The characterization of waste cathode-ray tube glass.

New re-use applications are needed to address the relatively large quantity of waste electronic products generated in the world. Cathode-ray tubes (CRTs) from computer monitors and TV sets are a large component of such waste. The three glass components of CRTs are the funnel, panel and neck, which are produced by various manufacturers and are now collected by asset-recovery centres. In this paper, we characterize waste funnel and panel glass from dismantled cathode-ray tubes with a view to assisting the development of new re-use applications. The heavy metal (lead, barium, and strontium) content of such glass represents an acute risk to the environment. Our results of the chemical composition for different kinds of waste CRT glass including black & white and color CRTs show that CRT glass from different producers have generally similar chemical compositions. In particular, the compositions of funnel and panel black & white CRT glass are similar, but are different to those of panel and funnel color CRT glass. We also measured the following specific properties of each type of CRT glass: density, glass transition temperature, and linear coefficient of thermal expansion. It was found that the coefficients of thermal expansion of CRT glass do not vary with their composition. In contrast, the measured densities and glass transition temperatures do vary with composition. On the basis of our experimental data and data found in the literature, we outline the main properties of several waste CRT glass currently in circulation. The aim of this study was to provide the data required to determine if this kind of waste could be entirely (or partially) re-used and to aid the search for promising methods of treatment.

Early detection of cacao swollen shoot virus using the polymerase chain reaction.

A polymerase chain reaction assay was developed which allows early detection of cacao swollen shoot virus (CSSV) in DNA extracts from cacao plantlets agroinoculated with the Togolese isolate Agou 1. The primers used were derived from badnavirus conserved sequences and nucleic acid was extracted with the Plant DNeasy extraction kit (Qiagen). CSSV genome was detectable between 6 and 20 days after inoculation. The first leaf symptoms appeared after 4 weeks and the first shoot swelling symptoms after 8 weeks.

Interaction between the open reading frame III product and the coat protein is required for transmission of cauliflower mosaic virus by aphids.

Transmission of cauliflower mosaic virus (CaMV) by aphids requires two viral nonstructural proteins, the open reading frame (ORF) II and ORF III products (P2 and P3). An interaction between a C-terminal domain of P2 and an N-terminal domain of P3 is essential for transmission. Purified particles of CaMV are efficiently transmitted only if aphids, previously fed a P2-containing solution, are allowed to acquire a preincubated mixture of P3 and virions in a second feed, thus suggesting a direct interaction between P3 and coat protein. Herein we demonstrate that P3 directly interacts with purified viral particles and unassembled coat protein without the need for any other factor and that P3 mediates the association of P2 with purified virus particles. The interaction domain of P3 is located in its C-terminal half, downstream of the P3-P2 interaction domain but overlapping a region which binds nucleic acids. Mutagenesis of P3 which interferes with the interaction between P3 and virions is correlated with the loss of transmission by aphids. Taken together, our results demonstrate that P3 plays a crucial role in the formation of the CaMV transmissible complex by serving as a bridge between P2 and virus particles.

The cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana.

The cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is involved in several aspects of the infectious cycle. P6 specifically controls the synthesis of other CaMV proteins by transactivating their expression from the polycistronic 35S RNA. By far-Western assays, we have demonstrated that P6 interacts with proteins from both healthy and CaMV-infected leaves of Arabidopsis thaliana. These proteins are found in ribosome-enriched extracts, suggesting that they participate in the translation process. One of these proteins, identified by microsequencing, corresponds to the 60S ribosomal subunit protein L18 (RPL18). Its cDNA was cloned and expressed in Escherichia coli, and the resulting RPL18 protein was shown to interact with the minimal region required for translational transactivation, designated the miniTAV domain of P6.

Aphid transmission of cauliflower mosaic virus requires the viral PIII protein.

