Cell-cycle, phase-specific activation of Maize streak virus promoters.

It is believed that geminiviral DNA replication is coupled to the cell-cycle regulatory complex of the plant cell and that the virus-early (complementary or C sense) gene products REP and REPA may be able to manipulate the regulation of the cycle. In this study, we examined expression from the promoters of Maize streak virus (MSV) in transgenic maize plants and cells to determine whether they showed cell-cycle specificity. Histochemical staining of plant roots containing "long and short" C-sense promoter sequences upstream of the GUS (beta-glucuronidase) reporter gene showed that promoter activity was restricted to the meristematic region of the roots and was enhanced by 2,4-dichlorophenoxy acetic acid (2,4-D) treatment. Analysis of reporter gene and cell-cycle-specific gene transcript levels coupled with flow cytometric data in synchronized transgenic maize cells revealed that all of the MSV promoters showed cell-cycle specificity. The coat protein gene promoter showed highest activity in early G2, whereas the C-sense promoter sequences produced two peaks of activity in the S and G2 cell-cycle phases.

Bean yellow dwarf virus RepA, but not rep, binds to maize retinoblastoma protein, and the virus tolerates mutations in the consensus binding motif.

It has previously been reported that complementary-sense gene products of wheat dwarf virus (WDV), a geminivirus of the genus Mastrevirus that infects monocotyledonous plants, bind to human and maize retinoblastoma (Rb) protein. Rb proteins control cell-cycle progression by sequestering transcription factors required for entry into S-phase, suggesting that the virus modifies the cellular environment to produce conditions suitable for viral DNA replication. Using a yeast two-hybrid assay, we have investigated whether the complementary-sense gene products of bean yellow dwarf virus, a mastrevirus that is adapted to dicotyledonous plants, also bind maize Rb protein. We demonstrate that whereas RepA binds to Rb protein, Rep does not, suggesting that RepA alone regulates host gene expression and progression of cells to S-phase. RepA mutants containing L --> I, C --> S, C --> G, and E --> Q mutations within the consensus Rb protein binding motif LXCXE retained the ability to bind to Rb, but with reduced efficiency. Most notably, the E --> Q mutation reduced binding by approximately 95%. Nonetheless, all LXCXE mutants were able to replicate in tobacco protoplasts and to systemically infect Nicotiana benthamiana and bean, in which they produced wild-type symptoms.

Adaptation of the geminivirus bean yellow dwarf virus to dicotyledonous hosts involves both virion-sense and complementary-sense genes.

Bean yellow dwarf virus (BeYDV) and maize streak virus (MSV) belong to the geminivirus genus Mastrevirus and have host ranges confined to dicotyledonous and monocotyledonous species, respectively. To investigate viral determinants of host range specificity, chimeras were constructed by exchanging their coding and non-coding regions. BeYDV chimeras containing MSV ORF V1, ORF V2 or small intergenic region sequences, either individually or in various sequential combinations, replicated and produced virus particles in Nicotiana tabacum protoplasts. BeYDV chimeras containing MSV ORFs C1 and C2 and/or the large intergenic region were unable to replicate. None of the chimeras was able to systemically infect either N. benthamiana or maize. Complementation experiments using BeYDV chimeras containing MSV ORF V1 and/or ORF V2 suggest that expression of MSV movement protein and/or coat protein prevents BeYDV movement. The results demonstrate that factors involved in both viral DNA replication and virus movement are exclusively adapted to either monocotyledonous or dicotyledonous host backgrounds.

Mutational analysis of bean yellow dwarf virus, a geminivirus of the genus Mastrevirus that is adapted to dicotyledonous plants.

Bean yellow dwarf virus (BeYDV) is an atypical member of the geminivirus genus Mastrevirus that infects dicotyledonous plants. BeYDV DNA contains six open reading frames (ORFs) with the capacity to encode proteins in excess of 10 kDa. Two virion-sense ORFs (V1 and V2) and two complementary-sense ORFs (C1 and C2) have homologues in all mastreviruses, while ORFs C3 and C4 are not conserved. To investigate their functions, each of the ORFs has been truncated by either frame-shifting or the introduction of a stop codon. We demonstrate that an ORF V1 mutant replicated efficiently in Nicotiana tabacum protoplasts but was unable to systemically infect Phaseolus vulgaris and Datura stramonium, consistent with a role for V1 protein in virus movement. However, the mutant was able to systemically infect Nicotiana benthamiana although the onset of symptoms was appreciably delayed in comparison with wild-type virus. Disruption of ORF V2, encoding the coat protein, prevented systemic infection of all three hosts but the mutant replicated in protoplasts. Both ORF C1 and ORF C2 were essential for replication in protoplasts. Modification of the complementary-sense splice donor and acceptor sequences also prevented replication. Removal of the intron prevented systemic infection, although the intronless mutant was able to produce functional replication-associated protein (Rep) and replicated efficiently in protoplasts. ORFs C3 and C4 were not required for systemic infection. Our results indicate that four ORFs are spatially and functionally conserved in mastreviruses that infect both monocotyledonous and dicotyledonous plants.

