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0023740032
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+ RNA and genomic DNA were purified using standard methods, and hybridizations were carried out under moderate stringency [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory Press, Boston, 1989)]. Defolliculated oocytes were injected with 50 ng RNA (50 nl), and recordings were carried out under continuous perfusion using a two-electrode voltage clamp [K. A. Eidne, in Membrane Protein Expression Systems, C. W. Gould, Ed. (Portland Press, London, 1994), pp. 1-27S], μP/μLAB amplifier, and interface ( WyeScience, Wye, Kent, UK).
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0023740032
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Cold Spring Harbor Laboratory Press, Boston
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+ RNA and genomic DNA were purified using standard methods, and hybridizations were carried out under moderate stringency [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory Press, Boston, 1989)]. Defolliculated oocytes were injected with 50 ng RNA (50 nl), and recordings were carried out under continuous perfusion using a two-electrode voltage clamp [K. A. Eidne, in Membrane Protein Expression Systems, C. W. Gould, Ed. (Portland Press, London, 1994), pp. 1-27S], μP/μLAB amplifier, and interface ( WyeScience, Wye, Kent, UK).
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(1989)
Molecular Cloning
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Sambrook, J.1
Fritsch, E.F.2
Maniatis, T.3
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7
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0023740032
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C. W. Gould, Ed. Portland Press, London
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+ RNA and genomic DNA were purified using standard methods, and hybridizations were carried out under moderate stringency [J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory Press, Boston, 1989)]. Defolliculated oocytes were injected with 50 ng RNA (50 nl), and recordings were carried out under continuous perfusion using a two-electrode voltage clamp [K. A. Eidne, in Membrane Protein Expression Systems, C. W. Gould, Ed. (Portland Press, London, 1994), pp. 1-27S], μP/μLAB amplifier, and interface ( WyeScience, Wye, Kent, UK).
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(1994)
Membrane Protein Expression Systems
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Eidne, K.A.1
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9
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84920308216
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note
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- current.
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10
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84920308215
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B. Leyman, D. Geelen, F. J. Quintero, M. R. Blatt, data not shown
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B. Leyman, D. Geelen, F. J. Quintero, M. R. Blatt, data not shown.
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11
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84920308214
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note
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The 5′ and 3′ strands were sequenced using an ABI Prism 310 sequencer (Perkin-Elmer). A full-length open reading frame was identified with methionine, upstream Kozak sequences, homology to known syntaxin sequences, and was confirmed by Northern (RNA) blot analysis.
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17
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0029949638
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M. Igarashi et al., J. Cell Biol. 134, 205 (1996); C. A. Kaiser, R. E. Gimeno, D. A. Shaywitz, in The Molecular and Cellular Biology of the Yeast Saccharomyces, J. R. Pringle, J. R. Broach, E. W. Jones, Eds. (Cold Spring Harbor Laboratory Press, Boston, 1997), p. 91; M. H. Lauber et al., J. Cell Biol. 139, 1485 (1997).
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Igarashi, M.1
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J. R. Pringle, J. R. Broach, E. W. Jones, Eds. Cold Spring Harbor Laboratory Press, Boston
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M. Igarashi et al., J. Cell Biol. 134, 205 (1996); C. A. Kaiser, R. E. Gimeno, D. A. Shaywitz, in The Molecular and Cellular Biology of the Yeast Saccharomyces, J. R. Pringle, J. R. Broach, E. W. Jones, Eds. (Cold Spring Harbor Laboratory Press, Boston, 1997), p. 91; M. H. Lauber et al., J. Cell Biol. 139, 1485 (1997).
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The Molecular and Cellular Biology of the Yeast Saccharomyces
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Kaiser, C.A.1
Gimeno, R.E.2
Shaywitz, D.A.3
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0031438153
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M. Igarashi et al., J. Cell Biol. 134, 205 (1996); C. A. Kaiser, R. E. Gimeno, D. A. Shaywitz, in The Molecular and Cellular Biology of the Yeast Saccharomyces, J. R. Pringle, J. R. Broach, E. W. Jones, Eds. (Cold Spring Harbor Laboratory Press, Boston, 1997), p. 91; M. H. Lauber et al., J. Cell Biol. 139, 1485 (1997).
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Lauber, M.H.1
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21
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84920308213
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note
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An Arabidopsis cDNA library in λYES (Clontech) was screened by colony hybridization with Nt-SYR1 cDNA. Positive inserts were sequenced, and a full-length open reading frame identified by homology with Nt-SYR1.
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22
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0028675704
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+-ATPase antibody was a gift of M. Boutry [Louvain-la-Neuve, A. D. Dexaerde et al., J. Biol. Chem. 270, 23828 (1995)]. Western blot analysis was carried out using anti-rabbit IgG-alkaline phosphatase conjugate for immunodetection [E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Boston, 1988), pp. 1-726].
