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R J Shepherd

Publications and source records attributed to R J Shepherd.

At least 19 recordsLinked to original sources
PubMed1998

Isolation and expression analysis of peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) promoter in transgenic plants.

A promoter fragment from peanut chlorotic streak caulimovirus (PClSV) full-length transcript (FLt) was identified and later modified to have duplicated enhancer domain. The FLt promoter with its single or double enhancer domains, fused with the GUS reporter gene to form chimeric gene constructs, showed a high level of expression of these genes in cells and transgenic plants. The FLt promoter with its double enhancer domain gives an average threefold greater expression of genes compared to the FLt promoter with its single enhancer domain in transgenic plants. In young seedlings the expression was in the order root > leaf > stem. The histochemical GUS assay in young seedlings showed more activity in root tips and leaf midribs, veins, and other vascular tissues. The expression from the PClSV FLt promoter was compared with that from the figwort mosaic virus promoter in transgenic plants. These constitutive promoters were comparable in respect to GUS expression level.

Caulimovirus↗
PubMed1998

Gene expression regulated by gene VI of caulimovirus: transactivation of downstream genes of transcripts by gene VI of peanut chlorotic streak virus in transgenic tobacco.

Here we document that the gene VI product of peanut chlorotic streak virus (PClSV), a newly characterized member of the group, transactivates the translation of dicistronic transcripts. Dicistronic expression units have been analyzed both in protoplast transient expression experiments and in transgenic tobacco plants. Transgenic plants containing a dicistronic transcription unit (PClSV-gene VII-GUS) under the control of PClSV full-length transcript promoter with its long leader sequence show a relatively high abundance of the expected transcript but very little, or no, GUS activity. However, high GUS activity is found when gene VI protein is then provided by subsequent infection with PClSV. The efficient translation of polycistronic mRNAs mediated by gene VI of caulimovirus has potential value in product engineering of plants.

Base Sequence↗
PubMed1997

Petunia vein-clearing virus: a plant pararetrovirus with the core sequences for an integrase function.

Petunia vein-clearing virus (PVCV) is a plant pararetrovirus that has some features of retrotransposons. It encapsidates dsDNA and has isometric particles and inclusion bodies similar to those of caulimoviruses. The PVCV genome of 7205 bp has two large ORFs in the transcribed strand and a methionine tRNA primer-binding site in its 663-bp intergenic region. The N-terminal position of the large protein (126 kDa) encoded by ORF I has similarity to the movement protein of caulimoviruses. Toward the C-terminus of this same polyprotein are the two distinctive sequence elements [HHCC and DD(35)E] of the integrase function of retroviruses and retrotransposons. ORF II of PVCV encodes a protein of 125 kDa with domains for an RNA-binding element, common to the gag gene of retroelements, followed by consensus sequences for an acid protease, reverse transcriptase, and ribonuclease H. Hence, the gag equivalent (capsid protein) and pol gene of PVCV are part of the same polyprotein. Phylogenetic comparison of the reverse transcriptase of PVCV with that of various other retroelements grouped PVCV between caulimoviruses and the Ty3/gypsy retrotransposons, suggesting that PVCV is a divergent member of the caulimoviruses.

Amino Acid Sequence↗
PubMed1997

Promoter/leader deletion analysis and plant expression vectors with the figwort mosaic virus (FMV) full length transcript (FLt) promoter containing single or double enhancer domains.

The boundaries required for maximal expression from the promoter/leader region of the full length transcript of figwort mosaic virus (FLt promoter) coupled to reporter genes were defined by 5' and 3' deletion analyses. In transient expression assays using protoplasts of Nicotiana edwardsonii, a 314 bp FLt promoter fragment sequence (-249 to +65 from the transcription start site) was sufficient for strong expression activity. Plant expression vectors developed with modified FLt promoters were tested with GUS or CAT as reporter genes in transgenic plants. The FLt promoter is a strong constitutive promoter, with strength comparable to or greater than that of the CaMV 35S promoter. The FLt promoter with its double enhancer domain linked to GUS or CAT reporter genes provides an average 4-fold greater activity than the FLt promoter with a single enhancer domain (-55 to -249 bp upstream fragment) in tests with transgenic plants and in protoplast transient expression assays.

Base Sequence↗
PubMed1997

Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus.

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.

Caulimovirus↗
PubMed1997

A case of relapsing psittacosis associated with a stroke.