Molecular characterization of a subgroup I geminivirus from a legume in South Africa.

A South African geminivirus for which we propose the name bean yellow dwarf virus (BeYDV) has been isolated from French bean (Phaseolus vulgaris cv. Bonus) showing stunting, chlorosis and leaf curl symptoms. A full-length cloned copy of the viral genome produced characteristic symptoms of the disease when reintroduced into French bean by agroinoculation, and was systemically infectious in Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum, Datura stramonium and Arabidopsis thaliana. BeYDV resembles subgroup I geminiviruses which infect monocotyledonous plants in having a single DNA component, two non-overlapping virion-sense (V1 and V2) and two overlapping complementary-sense (C1 and C2) coding regions, and an intron within the complementary-sense coding regions that is excised to produce a C1C2 fusion protein. It is most closely related to tobacco yellow dwarf virus from Australia, the only subgroup I geminivirus previously known to infect dicotyledonous plants, although it is sufficiently dissimilar (65% nucleotide sequence identity) to be considered a distinct virus.

Analysis of the nucleotide sequence of bean pod mottle virus middle component RNA.

The complete nucleotide sequence of the middle component RNA (M RNA) of the comovirus bean pod mottle virus (BPMV) has been determined. The sequence consists of 3662 nucleotides and contains a single long open reading frame sufficient to code for a protein of 113,353 Da. The proteolytic processing sites within this protein have been identified by comparison with the known three-dimensional structure of the virion and cleavage at these sites would lead to a range of products consistent with those observed during processing of the M RNA-encoded polyproteins in vitro. We have performed computer-aided searches for reiterated sequences within BPMV M RNA which might explain why ordered RNA is visible in the electron density map of BPMV middle component particles (Chen, Z., Stauffacher, C. V., Li, Y., Schmidt, T., Bomu, W., Kamer, G., Shanks, M., Lomonossoff, G., and Johnson, J. E., 1989, Science 245, 154-159). These searches revealed both the presence of overrepresented pentameric sequences and a consensus sequence which was repeated 15 times within the RNA sequence.

Histological and bacteriological studies of burn wounds treated with boiled potato peel dressings.

Histological and clinical studies have been made on comparable burn wounds covered with either boiled potato peels affixed to gauze bandages or gauze dressings alone; both dressings were applied over a thin layer of 5 per cent silver sulphadiazine. Compared with treatment with plain gauze dressings, the application of the potato peel dressing reduced or eliminated dessication, permitted the survival of superficial skin cells and hastened epithelial regeneration. Bacteriological studies showed that the potato peels had no intrinsic antibacterial activity, the wounds beneath both dressings showing either no growth or, on most occasions, the same bacterial species. The easy availability of potato peels and gauze bandages on to which they can be affixed, the simplicity of the preparation of this dressing, the ease of sterilization and its low cost of production make this the dressing of choice for burn wounds in our developing country.

Tangential excision of eschar for deep burns of the hand: analysis of 156 patients collected over 10 years.

Deep burns affecting the dorsum of the hand have been treated by tangential excision of the eschar in 156 patients involving 208 hands. From our 10-year experience we have concluded that: If the patient's general condition permits it all hands with deep partial and full thickness skin loss burns are suitable for early tangential excision of the eschar. The best time for the operation is within the first week after injury. If the burn is of limited extent and the requirement for autograft skin is small the operation may be carried out under nerve block anaesthesia. When the burn is more extensive (i.e. involving both hands) intravenous ketamine anaesthesia is recommended. During tangential excision sequential layers of tissue must be removed until the base of the burn appears porcelain white in colour, has a lustrous appearance with many small bleeding points and is firm in consistency. If there are deep burns of the finger webs they are incised or excised and then grafted. Postoperatively an absence of fever, pain or exudation from the wound indicates that inspection of the wound can be delayed for about 2 weeks. By this time the wound is usually healed and functional rehabilitation and physiotherapy can commence.

Synthesis of Sendai virus polypeptides by a cell-free extract from wheat germ.

The '18S' RNA population from Sendai virus-infected cells efficiently directed the synthesis of three virus-specific polypeptides P, NP and M, in wheat germ cell-free extracts. In agreement with previous results obtained with a reticulocyte extract, there was little or no production of virus glycopolypeptides. Analyses of tryptic peptides revealed close correspondence between the primary structures of NP and M made in vitro and the authentic virus polypeptides.

Translation of virus mRNA: synthesis of bacteriophage Q beta proteins in a cell-free extract from wheat embryo.

The RNA from bacteriophage Qbeta can be translated by cell-free extracts from wheat embryos. This translation, by 80S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qbeta proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.