Identification and initial characterization of the IR6 protein of equine herpesvirus 1.

The IR6 gene of equine herpesvirus 1 (EHV-1) is a novel gene that maps within each inverted repeat (IR), encodes a potential protein of 272 amino acids, and is expressed as a 1.2-kb RNA whose synthesis begins at very early times (1.5 h) after infection and continues throughout the infection cycle (C. A. Breeden, R. R. Yalamanchili, C.F. Colle, and D.J. O'Callaghan, Virology 191:649-660,1992). To identify the IR6 protein and ascertain its properties, we generated an IR6-specific polyclonal antiserum to a TrpE/IR6 fusion protein containing 129 amino acids (residues 134 to 262) of the IR6 protein. This antiserum immunoprecipitated a 33-kDa protein generated by in vitro translation of mRNA transcribed from a pGEM construct (IR6/pGEM-3Z) that contains the entire IR6 open reading frame. The anti-IR6 antibody also recognized an infected-cell protein of approximately 33 kDa that was expressed as early as 1 to 2 h postinfection and was synthesized throughout the infection cycle. A variety of biochemical analyses including radiolabeling the IR6 protein with oligosaccharide precursors, translation of IR6 mRNA in the presence of canine pancreatic microsomes, radiolabeling the IR6 protein in the presence of tunicamycin, and pulse-chase labeling experiments indicated that the two potential sites for N-linked glycosylation were not used and that the IR6 protein does not enter the secretory pathway. To address the possibility that the unique IR6 gene encodes a novel regulatory protein, we transiently transfected an IR6 expression construct into L-M fibroblasts alone or with an immediate-early gene expression construct along with a representative EHV-1 immediate-early, early, or late promoter-chloramphenicol acetyltransferase reporter construct. The results indicated that the IR6 protein does not affect the expression of these representative promoter constructs. Interestingly, the IR6 protein was shown to be phosphorylated and to associate with purified EHV-1 virions and nucleocapsids. Lastly, immunofluorescence and laser-scanning confocal microscopic analyses revealed that the IR6 protein is distributed throughout the cytoplasm at early times postinfection and that by 4 to 6 h it appears as "dash-shaped" structures that localize to the perinuclear region. At late times after infection (8 to 12 h), these structures assemble around the nucleus, and three-dimensional image analyses reveal that the IR6 protein forms a crown-like structure that surrounds the nucleus as a perinuclear network.     

A rapid assay for chloroplast-encoded triazine resistance in higher plants

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.     

Poly(amidoamine) dendrimer peripherally modified with 4-N,N-dimethylaminoethyleneamino-1,8-naphthalimide as a sensor of metal cations and protons.

A new poly(amidoamine) dendrimer from second generation whose periphery comprises sixteen fluorescent 4-N,N-dimethylaminoethylamino-1,8-naphthalimide units has been synthesized and characterized. In DMF, the dendrimer shows sensitivity to the presence of Cu(2+), Fe(3+) and protons. The changes in the fluorescence intensity of the material are in opposite directions if acids or metals are present. Fluorescence enhancements (FE from 5 to 9 depending on solvent) are recorded when the photoinduced electron transfer (PET) originating from the donating amine to the electron accepting naphthalimide is inhibited by the protonation of the N,N-dimethylamino groups. In the case of Cu(2+) cations, a fluorescence quenching (FQ of 6) is first observed, followed by fluorescence partial restoration. In the Fe(3+) case, the same behaviour is observed with a final FE of 2. The successive complexations of these cations by the dendrimer core and by the external rim of the dendrimer may explain the results.     

Freshwater Bacteria Can Methylate Selenium through the Thiopurine Methyltransferase Pathway

Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 μM sodium selenite and 10 μM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.     

Study of human erythrocyte membrane protein interactions by selective solubilization of Triton-skeletons

Summary— The membrane skeleton, responsible for shape and mechanical properties of the red cell, was purified by the Triton extraction procedure in presence of 5 mM, 150 mM or 600 mM NaCl. The proportion of spectrin, protein 4.1 and actin present in erythrocyte skeletons does not depend on the molarity of NaCl used. In contrast ankyrin, protein band 3 and protein 4.2 are removed from skeletons as the ionic strength increased. Solubilization assays of membrane skeletons were used to study protein interactions inside the skeleton. Solubilization was performed by Tris, a non-selective disruptive reagent, or by p-mercuribenzene sulfonic acid (PMBS), which principally release spectrin and actin. Tris action was assessed by calculation of the percentage of solubilized proteins, which increased proportionally with Tris molarity. PMBS action was kinetically determined as the decrease in skeleton turbidity. With these two reagents, we observed a lower dissociation of skeletons prepared with high ionic strength buffer. Erythrocyte pretreatment with okadaic acid, an inhibitor of serine-threonine phosphatases, revealed a phosphorylation-induced skeleton gelation and a better resistance to Tris-solubilization.     

On the Origin of Protein Synthesis Factors: A Gene Duplication/Fusion Model

Sequence similarity has given rise to the proposal that IF-2, EF-G, and EF-Tu are related through a common ancestor. We evaluate this proposition and whether the relationship can be extended to other factors of protein synthesis. Analysis of amino acid sequence similarity gives statistical support for an evolutionary affiliation among IF-1, IF-2, IF-3, EF-Tu, EF-Ts, and EF-G and suggests further that this association is a result of gene duplication/fusion events. In support of this mechanism, the three-dimensional structures of IF-3, EF-Tu, and EF-G display a predictable domain structure and overall conformational similarity. The model that we propose consists of three consecutives duplication/fusion events which would have taken place before the divergence of the three superkingdoms: eubacteria, archaea, and eukaryotes. The root of this protein superfamily tree would be an ancestor of the modern IF-1 gene sequence. The repeated fundamental motif of this protein superfamily is a small RNA binding domain composed of two α-helices packed along side of an antiparallel β-sheet. Received: 17 October 1996 / Accepted: 10 June 1997     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Evaluation of Steinernema carpocapsae Weiser for Control of Carrot Weevil Adults, Listronotus oregonensis (LeConte) (Coleoptera: Curculionidae), in Organically Grown Carrots

Under controlled conditions, application of Steinernema carpocapsae using a baiting method consistently reduced the oviposition of Listronotus oregonensis adults at all moisture regimens. At 80 and 95% relative humidity, soil applications at 10 4 infective juveniles (IJs) cm-2 and 10 5 infective stages (400 IJs cm-2) per pot reduced the survival and oviposition of L. oregonensis adults; soil applications were ineffective at relative humidity lower than 80%. When timed correctly, soil application at the rate of 2 105 IJs/linear meter of row (4.4 109 IJs ha-1) reduced damage by up to 59%.