Simon N. Covey

Transcription of cauliflower mosaic virus DNA. Detection of transcripts, properties, and location of the gene encoding the virus inclusion body protein

We have detected several cauliflower mosaic virus transcripts in infected turnip leaves by northern-blot hybridization. These RNAs ranged in size from 0.9 to about 8 kb. Two species, a heterogeneous 7-to 8-kb RNA and a 2.3-kb RNA, accumulated radioactivity when CaMV-infected leaves were labeled with [32P]orthophosphate 20 days postinoculation. An abundant 62,000 MW polypeptide was synthesized in a rabbit reticulocyte lysate programmed with RNA from infected plants, but this polypeptide was absent when RNA from noninfected plants was used to direct translation. The 62,000 MW polypeptide was also the major in vitro product specified by virus-specific poly(A)+RNA purified by hybridization with CaMV DNA immobilized on DBM paper. The in vitro-synthesised 62,000 MW polypeptide was shown to be very similar to the major protein component of virus inclusion bodies by peptide fingerprint analysis. The 2.3-kb transcript is the mRNA encoding the 62,000 MW inclusion body protein. Crossed-contact hybridization mapping of this messenger on cloned CaMV DNA revealed that it is transcribed from the contiguous EcoR1 fragments d and b. The 7- to 8-kb RNA hybridized to all EcoR1 fragments and is probably a full-length primary transcript of the alpha-DNA strand.

Aphid transmission and a polypeptide are specified by a defined region of the cauliflower mosaic virus genome

Infection of young turnip leaves with an aphid-transmissible isolate, Cabb B-JI, of cauliflower mosaic virus (CaMV) causes synthesis of an Mr 18 000 polypeptide (p18) which co-purifies with virus inclusion bodies. This polypeptide is not detectable in leaves infected with either of two aphid non-transmissible isolates. Campbell and CM4-184. Construction in vitro, of hybrid genomes between Cabb B-JI and Campbell isolates demonstrates that aphid transmissibility and presence of p18 is dependent on the small genome fragment from the BstEII site to the XhoI site. A deletion made in this fragment within open reading frame (ORF) II causes loss of aphid transmissibility and also terminates production of p18. We conclude that aphid transmissibility and the presence of p18 are related to the expression of ORF II of the CaMV genome.

Does cauliflower mosaic virus replicate by reverse transcription

Abstract Cauliflower mosaic virus (CaMV) has an encapsidated genome of double-stranded DNA which exhibits several unusual features. These and other observations have led us to propose a model for CaMV replication which includes a reverse transcription step.

Segregation of cauliflower mosaic virus symptom genetic determinants

We have created a series of hybrid cauliflower mosaic virus (CaMV) genomes between a severe virus strain (Cabb BJI) and a mild strain (Bari 1) to map the virus genetic loci responsible for specific systemic symptom characters produced in infected turnip plants. Recombinants were generated in vivo by recombinational rescue and in vitro by restriction enzyme fragment exchange. On infection, hybrids induced either parental (wild-type) symptoms or segregated parental characters. Some of the engineered hybrid genomes produced novel symptomatic effects not observed in either of the parental strains whilst others reverted to express parental symptom characters following passaging. Determinants defining differences between the two CaMV strains in respect of four specific symptom characters were delimited to separate genome regions. A locus involved in determining the rate of spread of systemic vein clearing symptoms mapped to a region containing part of gene VII and gene I (nts 109-780). This phenomenon is consistent with the putative involvement of the CaMV gene I product in mediating virus movement within infected plants. Determinants influencing the degree of leaf chlorosis were located in a separate genome domain encompassing part of gene VI together with the large intergenic region and part of gene VII (nts 6103-90). Determinants controlling timing of initial systemic symptom appearance were mapped to a region between nts 2150 and 4438 containing part of gene III, gene IV, and part of gene V. Plant stunting was influenced by loci in at least two separate regions, one containing parts of gene I and II, and a second within the reverse transcriptase gene (V). We conclude that symptoms produced by CaMV infection can be subdivided into individual characters, the genetic determinants of which segregate to different virus genetic loci and are not restricted to a single gene product.

Changes in the amount of ribosomal RNA and poly(A)-containing RNA during leaf development.

SummaryA rapid increase in RNA content of the primary leaves of Phaseolus aureus Roxb. begins between three and four days after germination. At about the same time there is a corresponding increase in the rate of cell division. The major part of the RNA consists of chloroplast and cytoplasmic rRNA. The accumulation of RNA proceeds more rapidly in the light but substantial amounts of both chloroplast and cytoplasmic rRNA are synthesised in the dark. Developing leaves also synthesise polydisperse RNA containing poly(adenylic) acid. The amount varies in leaves of different ages from 0.6 to 2.2^ of the total RNA. The poly (adenylic) acid regions of these molecules can be detected and measured by a hybridisation assay with [5-3H]poly(uridylic) acid. Leaves of dry seeds contain significant amounts of poly(adenylic) acid and additional sequences are synthesised during leaf growth in the dark and in the light. The maximum change, representing a five-fold increase in poly(adenylic) acid content, occurs during cell division in the light. The amount of poly(adenylic) acid declines rapidly soon after cell division ceases, in contrast to the rRNA which is much more stable.

A putative primer for the replication of cauliflower mosaic virus by reverse transcription is virion-associated

We have isolated, from a cauliflower mosaic virus (CaMV) preparation, an RNA species (75 nucleotides) covalently linked to the 5′‐end of the CaMV DNA fragment (sa‐DNA) which is complementary to the CaMV 35 S RNA transcript 5′‐end. Site‐specific cleavage located a unique m7G residue in the RNA moiety of sa‐DNA suggesting that it is host cell tRNAmet i sa‐DNA is reminiscent of retrovirus ‘strong‐stop“ DNA and is evidence fo the reverse transcription model of CaMV replication.

