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J Fütterer

Publications and source records attributed to J Fütterer.

32 records · Page 2Linked to original sources
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

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

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↗
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↗
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

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↗
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

In vitro expression of cauliflower mosaic virus genes.

The eight major open reading frames (ORFs) of cauliflower mosaic virus (CaMV) have been cloned for in vitro transcription and translation. All the ORFs could be translated. Using antisera against either purified virus or specific gene products, the translation products were screened by immunoprecipitation. The products of ORFs III, IV and V were confirmed as components of the virions. Molecular weights of primary translation products were determined and compared with those found in vivo. A further series of constructs was designed to test whether translation of adjacent ORFs is coupled in a relay-race fashion as proposed on the basis of earlier in vivo mutagenesis studies. Downstream genes on dicistronic RNAs could be translated, although inefficiently. In view of the similarity between the arrangement of the CaMV coat protein and reverse transcriptase genes and the corresponding genes of retroviruses, we asked whether the CaMV reverse transcriptase could be expressed in vitro as a fusion protein, e.g. by ribosomal frame shifting. No such fusion was observed, suggesting that the polymerase gene is translated from its own ATG.

Journal Article↗
PubMed1980

Initiation of adenovirus DNA replication.

In an attempt to study the mechanism of initiation of adenovirus DNA replication, an assay was developed to investigate the pattern of DNA synthesis in early replicative intermediates of adenovirus DNA. By using wild-type virus-infected cells, it was possible to place the origin of adenovirus type 2 DNA replication within the terminal 350 to 500 base pairs from either of the two molecular termini. In addition, a variety of parameters characteristic of adenovirus DNA replication were compared with those obtained in a soluble nuclear extract competent for viral DNA replication. It was observed that in vitro DNA replication, which is dependent on the exogenously added viral DNA-protein complex as its optimal template, occurs in a manner apparently indistinguishable from the situation in virus-infected cells. This includes the presence of proteinaceous material on the molecular termini of newly initiated viral DNA.

Adenoviruses, Human↗