Psittacosis, caused by Chlamydia psittaci, is mainly associated with an atypical pneumonia. We report a case of psittacosis where onset of respiratory symptoms was soon followed by the onset of focal neurological signs, a very uncommon feature. Despite adequate treatment with erythromycin, the patient relapsed when re-exposed to a sick pet bird. A prolonged course of doxycycline led to resolution of the pneumonia, but it is important to recall that successful management must also include the elimination of the precipitating cause of the infection.

Aged↗
PubMed1996

Efficient translation of distal cistrons of a polycistronic mRNA of a plant pararetrovirus requires a compatible interaction between the mRNA 3' end and the proteinaceous trans-activator.

Caulimoviruses, a type of plant pararetrovirus, employ a highly unusual mechanism to express the multiple cistrons of their pregenomic RNA. It involves translation of a polycistronic mRNA utilizing cis-acting viral RNA sequences and a transacting virus-encoded protein (P6). In addition to its role in polycistronic translation, the translational trans-activator protein P6 also activates its own expression from a monocistronic subgenomic RNA. Using Nicotiana Edwardsonii cell suspension protoplasts, we analyzed the ability of P6 proteins from three different caulimoviruses to activate viral RNA-based reporter constructs. Cis-acting elements present in figwort mosaic caulimovirus (FMV) are functional not only in the presence of the cognate P6 activator protein, but also in the presence of the heterologous activators from cauliflower mosaic caulimovirus (CaMV) and peanut chlorotic streak caulimovirus (PCISV). However, when 3' cis-acting elements essential for efficient polycistronic expression of FMV are replaced by their counterparts from PCISV, reporter gene expression is only observed in the presence of PCISV P6. Derepression of monocistronic reporter constructs tailed with FMV or CaMV 3' proximal sequences is less efficient in the presence of PCISV P6 than with either FMV or CaMV P6, but more efficient when the constructs contain a cognate PCISV 3' cis-element. Efficient expression of polycistronic and monocistronic caulimovirus mRNAs in plant cells thus requires compatible interactions between P6, a translational trans-activator, and its cognate cis-element at the 3' end of the mRNA.

Caulimovirus↗
PubMed1996

In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model.

Animals↗
PubMed1996

An atypical case of atypical pneumonia.

A 23-year-old man, previously fit and well, presented with an atypical pneumonia, associated with microangiopathic anaemia, thrombocytopenia, rhabdomyolysis and renal impairment. Despite administration of intravenous fluids and antibiotics, his condition rapidly deteriorated, and the possibility of an aggressive connective tissue disorder was raised. Thus he was treated with high-dose oral steroids and plasma exchange until autoantibodies were shown to be negative. At this stage it transpired that the patient had swallowed water from a stream three weeks earlier, and leptospira antibody titres were subsequently found to be elevated. Antibiotics were continued, and after a protracted course he made a full recovery. Leptospirosis should be remembered as a rare cause of atypical pneumonia, particularly if there is associated hepatic or renal impairment.

Adult↗
PubMed1995

Molecular analysis of the essential and nonessential genetic elements in the genome of peanut chlorotic streak caulimovirus.

The DNA genome of caulimoviruses contains a set of essential genes: I (movement gene), IV (major capsid protein gene), V (reverse transcriptase gene), and VI (gene coding for a post-transcriptional activator of the expression of other virus genes). In peanut chlorotic streak caulimovirus (PCISV), three ORFs, A, B, and C, are located between genes I and IV. They are dissimilar to other caulimovirus ORFs. ORF VII of PCISV is a homolog of ORF VII of soybean chlorotic mottle caulimovirus (SoCMV), but is not similar to the nonconserved ORF VII in other caulimoviruses. The sequence complementary to a portion of tRNA(Met), thought to be essential for the priming of minus-strand DNA synthesis in caulimoviruses, is located within the coding sequence of ORF A. To explore the functional significance of ORFs VII, A, B, and C, various mutations were engineered into an infectious DNA clone of PCISV. ORFs VII and B are shown to be dispensable, while ORFs A and C are essential. ORF C is a possible functional equivalent of gene III in other caulimoviruses. Sequences within ORF A that are required for efficient priming of minus-strand synthesis are likely to extend beyond the 12-bp tRNA-binding site. Complete deletion of ORF VII was correlated with severe symptoms, notably with the necrosis of apical meristems. Significance of these observations for the understanding of replication and pathogenesis of plant pararetroviruses and for the improvement of caulimovirus-based expression vectors is discussed.

Arachis↗
PubMed1995

Characterization of cassava vein mosaic virus: a distinct plant pararetrovirus.

