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21
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4243100222
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note
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32P-labeted DNA fragment amplified by polymerase chain reaction (PCR) corresponding to amino acids 1 to 53 of a partial open reading frame from S. cerevisiae (GenBank accession number P33331). The full-length gene has meanwhile been sequenced by the yeast genome sequencing project (GenBank accession number 603601).
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22
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4243195476
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Sequences were compared pairwise with the use of the Wilbur-Liprnan method, which showed 45.6% identity between yeast and Xenopvs p10 genes
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Sequences were compared pairwise with the use of the Wilbur-Liprnan method, which showed 45.6% identity between yeast and Xenopvs p10 genes.
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23
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4243139066
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The deletion of the p10 gene in strain W303 (38) was performed by integrative transformation with the use of the procedure of Rothstein (39), with modifications as described (38).
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The deletion of the p10 gene in strain W303 (38) was performed by integrative transformation with the use of the procedure of Rothstein (39), with modifications as described (38).
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24
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4243061298
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note
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To obtain recombinant p10, Nup36, or GST-ΔNup36 (29), corresponding genes were amplified with PCR primers carrying a 5′ flanking Bam HI site substituting for the initiation codon ATG and a 3′ Eco RI site. The further subctoning, expression, and protein purification steps were performed as described (18) 25. Indirect immunofluorescence was performed after 5 min of fixation of yeast spheroplasts in 3.7% formaldehyde as described elsewhere (38), with the use of an affinitypurified mouse antiserum generated against purified p10 (24), and yielded punctate nuclear peripheral staining.
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26
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4243093562
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note
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p10 (0.5 mg/ml) was dialyzed against 5 mM phosphate buffer (pH 6.0), added to a solution of 10-nm gold beads (pH 6.5) (Auro Beads G10, RPN476, Amersham) at a final concentration of 16 μg/ml, and loft for 5 mm at 20°C. Finally, the pH was adjusted to 9.0 and bovine serum albumin (BSA) was added to give a 1% final concentration. BSA-gold conjugates were prepared by means of the same protocol 28 Nuclear envelopes were prepared from S uvarum as described (24). Overlays were performed as described (13), except that dithiothreitol (DTT) was omitted from the washing buffer. p10-gold (27) was incubated with strips for 2 hours at 20°C at a 1-in-200 dilution in renaturing buffer (13) without DTT. For the competition experiment, p10 was added at a final concentration of 1.3 μM.
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28
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0027732054
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E. Coutavas, M Ren, J Oppenheim, P. D'Eustachio, M. Rush, Nature 366, 585 (1993).
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Coutavas, E.1
Ren, M.2
Oppenheim, J.3
D'Eustachio, P.4
Rush, M.5
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29
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4243188902
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unpublished results
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Nup36 is homologous to Nup2p and contains five FXFG peptide repeats, as well as a COOH-terminal RanBP1 homologous domain Nup36 was localized to the NPC (U. Nehrbass and G. Blobel, unpublished results). ΔNup36, a truncated version of Nup36, lacks the COOH-terminal RanBP1 domain amino acids 208 to 327
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Nehrbass, U.1
Blobel, G.2
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30
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4243195477
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Yeast Ran was purified and preloaded with nucleotides as described previously (20). Kap60α and Kap95β were prepared as described elsewhere (19). Liquid phase binding assays were essentially performed as described (19)
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Yeast Ran was purified and preloaded with nucleotides as described previously (20). Kap60α and Kap95β were prepared as described elsewhere (19). Liquid phase binding assays were essentially performed as described (19).
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31
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4243120415
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note
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In the absence of p10, no Ran-GDP was found in the bound fraction when added to docked karyophenn αβ. Use of GST-Nup1 (19) instead of GST-ΔNup36 gave similar results, in that Ran-GDP bound to docked karyopherin αβ in presence of p10. Accordingly, no Ran-GDP was found in the bound fraction in the absence of p10, as reported previously (19)
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32
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4243077157
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note
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Amounts of Ran in the bound fraction, as judged by the apparent intensity of the silver-stained band, decreased to about 50% of the initial value after 1 hour of incubation with 200 μM GTP. Some GST-ΔNup36 appeared to bleed off the glutathione resin (19). This corresponded to a small amount of bound karyopherin bleeding into the nonbound fraction. In addition, some of the bound karyophenn β may dissociate from the p10-β-nucleoponn complex and appear in the non-bound fraction. The quantitative release of karyophenn α with most of karyopherin β remaining bound cannot be attributed to bleeding from the column.
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33
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4243089013
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note
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This slight dissociation of both karyopherin α and β is likely to have been caused by small amounts of Ran-GTP that were present in the Ran-GDP preparation because of incomplete exchange. In the presence of p10, the contaminating Ran-GTP may not have been recruit ed into the pentamer and therefore might have been prevented from accessing the karyophenn heterodimer
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35
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0028222104
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F. R. Bischoff, C. Klebe, J. Kretschmer, A. Wittighofer, H. Ponstingl, Proc. Natl. Acad Sci. U.S A. 91, 2587 (1994).
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Bischoff, F.R.1
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Kretschmer, J.3
Wittighofer, A.4
Ponstingl, H.5
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36
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0028819762
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J. D Aitchison, M. P. Rout, M. Marelli, G. Blobel, R. W. Wozniak, J. Cell Biol 131, 1133 (1995)
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Aitchison, J.D.1
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Blobel, G.4
Wozniak, R.W.5
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38
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4243141355
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note
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We thank F Kessler and J. Aitchison for helpful discussion and A H. Bnvanlou and F Kessler for help in preparing the figures.
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