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T Hohn

Publications and source records attributed to T Hohn.

At least 91 records · Page 5Linked to original sources
PubMed1988

A viable mutation in cauliflower mosaic virus, a retroviruslike plant virus, separates its capsid protein and polymerase genes.

A viable strain of cauliflower mosaic virus is described which arose by illegitimate recombination of two lethal parents. In this strain, the normally overlapping open reading frames IV and V, corresponding to the retrovirus gag and pol genes, are separated by a short intergenic region, suggesting that in this virus and in contrast to retroviruses, fusion of gag and pol gene products is not obligatory.

Base Sequence↗
PubMed1987

Translation products of cauliflower mosaic virus ORF V, the coding region corresponding to the retrovirus pol gene.

Open reading frame (ORF) V of cauliflower mosaic virus (CaMV), the candidate for the reverse transcriptase gene, has been expressed in E. coli under control of the PR promoter of bacteriophage lambda either as an N-terminal polypeptide fused to beta-galactosidase or as the total ORF V without fusion. Antibodies against these proteins were used to analyze extracts from CaMV-infected plants by immunoblotting. ORF V-specific polypeptides of 80, 62, 58, 22, and 18 kD apparent molecular weights were detected, with the largest species corresponding to the full length translation product. The 62 and 22 kD species could be assigned to the N-terminus and the remaining two species to the C-terminus of the ORF.

Bacteriophage lambda↗
PubMed1987

Cauliflower mosaic virus coat protein is phosphorylated in vitro by a virion-associated protein kinase.

A protein kinase has been found to be associated with particles of the plant virus cauliflower mosaic virus. This protein kinase can phosphorylate endogenous viral capsid proteins in vitro and exchange substrates with casein kinase type II. The activity is not affected by cAMP but is enhanced considerably by ADP. The cofactor is either Mn(2+) or Mg(2+), and the phosphate donor is either ATP or GTP. Serine and threonine residues are phosphorylated.

Journal Article↗
PubMed1986

Expression in plants of two bacterial antibiotic resistance genes after protoplast transformation with a new plant expression vector.

Two bacterial antibiotic resistance genes, one coding for the neomycin phosphotransferase (NPT I) from Tn903, and the other coding for the chloramphenicol acetyltransferase from Tn9 were used as plant selectable markers. Both genes were introduced into the Nicotiana tabacum genome in a new plant expression vector, using the direct gene transfer method. The vector pDH51, used in these experiments contains a plant expression unit as a movable cassette, consisting of the strong cauliflower mosaic virus (CaMV) 35S RNA promoter and transcription terminator separated by a polylinker containing several unique restriction sites.

Acetyltransferases↗
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

Oligonucleotide directed mutagenesis of cauliflower mosaic virus DNA using a repair-resistant nucleoside analogue: identification of an agnogene initiation codon.

Mutation of the initiation codon of the dispensible open reading frame, ORF VII, of cauliflower mosaic virus (CaMV) delayed the appearance of disease symptoms, but the mutants reverted with high frequency. This suggests a role of this start codon in viral expression. Oligonucleotide-directed mutagenesis, utilizing a novel, repair-resistant deoxyguanosine analogue, 2'-deoxy-7-deazainosine (dDI), highly improved the yield of mutants.

Base Sequence↗
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↗
PubMed1985

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence.

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.

Cloning, Molecular↗
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

Initiation of translation of the cauliflower mosaic virus genome from a polycistronic mRNA: evidence from deletion mutagenesis.

Mutants with deletions in the open reading frame (ORF) VII to I region of the cauliflower mosaic virus genome were constructed in vitro and tested for infectivity by inoculation of plants. Sequences within ORF VII and the intergenomic region between ORFs VII and I are not required for viral infectivity. Nevertheless, the location of translation initiation and termination codons within this region governs the infectivity of the virus. Firstly, translation beginning at the initiation codon of ORF VII should reach an in-phase termination codon before the initiation codon of ORF I. Secondly, an initiation codon should be located in the vicinity of the sequence homologous to methionine tRNA, the proposed primer binding site for reverse transcription. Results suggest translation from a polycistronic mRNA by a ;relay race' mechanism whereby ribosomes after passing a termination codon re-initiate protein synthesis at the nearest AUG codon.

