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T Hohn

Publications and source records attributed to T Hohn.

At least 37 records · Page 2Linked to original sources
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

A dissection of the cauliflower mosaic virus polyadenylation signal.

Mutagenesis analysis of the polyadenylation [poly(A)] signal from the cauliflower mosaic virus (CaMV), a plant pararetrovirus, revealed striking differences to known vertebrate poly(A) signals. Our results show that (1) the AATAAA sequence is necessary for efficient cleavage at the poly(A) site, although the requirement for an authentic AATAAA might be less stringent in plant than in vertebrate cells; (2) surprisingly and in contrast to the majority of vertebrate poly(A) signals, the sequences downstream of the CaMV poly(A) site do not influence processing efficiency drastically although they affect the precision of cleavage; and (3) deletion of sequences upstream of the CaMV AATAAA sequence decreased processing at the CaMV site dramatically, suggesting the presence of one or several positively acting upstream elements. An oligonucleotide consisting of CaMV upstream sequences could induce the recognition of a normally silent exogenous poly(A) signal when inserted upstream of its AATAAA motif.

Base Sequence↗
PubMed1990

Proximity to the promoter inhibits recognition of cauliflower mosaic virus polyadenylation signal.

The replication of retroviruses and pararetroviruses (such as caulimo- and hepadnaviruses) involves the production of terminally redundant genomic-length RNA. The sequence repeated at both ends of the RNA (the R region) contains a polyadenylation signal, and for production of full-length RNA the version of this at the 5' end of the template must be bypassed by RNA polymerase, but the version at the 3' end must be recognized. This implies that the position of the polyadenylation signal determines its efficiency, and we report here experiments aimed at investigating the basis of this phenomenon. Our results with cauliflower mosaic virus suggest that proximity to the transcription initiation site inhibits messenger RNA 3'-end processing directed by polyadenylation signals.

DNA, Recombinant↗
PubMed1990

The reverse transcriptase gene of cauliflower mosaic virus is translated separately from the capsid gene.

Cauliflower mosaic virus (CaMV) possesses start codons at the beginning of its reverse transcriptase (RT) gene (ORF V) suggesting that, unlike in retroviruses and retrotransposons, it is translated independently from the capsid gene (ORF IV). To test this hypothesis a mutational analysis of the CaMV ORF IV/V overlapping region was performed. Mutants in which both ORFs are separated by stop codons in all three reading frames are viable and stable, while mutations affecting the first two AUG codons of ORF V are either lethal or unstable, giving rise to true and second site reversions. Mutants in which the AUG codons were replaced by ACG or AAG reverted only slowly and ACG could direct the synthesis of small amounts of reporter enzyme in transfected plant protoplasts, showing that this codon can act in plant cells as a weak start codon. CaMV has apparently developed a strategy for translation of the RT gene different from that in retroviruses and retrotransposons, but similar to that of hepadnaviruses, another group of pararetroviruses. The separate translation of the RT gene as a common feature of pararetroviruses might reflect the difference in their life cycle in comparison with retroviruses.

Base Sequence↗
PubMed1990

Positive and negative control of translation by the leader sequence of cauliflower mosaic virus pregenomic 35S RNA.

We have studied the influence of the 600 nt long leader sequence of cauliflower mosaic virus 35S RNA on downstream translation. Plant protoplasts were transfected with plasmids expressing a CAT reporter gene from a mRNA, containing wild-type or mutant forms of the 35S RNA leader. Deletion analysis revealed the presence of three separate stimulatory sequence regions, S1, S2 and S3. The latter two interact with each other to enhance downstream translation 5- to 10-fold. This enhancement was not observed in protoplasts from a non-host plant. In the absence of either S2 or S3, the region I2, located in between, exerts an inhibitory effect on downstream translation, probably due to the presence of short open reading frames. Expression of a reporter gene inserted into I2 increases 2-fold upon deletion of either S2 or S3. We propose that mRNA regions S2 and S3 form a complex with cellular factors that allows scanning ribosomes to bypass region I2.

Base Sequence↗
PubMed1990

Open reading frame VIII is not required for viability of cauliflower mosaic virus.