The open reading frame (ORF) III product (PIII) of cauliflower mosaic virus is necessary for the infection cycle but its role is poorly understood. We have used in vitro protein binding ('far Western') assays to demonstrate that PIII interacts with the cauliflower mosaic virus (CaMV) ORF II product (PII), a known aphid transmission factor. Aphid transmission of purified virions of the PII-defective strain CM4-184 was dependent upon added PII, but complementation was efficient only in the presence of PIII, demonstrating the requirement of PIII for transmission. Deletion mutagenesis mapped the interaction domains of PIII and PII to the 30 N-terminal and 61 C-terminal residues of PIII and PII, respectively. A model for interaction between PIII and PII is proposed on the basis of secondary structure predictions. Finally, a direct correlation between the ability of PIII and PII to interact and aphid transmissibility of the virus was demonstrated by using mutagenized PIII proteins. Taken together, these data argue strongly that PIII is a second 'helper' factor required for CaMV transmission by aphids.

In situ localization of cacao swollen shoot virus in agroinfected Theobroma cacao.

Cacao swollen shoot virus (CSSV) is a small non-enveloped bacilliform virus with a double-stranded DNA genome. A very restricted host range and difficulties in transmitting the virus, either mechanically or via its natural vector, have hindered the study of cacao swollen shoot disease. As an alternative to the particle-bombardment method previously reported, we investigated another approach to infect Theobroma cacao. A greater-than-unit length copy (1.2) of the CSSV DNA genome was cloned into the Agrobacterium binary vector pBin 19 and was transferred into young plants via Agrobacterium tumefaciens. Typical leaf symptoms and stem swelling were observed seven and eleven weeks post inoculation, respectively. Viral DNA, CSSV coat protein and virions were detected in leaves with symptoms. Agroinfected plants were used to study the in situ localization of CSSV and its histopathologic effects in planta. In both leaves and petioles, virions were only seen in the cytoplasm of phloem companion cells and of a few xylem parenchyma cells. Light microscopy showed that stem swelling results from a proliferation of the xylem, phloem and cortex cells.

Rice tungro bacilliform virus open reading frame 3 encodes a single 37-kDa coat protein.

Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus and a member of the Caulimoviridae family and closely related to viruses in the Badnavirus genus. The coat protein of RTBV is part of the large polyprotein encoded by open reading frame 3 (ORF3). ORF3 of an RTBV isolate from Malaysia was sequenced (accession no. AF076470) and compared with published sequences for the region that encodes the coat protein or proteins. Molecular mass of virion proteins was determined by mass spectrometry (matrix-assisted laser desorption/ionization-TOF) performed on purified virus particles from three RTBV isolates from Malaysia. The N- and C-terminal amino acid sequences of the coat protein were deduced from the mass spectral analysis, leading to the conclusion that purified virions contain a single coat protein of 37 kDa. The location of the coat protein domain in ORF3 was reinforced as a result of immunodetection reactions using antibodies raised against six different segments of ORF3 using Western immunoblots after SDS-PAGE and isoelectrofocusing of proteins purified from RTBV particles. These studies demonstrate that RTBV coat protein is released from the polyprotein as a single coat protein of 37 kDa.

Mapping regions of the cauliflower mosaic virus ORF III product required for infectivity.

The open reading frame (ORF) III product (PIII) of the pararetrovirus cauliflower mosaic virus (CaMV) has nucleic acid-binding properties in vitro, but its biological role is not yet determined. ORF III is closely linked to ORF II and overlaps ORF IV out of frame in the CaMV genome. A new CaMV-derived vector (Ca delta) devoid of ORF III and containing unique restriction sites between ORFs II and IV was designed. Introduction of the wild-type CaMV ORF III into Ca delta results in a clone (Ca3) infectious in turnip plants. Truncated or point-mutated versions of ORF III were then inserted into Ca delta and tested in vivo. Inoculation of the different mutants into turnip revealed that the four C-terminal amino acid residues of PIII are dispensable for infectivity as well as an internal domain (amino acids 61 to 80). Taken together the results show that PIII possesses a functional two-domain organization. Moreover, the CaMV PIII function(s) cannot be replaced either by the PIII protein of another caulimovirus, the figwort mosaic virus, or by the P2 protein of the cacao swollen shoot badnavirus, a member of the second plant pararetrovirus group.

