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10544246716
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note
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2-terminal hexahistidine tag and expressed in Escherichia coli strain BL21(DE3) (24). The recombinant protein was purified on nickel-nitrilotriacetic acid-agarose (Qiagen). Antisera were prepared in rabbits with the use of Ribi Adjuvant System (Sigma).
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19
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10544247613
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The whole cell extract from S. cerevisiae was prepared as described (13)
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The whole cell extract from S. cerevisiae was prepared as described (13).
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20
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10544230470
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note
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Immunodepletion of the ammonium sulfate fractionated extract was performed as follows. Fifty microliters of preimmune sera or antisera to CFT1 protein or to PTF1 protein were coupled to 40 μl of protein A-Sepharose (Sigma), equilibrated with buffer D (6) containing 0.01% NP-40 for 2 hours at 4°C. After three washings, the ammonium sulfate-fractionated extract (50 μl) was added to the resin and incubated for 4 hours at 4°C on a rotating wheel.
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23
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10544229165
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note
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Standard in vitro processing reactions were performed in a volume of 10 μl as described (6, 13, 25). Reactions were incubated for 30 min at 30°C. Plasmids used for the in vitro transcription of the 3′-terminal region of CYC1 (pSKCYC) and ADH1 (pKSADHwt) were as described (26).
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25
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10544251279
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note
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Ammonium sulfate-fractionated extract and partially purified Mono Q fractions 14 to 24 were separated on an 8% SDS-polyacrylamide gel and analyzed by immunoblotting. Proteins were detected with antisera and reagents in the enhanced chemilumenescence kit (Amersham).
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26
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0028914846
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27
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10544223069
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note
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Precleaved CYC1 RNA was obtained by gel purification of the upstream cleavage product of CYC1 pre-mRNA incubated for 45 min in an S. cerevisiae whole cell extract under standard processing conditions. To prevent polyadenylation of the upstream fragment, we replaced ATP with CTP, omitted magnesium, and included EDTA at a final concentration of 1 mM (6). Specific polyadenylation of precleaved RNA was assayed under the same conditions as for the standard cleavage and polyadenylation reaction (15) except that reactions were incubated for 60 min at 3O°C.
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29
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0029090065
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36
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10544250887
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note
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For the reconstitution of specific polyadenylation, immunodepleted extract (2 μl) was mixed with 1 μl of partially purified PAP (contained in fraction 11) and 1 μl of Mono Q fractions 19 to 27. The PAP-containing fraction was identified by an unspecific polyadenylation assay as described (6).
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37
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10544239796
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note
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We thank J. Hani for advice with the computer search, U. Sterzer for technical assistance, H. Würl for materials, and A. Goppelt and K. Wiebauer for discussions and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft.
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