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32P]orthophosphate (0.75 mCi/ml, 9000 Ci/mmol) for 15 min at 30°C, and treated with 0.4 M NaCl for 2 min before harvesting. GST-PBS2(K-M) was purified with glutathione-Sepharose (Pharmacia) [D. B. Smith and K. S. Johnson, Gene 67, 31 (1988)] followed by SDS-polyacrylamide gel electrophoresis (PAGE).
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1842343199
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note
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+ transformants were replica-plated onto YPD (yeast extract, peptone, and dextrose) plates containing 1.5 M sorbitol, and 20 osmoresistant colonies were selected. Plasmids recovered from the osmoresistant transformants revealed that 10 contained SSK2 and another 10 contained the complete STE11 open reading frame.
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17
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0029120399
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+ marker by the microhomology-mediated polymerase chain reaction (PCR) targeting method [P. Manivasakam, S. C. Weber, J. McElver, R. Schiestl, Nucleic Acids Res. 23, 2799 (1995)]. The structure of disrupted genes was verified by PCR analysis of chromosomal DNA with specific primers. For the STE gene disruptants, their sterile phenotype was also confirmed by the replica-plate mating method (18).
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Manivasakam, P.1
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Schiestl, R.4
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18
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1842349937
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unpublished observations
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F. Posas and H. Saito, unpublished observations.
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Posas, F.1
Saito, H.2
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1842353975
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note
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1173 to the COOH-terminus of Ssk2p (3).
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-
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27
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0028261404
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32P]ATP (50 Ci/mmol)]. Reactions were stopped by the addition of 2× SDS sample buffer (7), and samples were subjected to SDS-PAGE and autoradiography
-
32P]ATP (50 Ci/mmol)]. Reactions were stopped by the addition of 2× SDS sample buffer (7), and samples were subjected to SDS-PAGE and autoradiography.
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30
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1842362705
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unpublished data
-
170 to the COOH-terminus). Other constructs (GST-PBS2, PBS2HA, STE11HA, and HOG1HA) contain the full-length coding sequences of the respective genes.
-
-
-
Takekawa, M.1
Saito, H.2
-
31
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0028227829
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170 to the COOH-terminus). Other constructs (GST-PBS2, PBS2HA, STE11HA, and HOG1HA) contain the full-length coding sequences of the respective genes
-
170 to the COOH-terminus). Other constructs (GST-PBS2, PBS2HA, STE11HA, and HOG1HA) contain the full-length coding sequences of the respective genes.
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Foreman, P.K.1
Davis, R.W.2
-
32
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1842307718
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note
-
Cell extracts were prepared essentially as described (7). Protein concentration was determined with the Bio-Rad protein assay. Cell extracts (750 μg) were incubated for 5 hours at 4°C with 50 μl of glutathione-Sepharose beads in a buffer containing 150 mM NaCl. The beads were washed extensively with the same buffer, and bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, incubated with the indicated antibodies, and visualized by chemiluminescence.
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-
-
-
33
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1842313462
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note
-
We thank E. A. Witten for technical assistance, M. Takekawa and S. I. Reed for plasmids, and M. Streuli for comments on the manuscript. Supported by grants GM50909 and GM53415 from NIH (H.S.) and a postdoctoral fellowship from Ia Dirección General de Investigación Científica y Técnica of the Spanish government (F.P.).
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