Journal Article↗
PubMed1984

A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body.

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.

Journal Article↗
PubMed1984

Nucleotide sequence of the dihydrofolate-reductase gene borne by the plasmid R67 and conferring methotrexate resistance.

The complete nucleotide sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R67 was determined. The gene is 234 bp long and codes for 78 amino acids. The polypeptide deduced from the DNA sequence is in perfect agreement with the previously published amino acid sequence. Comparison of the nucleotide sequence with the one determined for the R388-encoded DHFR indicates that 75% of the nucleotides are conserved in the two genes. The 3' end of the R67 gene can be modified without altering significantly the activity of the enzyme.

Amino Acid Sequence↗
PubMed1983

Involvement of reverse transcription in the replication of cauliflower mosaic virus: a detailed model and test of some aspects.

A model is presented according to which cauliflower mosaic virus (CaMV) DNA is replicated via an RNA intermediate. The model explains the typical S1 nuclease-sensitive sites in mature CaMV DNA, the occurrence of the large, terminally redundant transcript, the local separation of CaMV transcription and CaMV assembly, the abundance of knotted CaMV DNA forms, and the high recombination frequency. A site of perfect homology to plant tRNA was detected. Extracts from a mixture of nuclei and inclusion bodies can be separated into fast-sedimenting complexes elongating endogenous CaMV RNA, and slow-sedimenting ones elongating endogenous CaMV DNA. The CaMV DNA synthesis can be partly inhibited both by RNAase and actinomycin D, suggesting the presence of a mixture of RNA- and DNA-templates.

DNA Replication↗
PubMed1983

Mutagenesis of cauliflower mosaic virus.

A series of insertion mutants of cauliflower mosaic virus (CaMV) DNA has been constructed in vitro. These insertions consist of a short DNA sequence (10 or 22 bp) containing a restriction endonuclease site (SmaI) not represented on the viral DNA. Viral infectivity was analyzed by inoculating plants with the mutated cloned viral DNA and observing symptoms. Insertions within ORFVII, and in one site within the large intergenic region, did not interfere with viral infectivity, whilst insertions within ORFII and at the end of ORFIV retarded the development of viral symptoms. All other insertion mutants analyzed were lethal. CaMV with a deletion of 105 bp within ORFVII was viable. Such viable mutants can be used to construct additional deletions or to insert foreign DNA into the viral genome.

Base Sequence↗
PubMed1982

In vivo recombination of cauliflower mosaic virus DNA.

LIGATION AND RECOMBINATION OF THE DNA OF CAULIFLOWER MOSAIC VIRUS (CAMV) IS DEMONSTRATED BY THE FOLLOWING EXPERIMENTS: (i) Ligation: Different noninfectious fragments of the CaMV genome (obtained after insertion into plasmid pBR322 followed by enzymatic excision) regained infectivity when mixtures of them were used to inoculate their host. The symptom appearance was delayed by comparison with a typical CaMV infection, and only the newly formed leaves were affected. (ii) Recombination: Pairs of noninfectious recombinant full-length CaMV genomes (integrated into pBR322 at different restriction endonuclease sites) regained infectivity upon simultaneous inoculation of a sensitive host. The symptomatology of the resulting infection was indistinguishable from that of a typical CaMV infection. We show that progeny DNA had the same characteristics (size, structure, restriction endonuclease digestion pattern) as bona fide CaMV DNA, and that the vector pBR322 had been completely eliminated. A cloned tandem dimer of CaMV DNA with a partial deletion similarly was infectious in the plant assays. This system should be useful to study the expression of mutant genomes, thus allowing characterization of the CaMV genes.

Journal Article↗