A short basic domain supports a nucleic acid-binding activity in the rice tungro bacilliform virus open reading frame 2 product.

Little is known about the features of badnavirus open reading frame 2 products (P2). So far, no consensus functional domain has been found in these proteins. However, they all have in common at their C-terminus amino acids which may have the capacity to bind nucleic acids. Such capacity has already been established for cacao swollen shoot virus protein P2. We have looked for such a binding capacity of rice tungro bacilliform virus (RTBV) ORF 2 product. For this purpose, the protein was expressed as full-length or truncated versions in Escherichia coli. When used in nucleic acid-binding assays, complete RTBV P2 was shown to bind both DNA and RNA. This property may be related to a basic sequence, PPKKGIKRKYPA, localized at its C-terminus. Mutations were introduced into this sequence and revealed that four of the five basic residues, including a crucial lysine, are required for the binding to nucleic acids. Moreover, this sequence can confer binding capacity when it is fused to the N-terminus of nonbinding proteins.

The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein.

The function of the open reading frame 2 product (p2) of cacao swollen shoot virus (CSSV) and of other badnaviruses is not yet determined. Their carboxyl-termini are lysine and proline rich and also contain alanine residues, amino acids present at the C-termini of histone-like proteins. Full-length CSSV p2 (132 amino acids) or versions truncated at the C-terminus (128, 113, 103, or 101 amino acids) were expressed in Escherichia coli and partially purified. When assayed in nucleic acid-binding tests, p2 was able to interact with CSSV and other double-stranded DNAs and with CSSV and other single-stranded RNA transcripts in sequence-nonspecific manner. Moreover, this binding activity was progressively lost as the C-terminus was gradually deleted.

Sequence of a cauliflower mosaic virus strain infecting solanaceous plants.

The complete nucleotide sequence (8031 bp) of the DNA of cauliflower mosaic virus (CaMV) strain B29 is reported. This strain is unusual, since it infects both cruciferous and solanaceous plants. So far, from data of sequence comparisons between B29 and other CaMV strains there is no evidence for any obvious correlation between host range and distinct sequence features.

The full-length product of cauliflower mosaic virus open reading frame III is associated with the viral particle.

The gene III product (P15) of cauliflower mosaic virus (CaMV) is a DNA binding protein in which the DNA binding activity is located on its C-terminal part. In previous work, a C-terminal processed form of P15 (P11) was detected in purified viral particles as a minor component. The full-length P15 was shown to be present and to be matured, possibly by a cysteine proteinase, in CaMV replication complexes isolated from infected turnip leaves. In this paper, we have shown that a virion-enriched fraction obtained from such replication complexes by size exclusion chromatography contained most of the P15 in its uncleaved form and was enriched in the activity responsible for its proteolysis. This enabled us to characterize better the proteinase activity (temperature and pH optimum; effect of specific inhibitors) responsible for P15 cleavage and to confirm that it corresponds to a cysteine proteinase. Based upon these observations, a purification procedure for CaMV particles was devised which impaired the cleavage of P15 into P11 and allowed the isolation of virions containing almost exclusively the noncleaved form. This finding supports our hypothesis that the CaMV gene III product could be involved in the folding of the viral genome during encapsidation.

Assaying synthetic ribozymes in plants: high-level expression of a functional hammerhead structure fails to inhibit target gene activity in transiently transformed protoplasts.

A hammerhead ribozyme designed against the mRNA coding for the Escherichia coli beta-glucuronidase (GUS) reporter enzyme was constructed. The synthetic ribozyme appeared able to correctly cleave in vitro the target RNA. This catalytic molecule was then assayed for in vivo activity in plant protoplasts. Plasmids coding either for the ribozyme or for the GUS target gene were cotransfected into the cells by the PEG-calcium procedure and GUS gene expression monitored following transient expression by measuring the intracellular GUS enzymatic activity. Expression of the ribozyme to high molar excess over the GUS transcript did not lead to any significant decrease of GUS activity in the transfected protoplasts. Insertion of the ribozyme sequence in the 3'-untranslated region of the GUS mRNA also had no detectable effect on GUS reporter gene expression whereas the corresponding RNA appeared able to self-cleave in vitro. These results indicate that the ability of ribozymes to perform catalytic cleavage of their substrate mRNA in vitro is essential but clearly not sufficient to ensure that efficient inhibition of the corresponding target gene will occur upon endogenous expression of this catalytic RNA in the plant cell.

