FIG. 4.

Expression screening of a cDNA library with the MCAT1 element yielded a partial PARP cDNA. (A) Tertiary screen of λgt11 cDNA expression library. A representative tertiary screen is shown. One of the phage clones that had survived two rounds of screening was plated out onto four plates, and the plaques were blotted onto membranes. The membranes were then probed with concatenated oligonucleotide containing either MCAT1, MCAT2, MCAT/SV40, or MCAT1mt as indicated. (B) Schematic representation of the functional domains of chicken PARP. The numbers refer to amino acid positions. The alignment of the PARP cDNA clone isolated in the library screen is shown above. (C) Gel shift analysis of the DNA binding activity of the PARP clone. Cell extracts were made from lysogens carrying an integrated copy of λgt11 (control) and the PARP fragment of one of the phage clones obtained from the library screen (PARP). MCAT1, MCAT2, MCAT/SV40, and MCAT1mt elements were radiolabelled, incubated with the extracts, and analyzed by gel retardation analysis. The method used for generating a lysogen from purified phage stock was that outlined in reference (12). The PARP-DNA complex is indicated by an arrow on the left of the figure.