Combinatorial control required for the specificity of yeast MAPK signaling

Science

Volumn 275, Issue 5304, 1997, Pages 1314-1317

  Madhani, Hiten D ^a   Fink, Gerald R ^a  

^a WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MITOGEN ACTIVATED PROTEIN KINASE; PHEROMONE; TRANSCRIPTION FACTOR;

ALLELE; ARTICLE; FUNGUS GROWTH; GENETIC TRANSCRIPTION; HAPLOIDY; PRIORITY JOURNAL; PROTEIN DOMAIN; PROTEIN FAMILY;

CA(2+)-CALMODULIN DEPENDENT PROTEIN KINASE; DNA-BINDING PROTEINS; ENHANCER ELEMENTS (GENETICS); FUNGAL PROTEINS; MAP KINASE KINASE KINASES; MITOGEN-ACTIVATED PROTEIN KINASE KINASES; MUTATION; PROTEIN KINASES; PROTEIN-SERINE-THREONINE KINASES; RETROELEMENTS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; SCHIZOSACCHAROMYCES POMBE PROTEINS; SIGNAL TRANSDUCTION; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC;

FUNGI;

EID: 0031042444     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5304.1314     Document Type: Article

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19. FRE(Ty1)::lacZ was constructed by replacing the Xho I fragment of pLG669-Z (22) with a double-stranded oligonucleotide corresponding to the Ty1 FRE (CATTCTTCTGTTTTGGAAGCTGAAACG) flanked by Xho I ends. The FRE extends from positions +60 to +86 with respect to the Ty1 δ sequence, β-galactosidase (β-Gal) assays were performed as described (8) on extracts of exponentially growing cells in liquid synthetic complete medium lacking uracil for haploid cells and liquid SLAD (synthetic low-ammonia dextrose) media (23) for diploid cells.
20. H. D. Madhani and G. R. Fink, unpublished observations.
21. The FRE(TEC1)::lacZ construct was made in the same manner as the FRE(Ty1)::lacZ construct, except that an oligonucleotide corresponding to the TEC1 FRE (TGAAACACGCACATTCC) was cloned between the Xho I sites of pLG669-Z. The TEC1 FRE extends from positions -383 to -399 with respect to the start codon.
22. The Eco RI-Xba I fragment containing the TEC1 promoter was cloned into YEp356R (2μ, URA3), which contains a promoterless E. coli lacZ gene. The PRE and TCS mutants were constructed by polymerase chain reaction (PCR) with the use of mutagenic primers and Pfu polymerase.
23. β-Gal assays were performed on cultures grown on SLAD plates for 3 days as described (8); for the TEC1::lacZ fusion, solid medium gave more reproducible results than liquid medium.
24. The Xba I-Sac I fragment containing the TEC1 coding sequence and 3′ flank was cloned in pRS306 (URA3). Wild-type and mutant promoter fragments from the TEC1::lacZ constructs were cloned up-stream of the Xba I site.
25. The TEC1 and STE12 coding sequences were amplified with Pfu polymerase and their 3′ ends were modified to include COOH-terminal FLAG tags. They were then cloned into the Bam HI and Eco RI sites of pMAL-c2 (New England Biolabs), respectively. Expression and purification were performed as described [C. M. Chiang and R. G. Roeder, Pept. Res. 6, 62 (1993)], with minor modifications. A portion of this material was purified further by chromatography on beaded amylose (New England Biolabs).
26. 32P-dCTP and Klenow fragment. The Ty1 competitor DMAs are identical except that the unpaired G residues are not present, and the PRE and TCS mutant competitors contain the appropriate base substitutions. The TEC1 competitor DNAs correspond precisely to the 17-bp TEC1 FRE with 8 bp of its native 5′ and 3′ flanking sequences. Protein-DNA complexes were allowed to form for 90 min at 24°C in binding buffer [20 mM tris-HCl (pH 8.0), 40 mM NaCl, 1 mM dithiothreitol, 5% glycerol, bovine serum albumin (1 mg/ml), and Hae III-digested salmon sperm DNA (100 μm/ml)] in a 20-μl volume. Competitor DNAs were added in a 100-fold molar excess before the addition of proteins. Samples were electrophoresed through a 5% (19:1) acrylamide gel for 3 hours at 13 V/cm, dried, and autoradiographed.
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30. We thank S. M. Noble and B. M. Cali for critically reading the manuscript and members of the laboratory and fifth floor for helpful discussions. H.D.M is a fellow of the Helen Hay Whitney Foundation. G.R.F. is an American Cancer Society Research Professor of Molecular Genetics.