Cassava vein mosaic virus (CVMV) was found to be widespread throughout the north-eastern region of Brazil. The complete sequence of CVMV was determined, and the genome was 8158 bp in size. A cytosolic initiator methionine tRNA (tRNA met1)-binding site that probably acts as a primer for minus-strand synthesis was present. The genome contained five open reading frames that potentially encode proteins with predicted molecular masses of 186 kDa, 9 kDa, 77 kDa, 24 kDa and 26 kDa. The putative 186 kDa protein had regions with similarity to the zinc finger-like RNA-binding domain that is a common element in the capsid proteins and similarity to the intercellular transport domain of the plant pararetroviruses. The predicted 77 kDa protein had regions with similarity to aspartic proteases, reverse transcriptase and RNase H of pararetroviruses. This gene order was confirmed by the amplification of similar PCR products from total DNA extracted from CVMV-infected cassava plants. The genomic organization of CVMV was different from the organization of either the caulimoviruses or badnaviruses. In comparisons of the regions with the reverse transcriptase motif, CVMV was grouped between the caulimoviruses and badnaviruses. It appears that CVMV is distinct from the other well-characterized plant pararetroviruses.

Amino Acid Sequence↗
PubMed1995

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants.

Amino Acid Sequence↗
PubMed1995

Genetic elements of plant viruses as tools for genetic engineering.

Viruses have developed successful strategies for propagation at the expense of their host cells. Efficient gene expression, genome multiplication, and invasion of the host are enabled by virus-encoded genetic elements, many of which are well characterized. Sequences derived from plant DNA and RNA viruses can be used to control expression of other genes in vivo. The main groups of plant virus genetic elements useful in genetic engineering are reviewed, including the signals for DNA-dependent and RNA-dependent RNA synthesis, sequences on the virus mRNAs that enable translational control, and sequences that control processing and intracellular sorting of virus proteins. Use of plant viruses as extrachromosomal expression vectors is also discussed, along with the issue of their stability.

DNA Replication↗
PubMed1993

The putative zinc finger of a caulimovirus is essential for infectivity but does not influence gene expression.

Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

Amino Acid Sequence↗
PubMed1992

Regulation of caulimovirus gene expression and the involvement of cis-acting elements on both viral transcripts.

In a further analysis of gene regulation of figwort mosaic virus (FMV), a caulimovirus, we studied transient gene expression with modified viral genomes in Nicotiana edwardsonii cell suspension protoplasts. The results demonstrated that the presence of the promoter for the full-length RNA interferes with expression from the separate downstream promoter for gene VI. In addition, expression of gene VI was inhibited by cis-acting sequences within gene VI itself. Both inhibitory effects could be partially relieved by coelectroporation with a plasmid that produces gene VI protein, demonstrating that expression of gene VI is transactivated by its own product. Subsequent expression studies with partially redundant FMV plasmids containing a reporter gene in frame with gene IV showed that efficient transactivation of CAT expression relies on a cis-acting element inside the downstream gene VI. Insertions of a transcriptional terminator upstream of the cis-acting element for premature termination of transcription showed that the cis-acting region is not a DNA element but is active only as a feature of the RNA transcript. We conclude that the cis-acting element, together with the transacting gene VI product, enhances expression of all major genes, including gene VI, from the polycistronic mRNA and the separate mRNA for gene VI.

Enhancer Elements, Genetic↗
PubMed1992

The full-length transcript of a caulimovirus is a polycistronic mRNA whose genes are trans activated by the product of gene VI.

Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

Cells, Cultured↗
PubMed1991

Requirement of gene VII in cis for the expression of downstream genes on the major transcript of figwort mosaic virus.

The six major conserved genes of figwort mosaic virus (FMV), a caulimovirus, appear in tandem array on an RNA transcript that spans the entire viral genome. Gene VI, the only cistron that appears as a separate subgenomic RNA, has been reported to transactivate the expression of downstream genes of the full-length transcript. This transcript has a long 5'-leader of about 600 nucleotides followed by a small nonconserved region (gene VII), a smaller intergenic region (57 nucleotides), and the major conserved genes in a closely spaced array. In our present experiments we have constructed expression units containing the promoter for the full-length transcript followed by the 5' leader region, gene VII, and a reporter gene. These have been tested for expression with and without gene VI as a separate plasmid by electroporation into plant protoplasts. A series of these expression units containing truncated versions of the 5' leader region placed upstream of a reporter gene (CAT) showed that gene VI transactivation occurred only when gene VII sequences were present in cis between the leader region and the reporter gene. In addition, a more complete version of the FMV genome containing the reporter gene further downstream (in viral gene IV) showed CAT expression only when gene VII sequences were present in an upstream position. A similar construct failed to express CAT activity when gene VII was absent.

Chloramphenicol O-Acetyltransferase↗