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.

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.

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.

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.

[Study of kinetoplast DNA of Trypansoma cruzi using restriction endonucleases]. / Etude de l'Adn kinétoplastique de Trypansoma cruzi à l'aide d'endonucléases de restriction

The cleavage of kinetoplastic DNA (kDNA) with the restriction endonucleases (Eco RI, Hind II + III, Hpa II) was studied. The quantitative data of electrophoretically separated fragments show that the kDNA (free circular genome units and complex network) is composed of distinct populations of circles. Kinetic studies of renaturation of kDNA previously cleaved by Hpa II into fragments having the length of one genome unit also indicate the presence of at least two and probably three populations of molecules.

Hydrolyse des aminoacyl-tARN par la phosphodiesterase du venin.

The influence of the esterification of the 3'(2') hydroxyl group of tRNA on the hydrolysis by venom phosphodiesterase (Crotalus adamanteus) was studied. It was shown that this esterification does not change the rate of hydrolysis. At pH 7-8, no significant differences were observed either when the alpha amino group of the attached amino acid was free or blocked, or when the aminoacid contained a free carboxyl group or a second amino group (glutamic acid or lysine). Aminoacyl-AMP or oligopeptidyl-AMP can be prepared by this method.

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.

Studies on the single-stranded discontinuities of the cauliflower mosaic virus genome.

The Cauliflower Mosaic Virus (CaMV) genome is a double-stranded DNA molecule of about 5 million daltons. Native DNA molecules appear heterogeneous when analysed by gel electrophoresis. We have examined the nature of this apparent heterogeneity. Besides, this genome is shown here to contain three single-stranded breaks, as revealed by different denaturation experiments: heating at 75 degrees C, treatment with NaOH or dimethyl sulfoxide (DMSO). Labelling with terminal transferase proves that the 3' ends at these interruptions all have free hydroxyl groups. Electron microscopy and alkaline gel electrophoresis indicate that these three discontinuities are shared by both strands, and that they are not randomly located. S1 nuclease is active on CaMV DNA and generates three fragments. The comparison between the sizes of these fragments and of the products of denaturation leads us to consider that S1 acts at the level of the interruptions. We have determined that two of them, distant by one third genome unit, are in the same strand; the other is in the opposite strand, distant by one sixth genome unit from the nearest other one. The combined use of restriction enzymes and S1 nuclease has enabled us to locate these three discontinuities on the restriction map of the CaMV genome that we have otherwise established.

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.

A simple and fast electrophoretic method for elution of nucleic acids from gels.

A very convenient electrophoretic procedure for DNA or RNA elution from agarose or polyacrylamide gels is described. The gel piece with nucleic acid to be eluted is contained in a dialysis bag filled with buffer and elution is carried out in a horizontal electrophoresis apparatus. The nucleic acid is recovered with a high yield and can be used, without prior treatment, in further enzymatic or chemical reactions. Results obtained with DNA are presented here.

Heterogeneity of the kinetoplast DNA molecules of Trypanosoma cruzi.

Kinetoplast DNA (kDNA) of the culture form of Trypanosoma cruzi is cleaved by restriction endonucleases (HpaII, HindII, EcoRI, and HaeIII). The analysis of the cleavage patterns proves that the minicircles (free circulargenome units) are heterogeneous in base sequences. The same results are obtained with the complex kDNA network which is composed of the association of minicircles and linear molecules. Kinetic studies of the renaturation of kDNA previously cleaved by HpaII into fragments of the genome unit size show at least two populations of molecules. About 75% of these molecules correspond to the fast renaturing population having the molecular complexity of the minicircles. The molecules of the slow renaturing population have a much higher molecular complexity than the minicircles and do not seem to be related to the majority of the long linear molecules.

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.

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.

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.

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.

Determination of genes, restriction sites, and DNA sequences surrounding the 6S RNA template of bacteriophage lambda.

A major product of the transcription of bacteriophage lambda DNA in vitro is the 6S RNA. This article presents a detailed mapping of restriction endonuclease cleavage sites about the region of the 6S RNA template within the lambda genome. Restriction fragments defined by these sites have been used to localize the 6S RNA template within the physical and genetic maps of the lambda genome. Nucleotide sequence analysis of one of these fragments has largely confimed the nucleotide sequence of the 6S RNA reported previously and has indicated the sequence of DNA that immediately follows the 6S RNA template. This article reports the nucleotide sequence following a known site of transcription termination by RNA polymerase of Escherichia coli.