Isolation and analysis of a genomic clone encoding a pokeweed antiviral protein.

Partial cDNAs encoding a pokeweed antiviral protein were obtained by polymerase chain reaction from the poly(A)+ RNA of seeds, leaves, and roots using two specific primers based on the amino acid sequence of a pokeweed antiviral protein from the seeds (PAP-S). Using the cDNAs as a radioactive probe, 17 and 39 positive plaques were isolated from libraries containing the genomic DNA of Phytolacca americana digested with Bam HI partially and completely, respectively. The plaques were grouped into nine types by Southern hybridization. The type alpha genomic clone encodes a protein of 294 amino acids. Its amino acid sequence is similar but not identical to that of PAP-S. A comparison of the two amino acid sequences suggested that the deduced protein contains extrapeptides of 24 and 9 amino acids at the NH2 and the COOH terminals, respectively. The putative protein was expressed in Escherichia coli and shown to depurinate the specific adenine of wheat 25S rRNA, indicating that the protein encoded by a type alpha genomic clone is a functional protein exhibiting RNA N-glycosidase activity.