Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.

Agrobacterium tumefaciens-mediated transformation of yeast.

Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes of A. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains. In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains. On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains. The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3'-methoxy groups, such as acetovanillone. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid. Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation.

Diogenis laertii de Clarorum philosophorum vitis, dogmatibus et apophthegmatibus libri decem : ex italicis codicibus...

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Diogenis Laertii De vitis, dogmatibus et apophthegmatibus clarorum philosophorum libri X. Graece et latine ad fidem optimorum librorum ... recensiti ... insertis XXVI. philosophorvm figuris aere incisis ... a Paullo Daniele Longolio.

Translated by A. Traversarius. cf. Brit. mus. Catalogue.

Diogenis Laertii De vitis, dogmatis & apophtegamatis eorum qui in Philosophia claruerunt libri decem : Hesichii illustrii de iisdem philosophis & de aliis scriptoribus liber.

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De vitis, dogm. & apoph. clarorum philosophorum, Libri X. Hesychii II. L. de iisdem philos. & de aliis scriptoribus liber. Pythagor. philosophorum fragmenta ... Omnia graece & Lat. ex editione II. Is. Casauboni notae ad lib. Diogenis, multo auctiores & emendatiores.

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Lexicon ecclesiasticum latino-hispanicum : ex sacris biblijs, concilijs, pontificum ac theologorum decretis, divorum vitis, dictionarijs, alijsque probatissimis scriptoribus concinnatum ... /

En p. 399 empieza, "Computus est talis propire dictus manualis, si nistra situatur ... Tablas reformadas, según el kalendario gregoriano, por Estevan Pujazol ..."

Prima [-secunda] pars Promptuarii iconum insigniorum a seculo hominum : subiectis eorum vitis per compendium ex probatissimis autoribus desumptis.

CCBE S. XVI,

Promtuarii iconum insigniorum à seculo hominum, subiectis eorum vitis, per compendiu[m] ex probatissimis autoribus desumptis : prima pars [-secunda]

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Vocabularium seu lexicon Ecclesiasticum, latino- hispanicum : ex sacris bibliis, conciliis, pontificum ac theologorum decretis divorum vitis dictionariis aliisque probatissimis scriptoribus, concinnatum /

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Diogenis Laertii De vitis, dogmatibus et apophthegmatibus clarorum philosophorum libri decem : Graece et Latine

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De vitis, sectis, et dogmatibus omnium haereticorum, qui ab orbe condito, ad nostra vsque tempora & veterum & recentium authoru[m] monimentis proditi sunt, elenchus alphabeticus ... compactus /

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Cornelius Nepos De vitis excellentium imperatorum : breuiariis, et notis hispanicis illustratus a P. Francisco Xaverio de Idiaquez Soc. Jesu. ad usum scholarum ejusdem societatis.

Palau,