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16044370274
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note
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2 and negatively stained cells by mixing them with an equal volume of a 2% aqueous solution of phosphotungstic acid (pH 7.4). Cells were spotted onto Formvar-coated grids. After 15 s, excess liquid was removed. Samples were viewed with a JEOL1200 EXII transmission electron microscope operated at 80 kV.
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P. J. Christie, J. E. Ward, S. C. Winans, E. W. Nester, J. Bacteriol. 170, 2659 (1988); N. Grimsley, B. Hohn, C. Ramos, C. Kado, P. Rogowsky, Mol. Gen. Genet. 217, 309 (1989); A Beijersbergen A. Den Dulk-Ras, R. Schilperoort, P. J. J. Hooykaas, Science 256, 1324 (1992).
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P. J. Christie, J. E. Ward, S. C. Winans, E. W. Nester, J. Bacteriol. 170, 2659 (1988); N. Grimsley, B. Hohn, C. Ramos, C. Kado, P. Rogowsky, Mol. Gen. Genet. 217, 309 (1989); A Beijersbergen A. Den Dulk-Ras, R. Schilperoort, P. J. J. Hooykaas, Science 256, 1324 (1992).
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16044365731
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note
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Nonpolar virB mutants, as described in (25), were as follows: A348ΔB1, A348ΔB2, A348ΔB3, A348ΔB5, A348ΔB6, A348B7, A348ΔB8, A348ΔB10, At12501, Ax42, and At10011.
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20
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16044375172
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thesis, University of Washington, Seattle
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K. J. Fullner, thesis, University of Washington, Seattle (1996). The virB promoter (PvirB), the virB operon, and the virD4 gene were cloned into high copy vectors to create pKJF91, pKJF72a, and pWD102, respectively. The nptll gene was first cloned adjacent to PvirB on pKJF91 and was carried through all cloning steps to provide selection of kanamycin resistance. The virD4 gene was placed under control of the virB promoter by insertion of PvirB upstream of virD4 on pWD102. PvirB-virD4 was moved onto pKJF72a adjacent to the virB operon. Both loci were moved as a single fragment onto pGP159 (26).
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38
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0023150549
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The 3.2-kb Bam HI fragment of pSW108 [S. C. Winans, P. Allenza, S. E. Stachel, K. E. McBride, E. W. Nester, Nucleic Acids Res. 15, 825 (1987)], which contains the virE operon of pTiA6, was cloned into the Bam HI site of the IncW vector pUFR047 (27) to create pWD200. The plasmid pWD200 complemented various virE mutants for tumorigenesis and production of the VirE2 protein.
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K, L. Piers, J D. Heath, X. Liang, K. M. Stephens, E. W. Nester, Proc. Natl. Acad. Sci. U.S.A. 93, 1613 (1996).
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Piers, K.L.1
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Stephens, K.M.4
Nester, E.W.5
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40
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16044365806
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note
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We thank W. Deng for constructing pWD200 and pWD102; P. Christie and A. Binns for providing strains; and C. Manoil, B. Traxler, and K. Hughes for reading the manuscript. Supported by NIH grant GM32618 and by a predoctoral fellowship to K.J.F. from the University of Washington Committee for Plant-Molecular Integration and Function.
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