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Plant Physiol.
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Daniels, M.J.1
Mirkov, T.E.2
Chrispeels, M.J.3
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23
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0040540829
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+-ATPase antibody was a gift of M. Boutry [Louvain-la-Neuve, A. D. Dexaerde et al., J. Biol. Chem. 270, 23828 (1995)]. Western blot analysis was carried out using anti-rabbit IgG-alkaline phosphatase conjugate for immunodetection [E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Boston, 1988), pp. 1-726].
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J. Biol. Chem.
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Louvain-la-Neuve1
Dexaerde, A.D.2
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24
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0003448569
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Cold Spring Harbor Laboratory Press, Boston
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+-ATPase antibody was a gift of M. Boutry [Louvain-la-Neuve, A. D. Dexaerde et al., J. Biol. Chem. 270, 23828 (1995)]. Western blot analysis was carried out using anti-rabbit IgG-alkaline phosphatase conjugate for immunodetection [E. Harlow and D. Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Boston, 1988), pp. 1-726].
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(1988)
Antibodies: A Laboratory Manual
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Harlow, E.1
Lane, D.2
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25
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0026703547
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+-acetate [R. Elble, Biotechniques 13, 18 (1992)] and selected on SD medium without uracil and containing 2% galactose. Complementation screening was carried out on YPD (containing 2% glucose) in 1% agar at 28°C.
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(1992)
Biotechniques
, vol.13
, pp. 18
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Elble, R.1
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27
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84920308212
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note
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2 and 2 mM PMSF. BotN/C or BotN/D (Sigma) were diluted to 2 μM in 20 mM NaCl and reduced with 10 mM dithiothreitol for 30 min at 37°C before adding 6 μg of membrane protein. The mix was incubated for 4 hours at 37°C before SDS-PAGE and Western blot analysis with anti-Sp2 antiserum (78).
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28
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0027413655
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T. Sollner et al., Nature 362, 318 (1993).
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Nature
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A. Grabov, J. Leung, J. Giraudat, M. R. Blatt, Plant J. 12, 203 (1997).
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Plant J.
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Grabov, A.1
Leung, J.2
Giraudat, J.3
Blatt, M.R.4
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31
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0032516044
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- currents with 15 mM tetraethylammonium chloride and 15 mM CsCl (24). Guard cells were loaded with BotN/C, BotN/D (100 nM) and Sp2 protein (20 or 100 μM) by diffusion from the microelectrode. Protein loads were estimated from diffusion assays and Western blot analysis to reach 50 nM for the toxins, and 10 μM for the Sp2 protein within 10 min, and all measurements were begun after this time.
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(1998)
Proc. Natl. Acad. Sci. U.S.A.
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Grabov, A.1
Blatt, M.R.2
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33
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A. Nishimune et al., Neuron 21, 87 (1998).
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Neuron
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Nishimune, A.1
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Y. Fujita et al., ibid. 20, 905 (1998).
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Neuron
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Fujita, Y.1
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E. F. Stanley and R. R. Mirotznik, Nature 385, 340 (1997); A. P. Naren, M. W. Quick, J. F. Collawn, D. J. Nelson, K. L. Kirk, Proc. Natl. Acad. Sci. U.S.A. 95, 10972 (1998).
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E. F. Stanley and R. R. Mirotznik, Nature 385, 340 (1997); A. P. Naren, M. W. Quick, J. F. Collawn, D. J. Nelson, K. L. Kirk, Proc. Natl. Acad. Sci. U.S.A. 95, 10972 (1998).
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Proc. Natl. Acad. Sci. U.S.A.
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Naren, A.P.1
Quick, M.W.2
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Nelson, D.J.4
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G. Thiel and N. Battey, Plant Mol. Biol. 38, 111 (1998); N. H. Battey and H. D. Blackbourn, New Phytol. 125, 307 (1993).
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Battey, N.2
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U. Homann, Planta 206, 329 (1998).
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Planta
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Homann, U.1
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45
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84920308211
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note
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Single-letter abbreviations for the amino acid rests dues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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46
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84920308210
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note
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We are grateful for support and comments from A. Grabov and G. Thiel and technical assistance from N. Grabov, B.L. was a Sainsbury Ph.D. student and was subsequently funded by UK Biotechnology and Biological Sciences Research Council grant P08406, D.G. was supported by Human Frontier Science Program grant RG303/95M, F.J.Q. was a Spanish Science Ministry Fellow and additional support came from European Commission Biotech grant CT96/0062. Nt-SYR1 and At-SYR1 are subject to patent applications UK9720784.9, UK9720785.6, and PCT/GB98/02937.
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