducing (Ti) plasmid of Agrobacterium, are integrated into host plant chromosomes and expressed. This transfer requires virulence (vir) genes that map outside the T-DNA on the Ti plasmid and that encode a series of elaborate functions that appear similar to those of interbacterial plasmid transfer. It remains a major challenge to understand how T-DNA moves from Agrobacterium into the plant cell nucleus, in view of the complexity of obstacles presented by the eukaryotic host cell. Specific anchoring of bacteria to the outer surface of the plant cell seems to be an important prelude to the mobilization of the T-DNA/protein complex from the bacterial cell to the plant cell. However, the precise mode of infection is not clear, although a requirement of wounded cells has been documented. By using a microinjection approach, we show here that the process of T-DNA transfer from the bacteria to the eukaryotic nucleus can occur entirely inside the plant cell. Such transfer is absolutely dependent on induction of vir genes and a functional virB operon. Thus, A. tumefaciens can function as an intracellular infectious agent in plants.

Journal Article↗
PubMed1995

Somatic intrachromosomal homologous recombination events in populations of plant siblings.

Intrachromosomal homologous recombination in whole tobacco plants was analyzed using beta-glucuronidase as non-selectable marker. We found that recombination frequencies were additive for transgenes in allelic positions and could be enhanced by treatment of plants with DNA-damaging agents. We compared the patterns of distribution of recombination events of different transgenic lines of tobacco and Arabidopsis with the respective Poisson distributions. Some lines showed Poisson-like distributions, indicating that recombination at the transgene locus was occurring in a random fashion in the plant population. In other cases, however, the distributions deviated significantly from Poisson distributions indicating that for specific transgene loci and/or configurations recombination events are not randomly distributed in the population. This was due to overrepresentation of plants with especially many as well as especially few recombination events. Analysis of one tobacco line indicated furthermore that the distribution of recombination events could be influenced by treating the seedlings with external factors. Our results suggest that different plant individuals, or parts of them, might exhibit different transient 'states' of recombination competence. A possible model relating 'recombination silencing' and transcription silencing to heterochromatization of the transgene locus is discussed.

Arabidopsis↗
PubMed1995

Vectors based on maize streak virus can replicate to high copy numbers in maize plants.

The genome of maize streak virus (MSV) consists of one molecule of circular, single-stranded DNA of 2.7 kb. A reporter gene (bar) coding for phosphinothricin acetyl-transferase was inserted into the small non-coding region of the MSV genome. The recombinant bar-containing MSV vectors were introduced into maize seedlings via agroinfection. The chimeric viral DNA was found to replicate to high copy numbers in maize leaves resistant to the application of the herbicide Basta. This establishes the usefulness of MSV as an efficient replicating vector in cells of maize plants.

Acetyltransferases↗
PubMed1994

Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus.

Transferred DNA (T-DNA) is transferred as a single-stranded derivative from Agrobacterium to the plant cell nucleus. This conclusion is drawn from experiments exploiting the different properties of single- and double-stranded DNA to perform extrachromosomal homologous recombination in plant cells. After transfer from Agrobacterium to plant cells, T-DNA molecules recombined much more efficiently if the homologous sequences were of opposite polarity than if they were of the same polarity. This observation reflects the properties of single-stranded DNA; single-stranded DNA molecules of opposite polarity can anneal directly, whereas single-stranded DNA molecules of the same polarity first have to become double stranded to anneal. Judging from the relative amounts of single- to double-stranded T-DNA derivatives undergoing recombination, we infer that the T-DNA derivatives enter the plant nucleus in their single-stranded form.

Journal Article↗
PubMed1994

Intrachromosomal homologous recombination in whole plants.

A system to assay intrachromosomal homologous recombination during the complete life-cycle of a whole higher eukaryote was set up. Arabidopsis thaliana plants were transformed with a recombination substrate carrying a non-selectable and quantitatively detectable marker gene. The recombination substrates contain two overlapping, non-functional deletion mutants of a chimeric beta-glucuronidase (uidA) gene. Upon recombination, as proven by Southern blot analysis, a functional gene is restored and its product can be detected by histochemical staining. Therefore, cells in which recombination events occurred, and their progeny, can be precisely localized in the whole plant. Recombination was observed in all plant organs examined, from the seed stage until the flowering stage of somatic plant development. Meristematic recombination events revealed cell lineage patterns. Overall recombination frequencies typically were in the range 10(-6)-10(-7) events/genome. Recombination frequencies were found to differ in different organs of particular transgenic lines.

Arabidopsis↗