Figure 1
Yeast two-hybrid bait constructs, prey libraries, and screens. (A) Bait constructs. TBP-FL, TBP-C, and TBP-N were expressed from the pDBLeu plasmid, which fused the Gal4 DNA-binding domain (DBD) upstream of TBP. N and C designate the vertebrate-specific N terminus and the pan-eukaryotic TBPCORE, respectively. (B) Three prey libraries were constructed and inserted into pPC86, which fused prey cDNAs downstream of the Gal4 activation domain (AD). Characteristics of each library are indicated. Libraries were constructed from oligo(dt)-primed placental and pregnant uteri RNA or from random-primed placenta + pregnant uteri RNA (p+u). Below is shown PCR analysis of arbitrary clones from both of the oligo(dT)-primed libraries using a primer pair that spans the multiple cloning site of the vector. Lane "λ" contained Hind III/Eco RI-cut λ-phage DNA markers. Landmark band sizes are indicated at the left of each gel; the asterisk denotes the size of the PCR product arising from empty prey vector. (C) The results of the two yeast two-hybrid screens performed are shown. In both screens, TBP-FL was used as bait to screen either the oligo(dT)- or random-primed placenta + uteri prey libraries for interacting proteins. TBP-interacting prey library clones were subsequently identified by sequencing.