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T Hohn

Publications and source records attributed to T Hohn.

At least 19 recordsLinked to original sources
PubMed1992

Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.

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

Transcriptional and translational control of gene expression in cauliflower mosaic virus.

Cauliflower mosaic virus sequences have developed as a powerful tool for the study of various aspects of gene expression in plants. Analysis of the promoter/enhancer region has led to the discovery of several transcription factors and factor-binding sites. Studies on RNA processing and polyadenylation reveal a viral strategy to obtain terminal redundancy of retrovirus pregenomic RNA. Striking differences between plant and vertebrate polyadenylation signals have been disclosed. The mechanisms for translation of the polycistronic 35S RNA are novel in the eukaryotic field and may give new insight to translational control in general.

Base Sequence↗
PubMed1992

Efficient initiation of translation at non-AUG triplets in plant cells.

The efficiency of translation initiation at triplets differing at one residue from AUG was tested by transient expression in protoplasts from two different plant species. All possible alternative codons were tested. Some triplets showed significant CAT activity, with CUG (30% of the AUG activity) being most active. Most others had between 5 and 15% of the activity obtained from AUG, whereas UUG and AUC yielded about 2% and the two composed only of purines, AAG and AGG, had no significant activity. Translation initiation from AUC, especially, responded to leader sequences outside the immediate context which did not affect translation initiation from AUG.

Base Sequence↗
PubMed1991

Translation of a polycistronic mRNA in the presence of the cauliflower mosaic virus transactivator protein.

Polycistronic mRNAs containing an upstream beta-glucuronidase (GUS) and a downstream chloramphenicol acetyltransferase (CAT) reporter open reading frame (ORF) were expressed in transfected plant protoplasts. CAT expression could be strongly induced by coexpression of the cauliflower mosaic virus encoded translation transactivator. Transactivation was abolished when an upstream ORF overlapped the CAT ORF for a long distance. No specific sequence elements were required for transactivation but the presence of a short ORF upstream of the GUS ORF strongly enhanced the process. The inhibitory effect of additional presumed stem structures inserted into various regions of the reporter mRNAs indicates that both ORFs are translated by ribosomes that associate with the RNA at the 5' end and reach the ORFs by a linear migration mechanism.

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

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 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↗