PubMed Health⌕ Search

Biomedical subjects

R J Shepherd

Publications and source records attributed to R J Shepherd.

At least 37 records · Page 2Linked to original sources
PubMed1991

A naturally occurring deletion mutant of figwort mosaic virus (caulimovirus) is generated by RNA splicing.

A naturally occurring deletion mutant is observed in plants infected with figwort mosaic virus (FMV), a caulimovirus. The encapsidated mutant genome is formed spontaneously in association with two different strains of FMV in four host plant species. The mutant also appears when cloned wild-type viral DNA is used as the inoculum. The deletion mutant alone is not infectious and it appears unable to replicate after its formation, even in the presence of wild-type virus. The gene for chloramphenicol acetyltransferase was inserted at different positions in the deletion mutant genome, and subsequent transient assays showed that gene expression of the mutant occurs despite the deletion. Sequence analyses of the mutant genome revealed a deletion of 1237-bp segment encompassing a major portion of the coat protein gene and the 5' end of the downstream reverse transcriptase gene. This deletion is associated with consensus signals for RNA splicing including the conserved 5' and 3' splice sites plus surrounding sequences, putative branch point(s) for lariat formation, and an extremely high adenosine content (41%) of the removed fragment. This suggests that splicing of the FMV full-length transcript has occurred prior to reverse transcription and this accounts for the presence and accumulation of encapsidated DNAs with the same deletion.

Base Sequence↗
PubMed1989

Gene VI of figwort mosaic virus (caulimovirus group) functions in posttranscriptional expression of genes on the full-length RNA transcript.

Experimental evidence for a molecular function for gene VI of the caulimoviruses is presented. Based on experiments with the figwort mosaic virus (FMV), it appears that gene VI has a role in the posttranscriptional expression of the closely packed genes (VII and I-V), which appear on the larger, full-length RNA transcript of this virus. Gene VI with its flanking 5'/3' expression signals included as a separate plasmid during electroporation of DNA into protoplasts of Nicotiana edwardsonii shows an unusual type of transactivation of a chloramphenicol acetyltransferase (CAT) gene fused at its 5' end to a small open reading frame (gene VII) of the long 5' leader of the full-length RNA transcript of the FMV genome. The level of activity of the CAT gene is increased up to 20-fold over the activity of control plasmids when gene VI is included in the electroporation mixture. Mutagenesis of the coding portions of gene VI of pGS1 RVI, a transactivating plasmid used in the electroporation experiments, demonstrated that it was probably the polypeptide product of gene VI that was responsible for the transactivating effect. Experiments with various portions of the 5' leader of the large, full-length RNA of FMV showed that the coding region of gene VII is necessary for the transactivation event. Clones of cauliflower mosaic virus (CaMV) or FMV with intact gene VI were found to reciprocally transactivate gene VII-CAT fusions (FMV) or gene I-CAT fusions (CaMV) located downstream of the 5' leader sequences of either viral genome.

Base Sequence↗
PubMed1988

Host range control of cauliflower mosaic virus.

Studies with recombinant genomes of cauliflower mosaic virus (CaMV) strains D4, CM1841, and Cabb-B have shown that a host range determinant of CaMV is encoded within the first half of region VI, a gene which codes for P62, an inclusion body protein. In order to further study the host specificity of CaMV, a fourth CaMV strain, W260, was chosen that has a host range that is intermediate between D4 and CM1841. To determine which portion of the W260 genome controls systemic spread, recombinant viruses made between this strain and CM1841 and D4 were tested for their ability to systemically infect several solanaceous plants (Datura stramonium, Nicotiana edwardsonii, and Nicotiana bigelovii). The first half of gene VI specified the type of local lesions and systemic spread of recombinant strains in D. stramonium. In N. edwardsonii, it was found that the first half of gene VI controlled the type of local lesion formed but systemic spread was dependent on the whole of gene VI. In N. bigelovii the number of genes that determined systemic spread of CaMV varied with the strain of CaMV. Systemic spread of D4 in N. bigelovii was dependent on the first half of gene VI. In contrast, systemic spread of W260 in the same host was dependent on the whole of gene VI and another locus which mapped within genes I-V. Consequently, it appears that other viral proteins may interact with P62 or that P62 may function well in some hosts only in compatible forms of other viral proteins.

Brassica↗
PubMed1987

Sequence of figwort mosaic virus DNA (caulimovirus group).

The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.

Base Sequence↗
PubMed1987

Gene I products of cauliflower mosaic virus detected in extracts of infected tissue.

The product of cauliflower mosaic virus (CaMV) gene I has been characterized from extracts of infected plants. Two size classes of this protein can be identified by the use of specific antiserum. The antiserum was induced against a chimeric protein produced in E. coli from a gene fusion between a fragment of the CaMV genome and the beta-galactosidase gene. A 36-kDa form of the gene I product is associated with virus particles. A 45-kDa form of this product was found only in the insoluble fraction of tissue homogenates. This insoluble fraction, the only fraction found to contain the product of viral gene VI, contains the virally induced inclusion bodies.

Genes, Viral↗
PubMed1987

Normal pressure hydrocephalus presenting as Parkinson's syndrome.

Although extrapyramidal features in normal pressure hydrocephalus (NPH) are not uncommon, presentations with Parkinson's syndrome as the predominant feature are rare and may give rise to diagnostic difficulties. Failure of patients with parkinsonism to respond to therapy, should alert one to the possibility of NPH.

