Double-label immunocytochemistry: combination of anterograde neuroanatomical tracing with Phaseolus vulgaris leucoagglutinin and enzyme immunocytochemistry of target neurons

By the neuroanatomical tracing technique based on uptake, transport, and immunocytochemical detection of injected Phaseolus vulgaris leucoagglutinin (PHA-L), fiber trajectories of labeled neurons can be followed with great accuracy to their termination areas. To further analyze the connectivity of these fibers, the target neurons must be chemically characterized. In vibratome and frozen sections of rat brain, we tried to visualize PHA-L-labeled fibers and, simultaneously, the target neuron-related antigen. As a model system we used the projection from the pre-frontal cortex to histaminergic neurons in the posterior hypothalamic region. We tested "sequential" and "pooled" immunocytochemical procedures. In the sequential procedure, the two antigens are detected by two successive and complete immunocytochemical staining procedures, with primary antibodies raised in different animal species and with different chromogens for the final visualization. In the pooled procedure, the sections are incubated with mixtures of primary and secondary antibodies, after which the procedure is similar to the sequential procedure. We obtained excellent results on vibratome sections with a sequential procedure using first conventional peroxidase immunocytochemistry (goat anti-PHA-L primary antibody) to visualize the transported PHA-L (brown reaction product), and subsequently alkaline phosphatase immunocytochemistry (rabbit anti-histidine decarboxylase primary antibody) to locate the histaminergic neurons (blue reaction product). The resulting preparations deteriorate, however, after 1-2 months of storage. Good results were also obtained with a double peroxidase procedure on frozen sections, using nickel-enhanced diaminobenzidine to visualize the PHA-L (dark blue reaction product), and diaminobenzidine (brown reaction product) to visualize the second antigen. The quality of these preparations is permanent.     

Structure of tobacco genes encoding thaumatin-like proteins

Two tobacco genes encoding thaumatin-like proteins were cloned and sequenced. Both genes are expressed after infection of tobacco with tobacco mosaic virus (TMV). Comparison of the upstream sequences of these genes with those of other TMV-inducible tobacco genes revealed limited regions of homology.     

Analysis of acidic and basic chitinases from tobacco and petunia and their constitutive expression in transgenic tobacco

cDNA clones of messenger RNAs for acidic and basic chitinases were isolated from libraries of tobacco mosaic virus-infected Samsun NN tobacco and petunia. The tobacco cDNA clones for acidic chitinase fell into two different groups, whereas all petunia cDNA clones had the same sequence. Also, tobacco genomic clones were isolated and one was characterized. This genomic clone, corresponding to one of the cDNA clones, showed that this acidic chitinase gene contains two introns. The amino acid sequences of the acidic chitinases from tobacco, as deduced from the cDNA clones, fully agreed with partial sequences derived from peptides obtained from purified tobacco-derived pathogenesis-related proteins PR-P and PR-Q. The deduced amino acid sequences showed that PR-P and PR-Q are 93 and 78%, respectively, identical to the petunia enzyme. All deduced chitinase sequences indicated the presence of an NH2-terminal, highly hydrophobic signal peptide. In addition, the polysaccharide-binding domain present at the NH2-terminus of basic chitinases from mature tobacco is not present in these acidic chitinases. Furthermore, the complete coding sequence for the petunia chitinase, constructed downstream of the cauliflower mosaic virus 35S promoter, was used to transform tobacco. The resulting chimeric gene was constitutively expressed, and the petunia enzyme was targeted to the extracellular fluid. In contrast, a basic chitinase of tobacco, expressed from a chimeric gene, was found in total leaf extracts but not in preparations of extracellular fluid.     

Molecular characterization of messenger RNAs for 'pathogenesis related' proteins la, lb and lc, induced by TMV infection of tobacco

A cDNA library was made to poly(A)-containing RNA from tobacco mosaic virus (TMV)-infected Samsun NN tobacco plants and clones corresponding to mRNAs for the `pathogenesis-related' (PR) proteins 1a, 1b and 1c were identified. One clone was found to contain a complete copy of PR-1b mRNA. The structural organization of this RNA is: a leader sequence of 29 nucleotides, an open reading frame of 504 nucleotides encoding a 30 amino acid long signal peptide and a 138 amino acid long mature protein, and a 3'-non-coding region of 235 nucleotides. Two other clones were found to contain partial copies of PR-1a and PR-1c mRNAs. The data indicate an ~90% homology between the amino acid sequences of PR-1a, -1b and -1c. Using one of the clones as probe it was shown that in the TMV-inoculated lower leaves and the non-inoculated upper leaves of a tobacco plant, the PR-1 mRNAs become detectable from 2 and 8 days after inoculation, respectively.     

