Note: All the protocols utilize an overlapping set of reagents Thus, all materials necessary for the three basic protocols are presented here Plasmids, strains, and libraries are most readily obtained by writing to Roger Brent, whose address is included at the end. In addition, a number of variants of the LexA-fusion plasmids have been created to facilitate work with proteins that are toxic or require unobscured amino-terminal segments. These also are detailed in the Notes section at the end.

1 pJK202 and pEG202, his3 plasmids for making LexA fusion protein with (JK202) or without (pEG202) an incorporated nuclear localization sequence Expression is from the constitutive ADH promoter

2 pJG4-5, TRP1 plasmid for making a nuclear localization sequence-activation domain-hemagglutinin epitope tag fusion to a unique protein or a cDNA library. Expression is from the GAL1 galactose-inducible promoter

3. pl840, pJK103, orpSH18-34, URA3 plasmids containing 1, 2, or 8 LexA operators upstream of the LacZ reporter gene, respectively.

4. pJKl01, URA3 UASGAI/LexA-operator/LacZ-reporter plasmid for repression assay 5 pSH 17-4, HIS3 plasmid encoding LexA-GAL4, a positive control for activation 6. pRFHM 1, HIS3 plasmid encoding LexA-bicoid, a negative control for activation and positive control for repression.

2.2. Strains

1. Yeast strain EGY48 (MATa ura3 trpl his3 3LexAop-leu2).

2. Yeast strain EGY191 (MATa ura3 trpl his3 lLexAop-leu2).

These strains are derivatives of the strain U457 (a gift of Rodney Rothstein) in which homologous recombination was used to replace the sequences

Pstl 2.65

Hindi« 1.10 Hindlll 1.26 Psil 1.46

Xbsl 3.86

Pstl 2.65

r EcoRf .XmalSamHI .Sail .Ncol .Notl Xhol .Sall.Pstl

Seal 9.10 Pstl 8.SO

Sphi 6.10

Hindi« 1.10 Hindlll 1.26 Psil 1.46

Xbsl 3.86

Fig. 2. Key Piasmids are shown. (A) pEG202 DBD-fusion plasmids. pEG202, a derivative of Lex202+PL, (18) uses the strong constitutive ADH promoter to express bait proteins as fusions to the DNA binding protein LexA. A number of restriction sites are available for insertion of coding sequences: those shown in bold type are unique. The reading frame for insertion is GAA TTC CCG GGG ATC CGT CGA CCA TGG CGG CCG CTC GAG TCG ACC TGC AGC. The sequence CGT CAG CAG AGC TTC ACC ATT G can be used to design a primer to confirm correct reading frame for LexA fusions. The plasmid contains the H1S3 selectable marker and the 2-pm origin of replica tion to allow propagation in yeast, and the ampicillin-resistance gene and the pBR origin of replication to allow propagation in E. coli. Endpoints of the HIS3 and 2-pm elements are drawn approximately with respect to the restriction data. pJK202 is identical, except that it incorporates nine amino acids corresponding to the SV40 T antigen nuclear localization motif between LexA and polylinker sequences. (B) JG4-5 AD-fusion plasmid, pJG4-5 (2) expresses cDNAs or other coding sequences inserted into the unique EcoRl and Xhol sites as transiational fusion to a cassette consisting of the SV40 nuclear localization sequence (PPKKKRKVA), the acid blob B42 (19), and the hemagglutinin (HA) epitope tag (YPYDVPDYA). Expression of sequences is under the control of the GAL1 galactose inducible promoter. The sequence CTG AGT GGA GAT GCC TCC can be used as a primer to identify inserts or confirm correct reading frame. The plasmid contains the TRP1 selectable marker and the 2-pm origin to allow propagation in yeast, and the ampicillin resistance gene and the pUC origin to allow propagation in E. coli. (C) LacZ reporter plasmids standardly used are derivatives of LR1A1 (a deletion of RY121 described in ref. 20). LexA operator sequences arc cloned into a unique Xhol site upstream of a minimal (noninducible) GAL1 promoter fused to the LacZ gene. The plasmid contains the URA3 selectable marker and the 2-pm origin to allow propagation in yeast, and the ampicillin resistance gene and the pBR322 origin to allow propagation in E. coli. 1 §40 (21) contains a single LexA operator (binds two LexA monomers), JK103 contains an "overlapping" operator found upstream of the colEI gene (3) that binds four LexA monomers, and SH18-34 contains four colEI operators and binds up to 16 LexA monomers.

upstream of the chromosomal LEU2 gene operators for LexA derived from the colEl gene (3). EGY48 contains three such operators, and can bind up to 12 LexA monomers: EGY191 contains one such operator, and can bind up to 4 LexA monomers.

3 Yeast strain RFY206 (MATa his3 D200 leu2-3 lys2D201 ura3-52 trplD. -hisG)

4 E coll strain KC8 (pyrF leuB600 trpC hisB463)

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