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B Hohn

Publications and source records attributed to B Hohn.

At least 19 recordsLinked to original sources
PubMed1992

The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals.

Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece of DNA, called T-DNA, into the nucleus of the plant cell. The virulence protein VirD2 of A. tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome. Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a single-cluster-type nuclear localization signal. The second signal is near the C terminus and is a bipartite-type nuclear localization signal. The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.

Amino Acid Sequence↗
PubMed1992

Mutational analysis of the 'conserved region' of maize streak virus suggests its involvement in replication.

Maize streak virus as well as other geminiviruses contain a potential hairpin structure with the conserved sequence TAATATTAC in the loop. We assessed the possible involvement of this structure in replication and symptom induction of the virus. A series of insertion and deletion mutants were analyzed by agroinfection. Deletion of the hairpin or insertions in the conserved sequence abolished symptom development. Viral DNA could not be detected in the infected tissue. However, a mutant with a point mutation in the 'conserved' sequence, isolated after inoculation of maize plants with an insertion mutant, was able to replicate and to induce symptoms.

Agrobacterium tumefaciens↗
PubMed1992

Mechanism of Ds1 excision from the genome of maize streak virus.

We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or "footprints" left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 bp duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 bp sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.

Base Sequence↗
PubMed1992

Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination.

Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene. The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences. In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants. The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.

Containment of Biohazards↗
PubMed1992

The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

Base Sequence↗
PubMed1992

Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared.

Chromosomes↗
PubMed1992

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.

Agrobacterium tumefaciens↗
PubMed1991

A transient assay in plant cells reveals a positive correlation between extrachromosomal recombination rates and length of homologous overlap.

An assay to monitor homologous recombination in plant cells has been established by cotransfecting Nicotiana plumbaginifolia protoplasts with different topological forms of plasmids of various deletion mutants of a non-selectable marker gene, the beta-glucuronidase (GUS) gene. Transient GUS enzyme activities were measured by a sensitive assay. In the nuclear DNA of the cotransfected protoplasts the recombined complete GUS gene could be detected by a specially modified PCR analysis. In comparison to the standard assay, which monitors homologous recombination by integration of a selectable marker, the described assay avoids position effects of gene expression, is fast, easy to handle and large numbers of samples can be processed simultaneously. We were able to demonstrate a positive correlation between the length of overlapping homology (up to 1200 base pairs) of the transfected supercoiled circular or linearized plasmids and the respective GUS activities. We found a significant drop in the recombination rates when the overlap of both substrates was reduced to 456 basepairs or less. The requirement for such a long stretch of homology for efficient recombination might ensure the stability of the rather repetitive plant genome.

Base Sequence↗
PubMed1991

Genomic homologous recombination in planta.

A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process.

DNA, Viral↗
PubMed1991

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Base Sequence↗
PubMed1991

The mechanism of extrachromosomal homologous DNA recombination in plant cells.

By cotransfecting plasmids carrying particular mutations in the beta-glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.

Base Sequence↗
PubMed1991

Mutational analysis of the small intergenic region of maize streak virus.

Maize streak virus (MSV) is a leafhopper-transmitted geminivirus containing one molecule of circular single-stranded DNA of about 2.7 kb. We have tested the infectivity of MSV mutants. A deletion of 29 bases in the small intergenic region (SIR) did not affect the infectivity of MSV. Mutants containing insertions of oligonucleotides of up to 32 bases at the AsnI site in SIR were also infectious. However, the infection efficiency of the insertion mutants decreased as the size of the oligonucleotides increased. This reduced virulence may be due to a decreased efficiency of 3'-end formation of the complementary sense mRNA(s). The stability of MSV mutants in infected maize plants was analyzed by polymerase chain reaction (PCR). Some oligonucleotide-insertion mutants were completely stable while others obtained deletions. Infectivity of the mutants, however, did not require deletion formation. Deletion rearrangement was shown to be dependent on the sequence of the inserted oligonucleotide. The AsnI site is the only site known to permit insertion of nonviral DNA without abolishing MSV infectivity. The system described may allow the use of MSV as a delivery vector to introduce at least small segments of DNA into maize.

Base Sequence↗
PubMed1989

Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation.

We show that upon induction of Agrobacterium tumefaciens, free linear double-stranded T-DNA molecules as well as the previously described T-strands are generated from the Ti plasmid. A majority of these molecules are bound to a protein. We show that this protein is the product of the virulence gene virD2. This protein was found to be attached to the 5' terminus of processed T-DNA at the right border and to the rest of the Ti plasmid at the left border. The protein remnant after Pronase digestion rendered the right end of the double-stranded T-DNA resistant to 5'----3' exonucleolytic attack in vitro. The protein-DNA association was resistant to SDS, mercaptoethanol, mild alkali, piperidine, and hydroxylamine, indicating that it involves a covalent linkage. The possible involvement of this T-DNA-protein complex in replication, transduction to the plant, nuclear targeting, and integration into the plant nuclear DNA is discussed.

Bacterial Proteins↗