Strand-specific viral DNA synthesis in purified viroplasms isolated from turnip leaves infected with cauliflower mosaic virus.

There is some evidence that two steps are involved in the DNA replication of cauliflower mosaic virus (CaMV): the first one may occur in the nucleus and the second one in the cytoplasm of infected cells. The latter would correspond to the reverse transcription step recently proposed in the model of the viral life cycle, and could occur in the viroplasms which are CaMV-induced cytoplasmic inclusion bodies. In order to test whether viroplasms are capable of DNA synthesis and to characterize the associated enzymatic activities, we developed an extensive purification method for these organelles. Such isolated viroplasms are indeed able to incorporate radioactive precursors into exclusively viral-specific sequences without added template primer. Hybridization of sequences labeled in viroplasms to cloned CaMV DNA shows that the DNA synthesis occurs throughout the whole viral genome and has marked strand specificity; neosynthesized molecules are of minus polarity, i.e., complementary to the large viral transcript (35 S RNA). Moreover, during the purification of viroplasms, the poly(rC)-directed DNA synthesis activity, which is specific to infected plants, is preferentially retained.

Cauliflower mosaic virus-induced viroplasms support viral DNA synthesis in a cell-free system.

Viroplasms are the main cytological modifications observed upon infection of Brassica cells by cauliflower mosaic virus (CaMV). Previous experiments suggested that the replication of viral DNA proceeded in two steps, starting in the nucleus and going on to the viroplasms. Recent evidence has been obtained on the role of the nuclear step of CaMV DNA replication. We have developed an in vitro system, derived from infected leaves, which is able to synthesize viral DNA and which contains nuclei and viroplasms, the putative sites of CaMV replication. In such a system, viroplasms are the sites of active DNA synthesis, and replicated viral DNA molecules are preferentially associated with them.

RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves.

Cauliflower mosaic virus (CaMV) is a plant DNA with an 8-kb circular double-stranded genome. CaMV-specific DNA and RNA molecules present in infected Brassica cells share some structural features with DNAs and RNAs of retroviruses and hepatitis B virus. This led to the hypothesis that CaMV replication occurs via reverse transcription of an RNA intermediate. Here we report the first characterization of a new DNA polymerase activity, specific to CaMV-infected tissues. A subcellular fraction of infected cells shows capacity to copy poly(C) and the heteropolymeric regions of natural mRNAs. Chromatographic isolation of the poly(C)-dependent activity clearly establishes that it is distinct from the classical gamma-like DNA polymerases previously described in plant cells. The significant homology observed between defined regions of the Moloney murine leukemia virus (MMLV) polymerase and CaMV unassigned gene V product favours the idea that the reverse transcriptase-like DNA polymerase detected in infected cells is a virus-encoded enzyme.

Alteration of the specificity of restriction endonucleases in the presence of organic solvents.

The specificity of XbaI, SalI, HhaI, PstI, BamHI and SstI is relaxed in the presence of an organic solvent, such as 20% glycerol or 8% dimethylsulfoxide (DMSO). This alteration, very pronounced in some cases, requires an excess of enzyme, varies from one kind of enzyme to another and is highly dependent on the pH, the ionic strength, the nature of the metallic cofactor and/or the presence of a second organic solvent. Preliminary data concerning XbaI and BamHI used under conditions where the relaxation of specificity is moderate, suggest that some of the new ("pseudo") sites correspond to shortened sequences derived from the normal recognition sequence cleaved under the standard conditions of the reaction.