Genome organization and expression of reverse transcribing elements: variations and a theme

Introduction. Until recently, reverse transcription was considered to be the sole prerogative of retroviruses. Over the last 4 or 5 years it has been recognized that members of two other virus groups, the hepadnaviruses and the caulimoviruses, undergo reverse transcription during replication. Furthermore, some vertebrate genetic elements, e.g. intracisternal A particle (IAP) genes (Ono et al., 1985), VL30 genes (Hodgson et al., 1983) and L1Md (Loeb et al., 1986), and transposable elements from other taxonomic groups [yeast Ty elements (Clare & Farabaugh, 1985; Hauber et al., 1985), Drosophila copia (Mount & Rubin, 1985) and copia-like elements (Saigo et al., 1984), Dictyostelium DIRS-1 element (Cappello et al., 1985), maize Bs1 element (Johns et al., 1985), and possibly maize Cin1 element (Shepherd et al., 1984)] have been shown to possess structural similarities to integrated retroviruses. The yeast Ty element transcript has recently been found to be contained within virus-like particles which have reverse transcriptase activity (Garfinkel et al., 1985; Mellor et al., 1985a).

Molecular Properties of Bari 1, a Mild Strain of Cauliflower Mosaic Virus

Summary We studied aspects of the structure and expression of the genome of Bari 1, a mild strain of cauliflower mosaic virus. Differences were observed between gene products of Bari 1 detected in inclusion body preparations and those of the more typically severe strain, Cabb B-JI. The most striking difference was the gel mobility of the Bari 1 gene VI polypeptide (apparent M r 70K) which contrasted with that of Cabb B-JI (M r 62K). This difference was also observed between products of in vitro translation of viral mRNA suggesting that it was not due to post-translational modification. The open reading frame in the nucleotide sequence of the Bari 1 gene VI region was very similar in size to that of other CaMV strains but corresponded to an amino acid sequence with a much lower overall homology and diverged greatly in a 40 base pair sequence in the 3′ region compared to gene VI sequences of other strains. The level of the Bari 1 aphid transmission polypeptide P18, the product of gene II, was much lower than that of Cabb B-JI. Some of the possible subcellular consequences resulting from the molecular properties of Bari 1 were examined by electron microscopy. Differences were observed in the composition and intactness of Bari 1 cytoplasmic inclusion bodies compared with those of a severe strain, and the presence of nuclear inclusions.

Hairpin DNAs of cauliflower mosaic virus generated by reverse transcription in vivo.

Cauliflower mosaic virus (CaMV) is a DNA plant virus which replicates by reverse transcription. During our examination of CaMV replication intermediates by 2‐D gel electrophoresis, we have discovered a population of bizarre linear double‐stranded hairpin DNAs. The largest hairpin is the size of the CaMV genome; hairpin loop ends of smaller molecules map to several sites around the genome but the open ends are all located close to the origin of reverse transcription at the primer binding site. We believe that the hairpin DNAs are generated in vivo by reverse transcription of CaMV RNA followed by self‐primed second strand synthesis. The accumulation of hairpin DNAs in vivo might represent a side reaction of the CaMV reverse transcriptase although an essential role for them in the virus replication cycle cannot be discounted. The structure of the hairpin DNAs provides further evidence for the location of the start site and of the polarity of reverse transcription in CaMV.

The properties and function of rapidly-labelled nuclear RNA.

SummaryNuclei were isolated from cultured cells of Acer pseudoplatanus L. previously pulse-labelled with [5-3H]uridine or [32P]phosphate and the properties of the rapidly-labelled RNA were studied. Polyacrylamide gel electrophoresis showed ribosomal RNA precursors and processing intermediates with molecular weights of 3.4, 2.5, 1.4 and 1×106 daltons, together with polydisperse RNA. The relative proportions of ribosomal RNA precursors and polydisperse RNA varied according to the length of the labelling period, but after 30 min approximately 90% of the radioactive RNA was polydisperse. The relationship between this polydisperse RNA and messenger RNA was investigated. The percentage of total nuclear RNA retained by chromatography on oligodeoxythymidylic acid-cellulose columns varied from 6% to 16% depending on the length of the labelling period. This RNA fraction, which has an adenylic acid content of approximately 45%, is assumed to represent RNA with polyadenylic acid sequences attached. A larger proportion of the nuclear polydisperse RNA lacked polyadenylic acid. Both types of polydisperse RNA were similar in size and during polyacrylamide gel electrophoresis migrated as broad peaks with an average molecular weight of approximately 106 daltons. The polydisperse nuclear RNA that lacks polyadenylic acid was found to be similar in nucleotide composition to ribosomal RNA and is assumed to represent growing chains of ribosomal precursor RNA. After short labelling times the majority of the radioactivity incorporated into nuclear RNA is present in molecules of this type. This suggests that the designation of pulse-labelled polydisperse RNA as messenger RNA or precursor to messenger RNA solely on the basis of rapid labelling and size heterogeneity is unsound. The average molecular weight of the polyadenylic acid-containing messenger RNA from the cytoplasm was less than that of the corresponding nuclear RNA (6 and 9×105 daltons respectively). This suggest either that the majority of the nuclear polyadenylic acid-containing RNA does not enter the cytoplasm, or if it does, that it first undergoes a reduction in size.