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Biomedical subjects

T Hohn

Publications and source records attributed to T Hohn.

At least 19 recordsLinked to original sources
PubMed1996

Interaction between cauliflower mosaic virus inclusion body protein and capsid protein: implications for viral assembly.

The cauliflower mosaic virus (CaMV) inclusion body protein (pVI) is able to specifically interact with the viral capsid precursor protein (pIV). By using the yeast two-hybrid system and a blot assay, the pIV region required for the recognition of pVI was mapped to the lysine-rich domain. This region of only 48 amino acids when fused to dihydrofolate reductase (DHFR) mediated pVI and DNA binding in vitro. Competition experiments confirmed that pVI and DNA bind to the same region of pIV. Since pVI is absent from the mature virus, models are discussed in which pVI plays an accessory role in CaMV assembly, in addition to its function in transactivating translation.

Amino Acid Sequence↗
PubMed1996

Position-dependent ATT initiation during plant pararetrovirus rice tungro bacilliform virus translation.

The expression of the rice tungro bacilliform virus open reading frame I was studied in transiently transfected protoplasts. Expression occurs despite the presence of a long leader sequence and the absence of a proper ATG initiation codon. Translation is initiated at an ATT codon. The efficiency of initiation in rice protoplasts depends strongly on the mechanism by which ribosomes reach this codon. From the effects of scanning-inhibiting structures inserted into different leader regions, it can be deduced that this mechanism is related to the ribosome shunt described for cauliflower mosaic virus 35S RNA. The process delivers initiation-competent ribosomes to the region downstream of the leader and is so precise that only the second of two potential start codons only 12 nucleotides apart is recognized. The ATT codon that is used when it is present downstream of the leader is hardly recognized as a start codon by ribosomes that reach it by scanning.

Base Sequence↗
PubMed1995

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.

Base Sequence↗
PubMed1994

The contribution of AAUAAA and the upstream element UUUGUA to the efficiency of mRNA 3'-end formation in plants.

The requirement for sequence specificity in the AAUAAA motif of the cauliflower mosaic virus (CaMV) polyadenylation signal was examined by saturation mutagenesis. While deletion of AAUAAA almost abolished processing at the CaMV polyadenylation site, none of the 18 possible single base mutations had a dramatic effect on processing efficiency. The effect of replacing all six nucleotides simultaneously varied depending on the sequence used, but some replacements were as detrimental as the deletion mutant. Taken together, these results confirm that AAUAAA is an essential component of the CaMV polyadenylation signal, but indicate that a high degree of sequence variation can be tolerated. A repeated UUUGUA motif was identified as an important upstream accessory element of the CaMV polyadenylation signal. This sequence was able to induce processing at a heterologous polyadenylation site in a sequence-specific and additive manner. The effect of altering the spacing between this upstream element and the AAUAAA was examined; moving these two elements closer together or further apart reduces the processing efficiency. The upstream element does not function to signal processing at the CaMV polyadenylation site if placed downstream of the cleavage site. Analysis of further upstream sequences revealed that almost all of the 200 nt fragment required for maximal processing contributes positively to processing efficiency. Furthermore, isolated far upstream sequences distinct from UUUGUA were also able to induce processing at a heterologous polyadenylation site.

Amino Acid Oxidoreductases↗
PubMed1994

Splicing in a plant pararetrovirus.

Analysis of expression in rice cells of plasmids in which an ATG-less chloramphenicol acetyl transferase ORF was placed in frame with the coding sequence of the pararetrovirus rice tungro bacilliform virus (RTBV) ORF IV, and which contained much of the upstream full-length transcript sequence of RTBV, gave evidence to suggest that a splicing event occurred. Reverse transcription/PCR of RNA from transfected protoplasts and from RTBV-infected plants yielded a product which confirmed that the 5' end of ORF IV was spliced in frame to a short ORF (sORF) in the RTBV leader sequence removing an intron of about 6.3 kb. Most of the translation is initiated at the ATG codon in the sORF with only about 10% at the ORF IV ATG codon. The efficiency of splicing appears to be inversely related to the length of the intron. The finding of splicing in a pararetrovirus blurs the differences between them and retroviruses but is in accord with the hypothesis that retroelements acquire genes and sequences which adapt them to specific niches.

Base Sequence↗
PubMed1994

Rice tungro bacilliform virus: transcription and translation in protoplasts.

Protoplasts from cell suspension cultures of Oryza sativa (monocot) and Orychophragmus violaceus (dicot) support transcription from the rice tungro bacilliform virus (RTBV) promoter and translation of the resulting mRNA despite the presence of a long leader sequence with strong secondary structure and 12 short open reading frames. Transcriptional elements located both upstream and downstream of the transcription initiation site are defined by deletion analysis and the functional TATA motif is determined. Expression of an open reading frame downstream of the entire leader is more efficient than that downstream of truncated derivatives. For optimal expression sequences in the 5' and 3' parts of the leader are required.

Base Sequence↗
PubMed1993

Nonlinear ribosome migration on cauliflower mosaic virus 35S RNA.

Cauliflower mosaic virus 35S RNA contains a 600 nt leader with several small open reading frames that by themselves inhibit translation of downstream coding regions. In the context of the whole leader and in certain plant cells, however, translation of downstream coding regions is allowed. This translation is dependent on the RNA 5' terminus and other elements of the leader. However, its central portion is dispensable or can be modified by insertion of an energy-rich stem-loop structure or long coding region with many internal AUG codons. We conclude that this region can be by-passed (shunted) by the scanning complex. Shunting was also observed in trans between two separate RNA molecules.

Base Sequence↗
PubMed1993

Molecular dissection of the cauliflower mosaic virus translation transactivator.

The cauliflower mosaic virus (CaMV) transactivator (TAV) is a complex protein that appears to be involved in many aspects of the virus life cycle. One of its roles is to control translation from the polycistronic CaMV 35S RNA. Here we report a molecular dissection of TAV in relation to its ability to enhance dicistronic translation in transient expression experiments. We have identified a protein domain that is responsible and sufficient for that activity. This 'MiniTAV domain' consists of only 140 of the 520 amino acids in the full-length sequence. A further domain located outside the MiniTAV, and therefore dispensable for transactivation, is probably involved in interactions with other molecules. This was identified by its ability to compete with wild-type TAV and some of its deletion mutants. We found, furthermore, that the TAV protein binds RNA. Two regions needed for RNA-binding properties were defined outside the MiniTAV domain and RNA binding seems not to be directly involved in the transactivation mechanism.

Base Sequence↗
PubMed1993

Transcription from the CaMV 19 S promoter and autocatalysis of translation from CaMV RNA.

The 19 S promoter of cauliflower mosaic virus was analyzed in host protoplasts. In spite of its weakness, it contains a fully functional core promoter. It can be strongly activated by 35 S enhancer elements, even when these elements are located downstream of it, comparable to the situation in the viral genome. The 19 S promoter also contains an element that can strongly enhance expression from a heterologous core promoter. A plasmid expressing the same CAT ORF from two overlapping transcription units, a dicistronic one under control of the 35 S promoter and a monocistronic one under control of the 19 S promoter, was constructed. While in the absence of the virus ORF VI product (pVI, "transactivator") only low levels of CAT activity deriving from the 19 S promoter were observed, in the presence of this protein high levels of CAT activity derived from the 35 S unit were observed in addition. This suggests autocatalytic activation of pVI expression during virus infection.

Catalysis↗
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↗