RNA 2 of tobacco rattle virus strain TCM encodes an unexpected gene.

The sequence of the 3'-terminal 1210 nucleotides of RNA 1 and the complete sequence of 3389 nucleotides of RNA 2 of tobacco rattle virus (TRV) strain TCM has been deduced. The sequence of the 3'-terminal 1099 nucleotides of RNAs 1 and 2 was found to be identical. Thus the genome of this TRV strain is partially diploid, encoding a 16K protein in both RNA 1 and RNA 2. The sequence that is unique to RNA 2 contains two open reading frames: the coat protein cistron and a cistron for a 29.1K protein, which shows no homology with the RNA 1 encoded 28.8K protein. cDNA probes corresponding to these two open reading frames cross-hybridized to pea early-browning virus RNA 2, but not to RNA 2 of five other tobraviruses tested.     

Complete nucleotide sequence of alfalfa mosaic virus RNA 2.

Double-stranded cDNA of in vitro polyadenylated alfalfa mosaic virus (AlMV) RNA 2 has been cloned and sequenced. The use of an oligodeoxyribonucleotide corresponding to the known sequence of the 5'-end of RNA 2 to prime second-strand DNA synthesis, enabled us to construct the complete primary structure of AlMV RNA 2. The sequence of 2,593 nucleotides contains a long open reading frame for a protein of Mr 89,753 starting at the first AUG codon from the 5'-end. This coding region is flanked by a 5'-terminal sequence of 54 nucleotides and a 3'-noncoding region of 166 nucleotides which includes the sequence of 145 nucleotides the three genomic RNAs of AlMV have in common.     

A propos de quelques groupements végétaux observés entre monaco et gênes

Résumé Le climat chaud et humide de la Ligurie méditerranéenne détermine la réapparition de quelques groupements végétaux connus de la Catalogne et rares ou nuls en Languedoc: Inulo-Oryzopsietum miliaceae, Cichorio-Sporoboletum poiretii, Hyparrhenietum hirto-pubescentis, Rubo-Coriarietum.Le caractère particulier de la végétation ligure a été exprimé par des groupements spéciaux: Pistacio-Rhamnetum alaterni, qui remplace le Quercetum cocciferae, groupements à Ostrya carpinifolia et à Brachypodium pinnatum des endroits frais, groupements buissonnants à Erica arborea et Genista germanica, à Rosmarinus et Genista cinerea, Asplenio-Campanuletum macrorhizae des rochers, etc.

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Summary The warm and humid climate of the Mediterranean Liguria determines the reappearance of some communities, known in Catalonia, and seldom or not existing in Languedoc: Inulo-Oryzopsietum miliaceae, Cichorio-Sporoboletum poiretii, Hyparrhenietum hirto-pubescentis, Rubo-Coriarietum.The particular character of the Liguric vegetation is expressed in special communities: Pistacio-Rhamnetum alaterni, which replaces the Quercetum cocciferae, communities with Ostrya carpinifolia and with Brachypodium pinnatum in the fresh places, bushy communities with Erica arborea and Genista germanica, with Rosmarinus and Genista cinerea, Asplenio-Campanuletum macrorhizae of the rocks, and so on.

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Zusammenfassung Das warme und feuchte Klima der mediterranen Ligurie bestimmt das Wiedererscheinen einiger Pflanzengesellschaften, die in Katalonien bekannt sind, aber selten oder nicht in Languedoc vorkommen: Inulo-Oryzopsietum miliaceae, Cichorio-Sporoboletum poiretii, Hyparrhenietum hirto-pubescentis, Rubo-Coriarietum.Der eigenartige Charakter der ligurischen Vegetation ist in besonderen Pflanzengesellschaften ausgedrückt: Pistacio-Rhamnetum alaterni, das das Quercetum cocciferae ersetzt, Gesellschaften mit Ostrya carpinifolia und mit Brachypodium pinnatum der frischen Standorte, Erica arborea-Genista germanica-Silikatbodengebüsche, Rosmarinus-Genista cinerea-Kalkbodengebüsche, Asplenio-Campanuletum macrorhizae der Felsen usw.

     

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.     

Medium design based on stoichiometric analysis of microbial transglutaminase production by Streptoverticillium mobaraense

A stoichiometric model was developed for the application of medium design in microbial transglutaminase production by Streptoverticillium mobaraense. The model avoids dealing with all the metabolic reactions involved by simply lumping them into a single reaction. With the help of measurement results, an analysis of the nutrients' roles, and biochemical knowledge of the microorganism, all stoichiometric coefficients in the model were calculated. These coefficients were used for medium design. With this designed medium, microbial transglutaminase activity was increased fourfold, compared to that in the basal medium. (c) 1996 John Wiley & Sons, Inc.