Aged↗
PubMed1986

Region VI of cauliflower mosaic virus encodes a host range determinant.

A domain of cauliflower mosaic virus (CaMV) which controls systemic spread in two solanaceous hosts (Datura stramonium and Nicotiana bigelovii) was mapped to the first half of open reading frame 6. Whereas ordinary strains of CaMV are unable to infect solanaceous species except to replicate locally in inoculated leaves, a new CaMV strain (D4) induces chlorotic local lesions and systemically infects both D. stramonium and N. bigelovii. To determine which portion of the CaMV genome controls systemic spread of the virus in solanaceous hosts, nine recombinant genomes constructed between D4 and two ordinary strains of the virus were tested for their ability to infect solanaceous hosts. A 496-base-pair DNA segment comprising the first half of open reading frame 6 specified the type of local lesions and systemic spread of the virus in solanaceous hosts. Exchange of this segment of the genome between strains of CaMV converted a compatible host reaction to an incompatible (hypersensitive) one in response to infection. This suggests that the gene VI protein interacts with the plant to suppress hypersensitivity, the normal response of solanaceous hosts to CaMV infection.

DNA, Viral↗
PubMed1984

Expression of disease symptoms in cauliflower mosaic virus genomic hybrids.

In an effort to determine if particular regions of the cauliflower mosaic virus (CaMV) genome could be associated with particular phenotypic characters, strains of CaMV differing markedly in biological properties were recombined to produce hybrids. DNA from pairs of (infectious) genomic clones was cleaved with restriction endonucleases, then mixed and ligated. Recombinants were found by screening transformants in E. coli, or by selection in vivo for infectious hybrids. Recombinants in infected turnip plants were characterized by restriction endonuclease mapping of their DNA to confirm the hybrid genotype. New hybrid strains that induced less severe disease, or conversely, more severe disease than either parent were observed. The experiments revealed that typical disease expression, consisting of leaf chlorosis and mottling, mapped to a genome segment containing open reading frame VI (ORF VI) and the full-length promoter. This basic disease symptom was found to be influenced by other regions of the genome. Insect transmissibility mapped to ORF II. The ability to develop generalized infections in solanaceous plants was tested in hybrids between CaMV CM1841 and a variant that infects Datura stramonium systemically. In this case the systemic mobilization of virus appeared to be controlled by ORF VI, suggesting that this gene may function in cell-to-cell movement of virus.

DNA, Recombinant↗
PubMed1983

Insertional mutagenesis of the cauliflower mosaic virus genome.

A series of small insertions has been introduced into the various translational reading frames of the DNA of a "severe" strain of cauliflower mosaic virus (CaMV). A selectable gene (the kanamycin phosphotransferase gene of Tn903), flanked by a series of symmetrically arranged cloning sites taken from M13mp7, was used to prepare the site-specific mutants. In-phase insertions of 12 or 30 bp, which introduced unique SalI sites into reading regions I, III, IV, V and into the amino-proximal portion of region VI, destroyed infectivity. Insertions in the amino-distal portion of region VI, in the large intergenic region, and in region II retained infectivity. The amino-distal insertions in region VI reduced the severity of symptoms in plants. The insertion in region II destroyed aphid transmissibility. Longer DNA segments when inserted into region II or into the amino-distal portion of region VI destroyed infectivity, but similar insertions in the intergenic region were without effect on virus infection or development.

Animals↗
PubMed1982

Cloning and sequencing of restriction fragments generated by Eco RI*.

Thirty-four Eco RI* sites have been identified on the nucleotide sequence of CaMV, following cloning of Eco RI* fragments in M13mp2. From this sequencing data, we have deduced that Eco RI* recognizes sites that differ in a single position from the canonical Eco RI sequence, GAATTC. Any substitution can occur at any one of the six positions in the recognition site, with the exception of A leads to T or T leads to A changes within the central tetramer. The Eco RI* restriction patterns of phi x174 and pBR322 are consistent with these recognition criteria. Similarly, Bam HI* cleavage of phi x174 and SV40 (George et al., 1980) produces restriction patterns that are consistent with single-position degeneracy in the canonical Bam HI recognition site. Cohesive termini produced by Eco RI* cleavage were ligated into the Eco RI site of M13mp2, even when there was a base pair mismatch within the four nucleotide overlap. Mismatches were corrected asymmetrically during subsequent replication of M13 in E. coli.

Bacteriophage phi X 174↗
PubMed1981

The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing.

We have determined the complete primary structure (8031 base pairs) of an infectious clone of cauliflower mosaic virus strain CM1841. The sequence was obtained using the strategy of cloning shotgun restriction fragments in the sequencing vector M13mp7. Comparison of the CM1841 sequence with that published for another caMV strain (Strasbourg) reveals 4.4% changes, mostly nucleotide substitutions with a few small insertions and deletions. The six open reading frames in the sequence of the Strasbourg isolate are also present in CM1841.

Base Sequence↗
PubMed1977

Dielectric dispersion of DL-alpha-valine in aqueous solution.

The complex permittivity (epsilon = epsilon - jepsilon) of an (0.5 M) aqueous solution of DL-alpha-valine was measured at microwave frequencies over the temperature range 0.3 - 40 degrees C. The magnitude and relaxation time of the dispersions were determined, and the results interpreted in terms of the molecular structure of the valine molecule in solution. The peak in the dielectric loss due to the relaxation of the valine molecules occurs at approximately 2.6 GHz at 37 degrees C.

Electric Conductivity↗