Open reading frame (ORF) VIII of cauliflower mosaic virus (CaMV) was analyzed by site-directed mutagenesis in order to investigate its potential function for the viral life cycle. Removal of either the start or the stop codon of ORF VIII, as well as interruption of ORF VIII by a new stop codon, did not affect infectivity. Unlike certain ORF VII mutants all three ORF VIII mutants are stable. Hence the ORF VIII product is not essential and ORF VIII mutations do not have deleterious polar effects on the expression of the downstream ORF V, which codes for the viral protease/reverse transcriptase.

Codon↗
PubMed1989

Posttranscriptional trans-activation in cauliflower mosaic virus.

The ability of plant cells to translate dicistronic mRNAs that mimic a segment of the polycistronic 35S RNA from cauliflower mosaic virus has been tested. The chloramphenicol acetyltransferase and beta-glucuronidase open reading frames (ORFs) were fused in-frame to the second viral cistron (ORF I). Efficient reporter expression from the corresponding plasmids in plant protoplasts was observed only upon cotransfection with viral DNA. The trans-activating gene maps at ORF VI, which is expressed from a separate, monocistronic messenger (19S RNA). Deletion analysis shows that trans-activation selectively enhances downstream gene expression; the high expression of the upstream ORF is not further increased. The major reporter transcript remained bicistronic upon trans-activation, and its abundance varied only to a limited extent. Results indicate that trans-activation enhances the translation of downstream ORFs on polycistronic mRNAs derived from cauliflower mosaic virus.

Base Sequence↗
PubMed1989

Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed.

Amino Acid Sequence↗
PubMed1989

Differential inhibition of downstream gene expression by the cauliflower mosaic virus 35S RNA leader.

The effect of the 600 nucleotide-long CaMV 35S RNA 5' leader sequence on the expression of downstream genes was analyzed both in plant protoplasts and in vitro. For transient expression studies in protoplasts derived from host and nonhost plants, the bacterial chloramphenicol acetyl transferase (CAT) gene was fused to the initiation codon of ORF VII. The leader sequence reduced CAT expression two- to four-fold in protoplasts derived from three host species, but 10- to 50-fold in protoplasts derived from three different nonhost species. For in-vitro studies the 35S promoter was replaced by the SP6 promoter. The leader reduced in-vitro translation of SP6 transcripts approximately six-fold, indicating that at least part of the inhibition observed in protoplasts is directly due to the interference of the leader sequence with translation. Other steps in gene expression that may also be affected are discussed.

Chloramphenicol O-Acetyltransferase↗
PubMed1989

The expression, localization, and effect of a human interferon in plants.

The ORF II of Cauliflower mosaic virus (CaMV) DNA was replaced with the human IFN alpha D coding sequence to yield a stable CaMV strain designated Ca524i. Inoculation of turnip (Brassica rapa cv "Just Right") with strain Ca524i DNA excised from plasmid pCa524i resulted in the production of biologically active IFN alpha D in infected plants. This was also true for its mutant (Ca562i) where one of the CYS codons was deleted. IFN alpha D produced in planta did not hamper superinfection with a single-stranded (+) sense RNA plant virus, turnip yellow mosaic virus (TYMV). Antibody gold labeling techniques and electron microscopy of infected plants showed that IFN was localized in the CaMV inclusion bodies.

Cell Compartmentation↗
PubMed1988

The leading sequence of caulimovirus large RNA can be folded into a large stem-loop structure.

The 600 nt long sequences preceeding the first large ORFs (ORF VII) of three caulimoviruses, although varying in primary sequence, can be folded into a large stem/loop structure centered around a conserved stretch of 36 nucleotides. Deletions of the conserved sequence delay symptom appearance considerably, but do not affect expression of a reporter gene in plant protoplasts. Another striking similarity between the leaders concerns the number and distribution of small open reading frames (sORF) they carry. Expression of two of these sORFs was tested by fusion of a reporter gene: both were expressed in plant protoplasts.

Base Sequence↗
PubMed1988

The instability of a recombinant plasmid, caused by a prokaryotic-like promoter within the eukaryotic insert, can be alleviated by expression of antisense RNA.

A region of the cauliflower mosaic virus genome was found to direct the expression of a nucleic-acid-binding protein in Escherichia coli. This protein is apparently toxic for the bacteria and leads to a destabilization of plasmids containing that region. Antisense RNA was used to diminish the unwanted expression and to stabilize the respective recombinant plasmids. The approach described may prove useful in other cases where problems with cloning of eukaryotic DNA arise.

DNA Restriction Enzymes↗
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↗
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

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↗
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↗