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B Hohn

Publications and source records attributed to B Hohn.

At least 73 records · Page 4Linked to original sources
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

Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer.

A system for the analysis of independent T-DNA transfer events from Agrobacterium to plants is described. The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant, a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to the ends of T-DNA molecules. A rather conserved right border remnant of three nucleotides was found, whereas the sequences remaining at the left end were more variable. A point deletion in the left 25 bp sequence results in even less precise processing at the left end. In addition, many rescued T-DNA molecules carry small direct repeats between the joined T-DNA ends; linear T-DNA molecules are therefore transported to the plant.

Base Sequence↗
PubMed1989

DNA transfer from Agrobacterium to Zea mays or Brassica by agroinfection is dependent on bacterial virulence functions.

DNA transfer from Agrobacterium tumefaciens, a soil bacterium, to the non-host graminaceous monocotyle-donous plant Zea mays, was analysed using the recently developed technique of agroinfection. Agroinfection of Z. mays with maize streak virus using strains of A. tumefaciens carrying mutations in the pTiC58 virulence region showed an almost absolute dependence on the products of the bacterial virC genes. In contrast, agroinfection of the control host Brassica rapa with cauliflower mosaic virus was less dependent on the virC gene products. In other respects, the basic mechanism of the plant-bacterium interaction was found to be similar. While intact virA, B, D and G functions were absolutely necessary, mutants in virE were attenuated. Agroinfection of maize was effective in the absence of an exogenously supplied vir gene inducer, and indeed wounded Z. mays tissues were found to produce substance(s) which induced the expression of A. tumefaciens vir genes. These findings are discussed in the light of current knowledge about the function of Agrobacterium vir genes.

DNA, Bacterial↗
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↗
PubMed1988

Cosmids.

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Cloning, Molecular↗
PubMed1988

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.

Aerobiosis↗
PubMed1987

Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA.

Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.

Base Sequence↗
PubMed1986

Recombination in a plant virus: template-switching in cauliflower mosaic virus.

A hybrid plasmid, containing tandemly arranged pieces of two different but well-defined cauliflower mosaic virus (CaMV) genomes, was used to study the mechanism by which infectious viral DNA can escape from transforming DNA. Systemic viral infection followed inoculation of Brassica plants with a strain of Agrobacterium tumefaciens containing the hybrid plasmid in its T-DNA. Restriction mapping of uncloned viral DNA from these plants, and sequencing of relevant portions of cloned viral DNA, showed that the majority of viral progeny were probably descendants of DNA produced by transcription/reverse transcription of the viral genome, thus providing further evidence for the hypothesis that this process is normally involved in viral replication. The reverse transcriptase enzyme, which is thought to undergo an intramolecular template switch during viral replication, is shown to move very close to the 5' end of the terminal repeat on the 35S RNA molecule before switching templates. The remaining minority of viral genomes can best be explained as arising from products of recombination between homologous regions of CaMV DNA.

Journal Article↗
PubMed1986

Splicing of an intervening sequence from hybrid cauliflower mosaic viral RNA.

A DNA sequence encompassing intervening sequence 2 of the soybean leghemoglobin gene was inserted into the cauliflower mosaic virus (CaMV) genome. Upon passage through host plants progeny virus slowly accumulated that had lost the intron, apparently by exact splicing of the RNA form of the genome at the exon/intron borders. These findings suggest that reverse transcription from (spliced) RNA to DNA is a normal step in CaMV replication. S1 nuclease mapping revealed a mixed population of unspliced and spliced viral RNAs. The inefficiency of the splicing process is discussed.

Journal Article↗
PubMed1986

"Agroinfection," an alternative route for viral infection of plants by using the Ti plasmid.

Most plant viruses are transmitted by insect vectors. We present an alternative method for the introduction of infectious viral DNA that uses the ability of Agrobacterium to transfer DNA from bacterial cells to plants. Cauliflower mosaic virus was chosen to develop this method because it is the best characterized plant DNA virus and can be introduced into plants via aphids, virus particles, viral DNA, or suitably treated cloned DNA. We show that systemic infection of turnips results from wounding and inoculation with strains of Agrobacterium tumefaciens in which more than one genome of cauliflower mosaic virus have been placed tandemly in the T-DNA of the tumor-inducing plasmid. Thus such constructions allow escape of the viral genome from the T-DNA once inside the plants. The combined use of the tumor-inducing plasmid and viral DNA opens the way to molecular biological approaches that are not possible with either system alone.

Journal Article↗
PubMed1984

Direct gene transfer to plants.

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F(1) generation.

Journal Article↗
PubMed1984

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Carcinoma↗