Isolation and cloning of the Phytolacca americana anti-viral protein PAP-I gene
Abstract
The pokeweed antiviral protein (PAP) isolated from Phytolacca americana and Phytolacca acinosa plants, inhibits protein translation by catalytically removing a specific adenine residue from the large rRNA of the 60S subunit of eukaryotic ribosomes. In this study, the P. americana PAP-I gene was isolated and sequenced, and further compared to other ribosome-inactivating proteins (RIPs) genes previously reported in GenBank(r). Total DNA was extracted from the late summer leaves of P. americana. A polymerase chain reaction (PCR) 868 bp-long DNA product was obtained, using gene specific primers, based on the expected gene size. The eluted product was purified and cloned into the pTZ57R/T vector, and mobilized into the Escherichia coli strain DH5a. After sequencing, the analysis of the PAP-I PCR product showed 98 to 82 % nucleotide and amino acid 94 to 26 % homologies, respectively, compared to previously reported RIPs. A phylogenetic analysis confirmed that the amplified PAP-I gene corresponds to the single chain Type-I RIP (PAP-I).
