Screening for protein interaction in yeast can lead to the identification of many cDNAs that are false positives. For example, the isolated cDNA encoding proteins may activate the reporter genes independent of the presence of the 'bait' protein. It is therefore important to have corroborative assays to distinguish the cDNAs of interest. The original 1-hybrid screen performed by Dalton and Treisman (1992) which resulted in the isolation of ternary complex factor SAP-1 had the advantage that there was biochemical evidence for the existence of an accessory protein, and therefore it was
relatively easy to eliminate false positives by examining the interaction in vitro. In
contrast, there is no such assay for the putative factor mediating the RhoA-dependent signalling pathway. It was therefore important to design the screen in such a way as to filter out as many false positives as possible from the initial screen.
Figure 4.1. Strategy of the 1-hybrid Screen
The 1-Hybrid screen was based on SRF bound to DNA via its own DNA binding domain expressed constitutively in the UlHis3 strain. (A) The UlHis3 strain carries an integrated SRE-His3 reporter and an episomal 4SRE-LacZ reporter. UlHis3 cells
expressing full length SRF1.508 (from plasmid ADHSRF1.508) are auxotrophic for His3
and the colonies are white on a filter LacZ assay. This strain was transformed with a Gal4 activation domain tagged cDNA library from mouse spleen. (B) Three different
types of His3 positives could have been isolated: ones that interact with SRF1-508
directly; those that interact with SRF but require some additional DNA sequences; or those that activate the His3 reporter even in the absence of SRF. (C) The LacZ reporter using the colony colour assay would have only selected for clones that interact directly with SRF, since the sequences surrounding the SREs in the two reporters are different. Clones that activated the two reporters even in the absence of SRF would be lost through the 'curing' procedure. SRF is shown as dark grey ellipse, MCMl is shown as grey circle. Gal4 activation domain tagged proteins are shown as an open box with a black flag.
M CM l SRF R e p o r t e r 1 ] His- SRE (Integrated) R e p o r t e r 2 4XSRE LacZ (Episomal) I WHITE
Library of activator-tagged fusion proteins
B SRE HIS3 (Integrated) ] OR SRE H1S3 (Integrated) ] OR H is + His+
*
4XSRE LacZ (Episomal)I
P i c k e d a s p o s i t i v e s&
SRE HIS3(Integrated) H is + 4XSRE LacZ (Episomal)
I
D i s c a r d e d t h r o u g h c u r i n g p r o c e d u r e4.2.1 Design of the initial 1-hybrid screen
The yeast screen was designed based on the model proposed by Treisman and colleagues which suggests that serum-induced transcriptional activation by SRF requires the interaction of another factor/s with its DNA binding domain (Hill et al., 1994) (Figure 1.11). The yeast screen was therefore set up so that SRF was bound to
SRE via its own DNA binding domain. For this purpose full length SRF (SRF1.508)
was expressed constitutively from the ADHl promoter. Constitutive expression of SRF has two advantages. First it ensures that SRF will be bound to its site via its own DNA binding domain at all times during the screen. Secondly it allows the transformed yeast cells to be plated on glucose as a carbon source during the screen. This not only results in higher efficiency of transformation but also allows the positive colonies to be picked directly. This is in contrast to the screen performed by Dalton and Treisman (1992) in which SRF was expressed on a galactose inducible plasmid in which case the transformed yeast cells were first plated on glucose plates followed by transfer of the colonies to plates containing galactose as the carbon source to induce SRF expression.
Two reporter genes His3 and LacZ fused to SRF binding sites upstream of a minimal CYCl promoter were used. The strategy of using two reporter genes was to increase the stringency of the screen because the positive clones were picked only if they activated both reporters, unlike the screen performed by Dalton and Treisman (1992) where only one reporter (LacZ) was used (Figure 4.1). The screen was carried out in a haploid strain UlHis3 which was kindly provided by Gunvanti Patel (Figure 4.2). UlHis3 cells are auxotrophic for His3 and require an activator to induce His3 gene expression. This strain carries SRE-His3 as an integrated reporter under the
control of a derivative of the c-fos SRE (Act.L), in which the core SRF binding site,
TCCATATTAGGA, is replaced by the sequence CCCATATATGGG, that can bind SRF and form a ternary complex with TCFs, but which cannot be bound by the endogenous yeast activator M CMl. The same SRF binding site was previously used upstream of a LacZ reporter gene (Figure 4.2) to isolate ternary complex factor SAP-1 (Dalton and Treisman, 1992). As the second reporter 4SRE-LacZ containing 4 SRE elements upstream of a LacZ gene was used. Unlike the His3 reporter, the LacZ reporter was used as an episomal low copy number plasmid. The SRF binding sites in this reporter are identical to the one in the SRE-His3 reporter, however, it does not contain the additional TCF binding site present in the Act.L sequence (Figure 4.2). This eliminates the possibility of isolating ternary complex factors as positive clones.
Figure 4.2. Sequence of the activator binding sites used in the screens
(A) Reporters in the UlHis3 and S62L strains containing SRF binding sites used in this study are shown with the SRE sequence underlined with thick line. Sequence of the TCF binding site where present is shown underlined in thin line. (B) Reporters in the HF7c strain containing Gal4 binding sites are shown with Gal4 sites underlined with thick line.