MAKING BAITS

LexA fusions proteins ("baits") are typically expressed from pEG202.
Protein coding sequences can be inserted into this plasmid in a
polylinker downstream of the region encoding full length LexA (amino
acids 1 to 202, including the DNA-binding domain and the dimerization
domain).  Although LexA does not contain a functional yeast nuclear
localization signal, it and most fusions to it will be equally
partitioned between the cytoplasm and nucleus (Silver, et al. (1986)
MCB 6:4763-4766) (see TESTING BAITS).  Thus, most baits will occupy
LexA operators in the yeast nucleus (Golemis and Brent, 1992 MCB
12:3006-3014).  A small minority of baits are excluded from the
nucleus (for example, LexA-Oskar; Breitwieser and Ephrussi, personal
communication).  For these rare baits, we have available a close
relative of pEG202, a gift of Bert Vogelstein and his coworkers, which
carries the SV40 T nuclear localization signal between LexA and the
polylinker.  We also have a pEG202-related plasmid for making fusions
to the N-terminus of LexA, a gift of Manuel Sainz and Vicki Chandler.
This vector can be used in those cases where it is suspected that the
N-terminal region of a protein may be important for interaction.


LexA-Fusion Expression Plasmid, pEG202.
Erica A. Golemis

        pEG202 is a derivative of the plasmid LexA202+PL (Ruden et
al., Nature 350:250-252 (1991)) which increases the number of unique
polylinker sites available for cloning.  To create this plasmid,
LexA202+PL was cut at the unique SalI cloning site downstream of LexA
in the polylinker, and a 22mer with the sequence

                5'      TCGACCATGGCGGCCGCTCGAG
                            GGTACCGCCGGCGAGCTCAGCT  5'

was inserted (note that this double stranded oligo has sticky 5'GATC
Sal ends; to see this, you may have to display this using a
constant-spaced font) .  This oligo thus recreates flanking SalI
sites, and adds novel unique sites for NcoI, NotI, and XhoI.  The
total pEG202 polylinker sequence reads in frame from the last codon of
LexA as follows:


  CTG  GAA TTC CCG GGG ATC CGT CGA CCA TGG CGG CCG CTC GAG TCG ACC 
 LexA   ECORI  SmaI  BAMHI  SALI   NCOI    NOTI  XHOI   SALI

TGC AGC C.
PstI

        All sites indicated in capital letters are usable for
inserting fusion sequences (PstI and SmaI are not unique). This
plasmid contains the 2um origin and a HIS3 selectable marker and the
E.coli amp resistance gene.  Expression of the LexA-fusion cassette is
from the strong constitutive ADH1 promoter.  Primers for sequencing
and/or PCR can be constructed from the LexA and ADH1 terminator
sequences flanking the polylinker sites (see SEQUENCES).  There are
stop codons present in all reading frames approximately 20 codons
downstream of the PstI site.

Erica Golemis
ea_golemis@fccc.edu

June 1992
revised September 1993